CN118451197A - Methods and compositions for rolling circle amplification - Google Patents

Abstract

The present disclosure relates in some aspects to methods and compositions for detecting and quantifying multiple analytes present in a biological sample by Rolling Circle Amplification (RCA). In some aspects, the methods and compositions provided herein address the problems associated with controlling the signal spot size and intensity of RCA products (RCPs) in a sample. In some aspects, provided herein are methods and compositions (e.g., probes and/or reaction mixtures) for slowing RCA and/or reducing the size of RCA products. In some embodiments, a nucleotide or nucleotide analog having a hydrophobic group (e.g., a hydrophobic modification on a base) is included in a nucleotide for RCA and incorporated into the RCP product, resulting in a reduction in the size of the RCP without cross-linking the incorporated hydrophobic nucleotide or nucleotide analog to the RCP itself or another molecule.

Description

Methods and compositions for rolling circle amplification

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/294,037, filed on December 27, 2021, entitled “METHODS AND COMPOSITIONS FOR SLOWING ROLLING CIRCLE AMPLIFICATION,” and U.S. Provisional Patent Application No. 63/320,645, filed on March 16, 2022, entitled “METHODS AND COMPOSITIONS FOR ROLLING CIRCLE AMPLIFICATION SLOWING OR COMPACTION,” each of which is incorporated herein by reference in its entirety for all purposes.

Technical Field

The present disclosure generally relates to methods and compositions for rolling circle amplification (RCA), such as in situ detection of analytes by RCA.

Background technique

Using microscopic imaging to analyze genome, transcriptome and proteome of cell and tissue samples can simultaneously analyze multiple analytes of interest, thereby providing valuable information about analyte abundance and in situ positioning. Therefore, in situ determination (e.g., method based on rolling circle amplification (RCA)) is an important tool for, for example, understanding the molecular basis of cell identity and developing disease treatment. However, the size of the detected RCA product may limit the ability to resolve the individual RCA product corresponding to the target analyte. Large signal spots may overlap each other and/or cover adjacent smaller signal spots, making the signal spots indistinguishable. In addition, some analytes may be associated with bright signal spots (e.g., due to high analyte abundance and/or preferential signal amplification), while other analytes may be associated with signal spots that are too dark to be detected simultaneously with bright signal spots. New and improved in situ determination methods are needed. The present disclosure solves these and other needs.

Summary of the invention

In some aspects, provided herein is a method for analyzing a biological sample, the method comprising: (a) contacting the biological sample with a reaction mixture comprising one or more modified nucleotides or nucleotide analogs, wherein the one or more modified nucleotides or nucleotide analogs comprise modified nucleotides or nucleotide analogs having a hydrophobic modification, (b) performing rolling circle amplification (RCA) on a circular nucleic acid template in the biological sample using a polymerase, thereby generating an RCA product incorporating the one or more modified nucleotides or nucleotide analogs, wherein the RCA product is not cross-linked by the one or more modified nucleotides or nucleotide analogs incorporated into the RCA product, and (c) detecting an RCA product at a certain position in the biological sample that is not cross-linked by the one or more modified nucleotides or nucleotide analogs.

In some embodiments, the RCA product is not cross-linked to another molecule via one or more modified nucleotides or nucleotide analogues incorporated into the RCA product. In some embodiments, the RCA product is not cross-linked to itself, another molecule in the biological sample, or a matrix in which the biological sample is embedded, via one or more modified nucleotides or nucleotide analogues incorporated into the RCA product.

In some embodiments, the hydrophobic modification is a base modification. In some embodiments, the hydrophobic modification includes a carbon chain and/or a hydrocarbon ring. In some embodiments, the hydrophobic modification includes a triple bond. In some embodiments, the hydrophobic modification includes a vinyl or ethynyl group. In some embodiments, the modified nucleotide or nucleotide analog comprising a hydrophobic modification is ethynyl-dUTP or vinyl-dUTP. In some embodiments, the modified nucleotide or nucleotide analog comprising a hydrophobic modification is 5-ethynyl-dUTP or 5-vinyl-dUTP.

In some embodiments, the diameter of an RCA product generated using one or more modified nucleotides or nucleotide analogues is smaller than a reference RCA product produced using the same template without including the one or more modified nucleotides or nucleotide analogues in the reaction mixture.

In some embodiments, a modified nucleotide or nucleotide analog comprising a hydrophobic modification is added to a biological sample at a concentration of at least 1 μM, at least 1.25 μM, at least 2.5 μM, at least 5 μM, at least 10 μM, at least 40 μM, at least 80 μM, or at least 100 μM. In some embodiments, the modified nucleotide or nucleotide analog is a modified dUTP, and the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 1:99. In some embodiments, the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 40:60. In some embodiments, a modified nucleotide or nucleotide analog comprising a hydrophobic modification is added to a biological sample at a concentration of about 50 μM to about 100 μM. In some embodiments, the modified nucleotide or nucleotide analog comprising a hydrophobic modification is added to the biological sample at a concentration of about 80 μM to about 100 μM.

In some embodiments, the median diameter of the RCA products generated using one or more modified nucleotides or nucleotide analogues is less than the median diameter of a reference RCA product generated using the same template without including the one or more modified nucleotides or nucleotide analogues in the reaction mixture. In some embodiments, the median diameter of the RCA products generated using one or more modified nucleotides or nucleotide analogues is less than 500 nm. In some embodiments, the median diameter of the RCA products generated using one or more modified nucleotides or nucleotide analogues is no more than 90% or no more than 80% of the median diameter of the reference RCA product generated using the same template without including the one or more modified nucleotides or nucleotide analogues in the reaction mixture.

In some embodiments, the average intensity, average signal-to-noise ratio and/or density of RCA products incorporating one or more modified nucleotides or nucleotide analogs are not significantly different from the average intensity, average signal-to-noise ratio and/or density of a reference RCA product produced using the same template without including one or more modified nucleotides or nucleotide analogs in the reaction mixture.

In some embodiments, incorporating one or more modified nucleotides or nucleotide analogs into the RCA product increases the overall hydrophobicity of the RCA product. In some embodiments, incorporating one or more modified nucleotides or nucleotide analogs into the RCA product promotes base stacking interactions between nucleotides in the RCA product.

In some embodiments, one or more modified nucleotides or nucleotide analogs incorporated into the RCA product do not include a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, detecting the RCA product includes contacting the biological sample with a nucleic acid probe that can hybridize with the RCA product. In some embodiments, the nucleic acid probe includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, the nucleic acid probe is an intermediate probe, and the method includes contacting the biological sample with a detectably labeled probe that can hybridize with the intermediate probe.

In some aspects, provided herein is a method for analyzing a biological sample, the method comprising: (a) contacting the biological sample with a reaction mixture comprising one or more modified nucleotides or nucleotide analogs, (b) performing rolling circle amplification (RCA) on a circular nucleic acid template in the biological sample using a polymerase to generate an RCA product, wherein the one or more modified nucleotides or nucleotide analogs comprise: (i) an incorporable nucleotide or an analog thereof, which is not incorporated by the polymerase, and/or (ii) an incorporable modified nucleotide or nucleotide analog, which is configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate, and (c) detecting an RCA product at a certain position in the biological sample that is not cross-linked by the modified nucleotide or nucleotide analog.

In some embodiments, the RCA product is not cross-linked to the RCA product itself, another molecule in the biological sample, or a matrix in which the biological sample is embedded, by any modified nucleotide or nucleotide analogue incorporated into the RCA product. In some embodiments, one or more modified nucleotides or nucleotide analogues incorporated into the RCA product do not comprise a detectable label. In some embodiments, the detectable label is a fluorophore.

In some embodiments, detecting the RCA product comprises contacting the biological sample with a nucleic acid probe that can hybridize to the RCA product. In some embodiments, the nucleic acid probe comprises a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, the nucleic acid probe is an intermediate probe, and the method comprises contacting the biological sample with a detectably labeled probe that can hybridize to the intermediate probe.

In some embodiments, the presence of one or more modified nucleotides or nucleotide analogs in the reaction mixture reduces the polymerization rate of the polymerase and/or the size of the RCA product compared to a reference reaction mixture without the one or more modified nucleotides or nucleotide analogs. In some embodiments, the reference reaction mixture comprises only unmodified dATP, dTTP and/or dUTP, dCTP and dGTP.

In some aspects, provided herein is a method for analyzing a biological sample, the method comprising: (a) contacting the biological sample with a circular probe or a circularizable probe or a probe set comprising one or more modified nucleotide or nucleotide analog residues, wherein the circular probe or the circularizable probe or the probe set comprises a hybridization region that hybridizes to a target nucleic acid in the biological sample and wherein the one or more modified nucleotide or nucleotide analog residues are outside the hybridization region, and (b) performing rolling circle amplification (RCA) on the circular probe or the circularized probe generated by the circularizable probe or the probe set using a polymerase to generate an RCA product, wherein the presence of the one or more modified nucleotide or nucleotide analog residues reduces the polymerization rate of the polymerase on the circular or circularized probe and/or the size of the RCA product compared to a reference circular template without the one or more modified nucleotide or nucleotide analog residues.

In some embodiments, one or more modified nucleotides or nucleotide analog residues comprise modified deoxyribonucleotides (DNA) or DNA analog residues. In some embodiments, one or more modified nucleotides or nucleotide analog residues comprise modified ribonucleotides (RNA) or RNA analog residues. In some embodiments, the method includes detecting an RCA product at a position in a biological sample.

In some aspects, a method for analyzing a biological sample is provided herein, the method comprising: (a) contacting the biological sample with a circular probe or a circularizable probe or a probe set comprising one or more modified nucleotides or nucleotide analog residues, wherein the circular probe or the circularizable probe or the probe set is hybridized with a target nucleic acid in the biological sample, and (b) using a polymerase to perform rolling circle amplification (RCA) on the circular probe or the circularized probe generated by the circularizable probe or the probe set, thereby generating an RCA product, wherein the presence of one or more modified nucleotides or nucleotide analog residues reduces the polymerization rate of the polymerase on the circular or circularized probe compared to a reference circular template without one or more modified nucleotides or nucleotide analog residues. In some embodiments, the one or more modified nucleotides or nucleotide analog residues may comprise sugar-modified nucleotides. In any of the foregoing embodiments, the one or more modified nucleotides or nucleotide analog residues may comprise C2' modified nucleotides. In any of the foregoing embodiments, the one or more modified nucleotides or nucleotide analog residues may have a C3' endo pucker conformation.

In any of the foregoing embodiments, one or more modified nucleotides or nucleotide analog residues may include one or more modified ribonucleotide residues. In some embodiments, the circular probe or circularized probe does not include unmodified ribonucleotide residues. Alternatively, in some embodiments, the circular probe or circularized probe may include one or more unmodified ribonucleotide residues. In any of the foregoing embodiments, one or more modified nucleotides or nucleotide analog residues may be selected from the group consisting of the following items: 2'-O-methyl ribonucleic acid (2'-OMeRNA), locked nucleic acid (LNA) nucleotides, 2'-fluoro ribonucleic acid (2'-F RNA) and combinations thereof. In any of the foregoing embodiments, one or more modified nucleotides or nucleotide analog residues may include 2'-OMeRNA. In any of the foregoing embodiments, one or more modified nucleotides or nucleotide analog residues may include modified deoxyribonucleotide residues. In any of the foregoing embodiments, the circular probe or circularized probe may be mainly composed of deoxyribonucleotide residues. In some embodiments, the circular probe or circularized probe may be composed of deoxyribonucleotide residues. In any of the foregoing embodiments, the circular probe or cyclization probe may contain no more than 20%, no more than 10%, no more than 5% or no more than 1% of modified nucleotides or nucleotide analog residues and/or modified or unmodified ribonucleotide residues. In any of the foregoing embodiments, the circular probe or cyclization probe may contain no more than two, no more than three, no more than four or no more than five consecutive modified nucleotides or nucleotide analog residues. In any of the foregoing embodiments, the circular probe or cyclization probe may contain no more than two, no more than three, no more than four or no more than five consecutive modified or unmodified ribonucleotide residues. In any of the foregoing embodiments, one or more modified nucleotides or nucleotide analog residues may contain one or more triazole or thiol bonds instead of phosphodiester groups. In any of the foregoing embodiments, the cyclic or cyclizable template may contain one or more thiol bonds between residues, and the one or more thiol bonds are selected from thiophosphate and phosphorothioate bonds. In any of the foregoing embodiments, the method may include contacting the biological sample with a circularizable probe or probe set comprising one or more thiol bonds, wherein the one or more thiol bonds are not located at the 5' or 3' end of the circularizable probe or probe set. In any of the foregoing embodiments, the circular probe or circularizable probe or probe set may comprise two or more triazole bonds. In any of the foregoing embodiments, the circular probe or circularizable probe or probe set may comprise two or more thiol bonds. In any of the foregoing embodiments, at least two of the two or more triazole bonds or the two or more thiol bonds may be separated by less than 100 nucleotides. In any of the foregoing embodiments, the circular probe or circularizable probe or probe group may be a first circular probe or circularizable probe or probe group hybridized with a first target nucleic acid in a sample, and the method may further include: contacting the biological sample with a second circular or circularizable probe or probe group, wherein the second circular or circularizable probe or probe group hybridizes with a second target nucleic acid in the biological sample; and performing rolling circle amplification (RCA) on the second circular probe or the second circularized probe generated by the second circularizable probe or probe group using a polymerase, thereby generating a second RCA product. In some embodiments, the second circular probe or circularizable probe or probe group may not include modified nucleotides or nucleotide analog residues. In some embodiments, the second circular probe or circularizable probe or probe group may include less modified nucleotides or nucleotide analog residues than the first circular probe or circularizable probe or probe group. In some embodiments, the second circular probe or circularizable probe or probe group may include modified nucleotides or nucleotide analog residues different from the first circular probe or circularizable probe or probe group. In some embodiments, the second circular probe or circularizable probe or probe group does not include modified nucleotides or nucleotide analog residues. In any of the foregoing embodiments, the polymerization rate of the polymerase on the first circular or circularized probe can be slower than the polymerization rate of the polymerase on the second circular or circularized probe. In any of the foregoing embodiments, the method may include contacting the biological sample with a reaction mixture comprising one or more nucleotides and/or nucleotide analogs, wherein the one or more nucleotides and/or nucleotide analogs include: (i) non-incorporable nucleotides or nucleotide analogs, which are transiently bound to the polymerase but not incorporated by the polymerase, and/or (ii) incorporable nucleotides or nucleotide analogs, which are incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphates.

In some aspects, a method for analyzing a biological sample is provided herein, the method comprising: (a) contacting the biological sample with a reaction mixture comprising one or more nucleotides or their analogs, and (b) performing rolling circle amplification (RCA) on a circular nucleic acid template in the biological sample using a polymerase to generate an RCA product, wherein the one or more nucleotides or their analogs comprise: (i) a non-incorporable nucleotide or its analog, which is transiently bound to the polymerase but not incorporated by the polymerase, and/or (ii) an incorporable nucleotide or its analog, which is configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate. In some embodiments, the one or more nucleotides or their analogs may comprise: a non-incorporable nucleotide or nucleotide analog, which is transiently bound to the polymerase but not incorporated by the polymerase; and an incorporable nucleotide or nucleotide analog, which is configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate. In any of the foregoing embodiments, the average open time for the polymerase to incorporate an incorporable nucleotide or its analog during polymerization can be longer than the average open time for incorporating a corresponding nucleotide triphosphate (e.g., an unmodified dNTP, such as dATP, dTTP, dUTP, dCTP, or dGTP). In some embodiments, the average open time for the polymerase to incorporate an incorporable nucleotide during polymerization is at least 125%, 150%, 200%, 225%, or 250% of the average open time for incorporating a corresponding nucleoside triphosphate. In any of the foregoing embodiments, the incorporable nucleotide or its analog can comprise an α-thiol nucleotide. Additionally or alternatively, in any of the foregoing embodiments, the incorporable nucleotide or its analog can comprise a diphosphate nucleotide. Additionally or alternatively, in any of the foregoing embodiments, the incorporable nucleotide can comprise an azide-modified nucleotide or nucleotide analog and/or an alkyne-modified nucleotide or nucleotide analog. In some embodiments in which one or more incorporable nucleotides comprise a click-functionalized nucleotide, the method does not include performing a click reaction. In any of the foregoing embodiments, the reaction mixture may include one or more incorporable nucleotides or incorporable nucleotide analogs, wherein the incorporable nucleotides or incorporable nucleotide analogs are selected from alpha thiol nucleotides, diphosphate nucleotides, azide-modified nucleotides or nucleotide analogs, alkyne-modified nucleotides or nucleotide analogs, and combinations thereof. In some embodiments, the reaction mixture does not include unmodified deoxyribonucleotide triphosphates. In some embodiments, the method does not include contacting the biological sample with unmodified deoxyribonucleotide triphosphates. In some embodiments, no more than 50%, 40%, 30%, 20%, 10%, 5% or 1% of the nucleotides or their analogs in the reaction mixture are non-incorporable nucleotides or their analogs and/or incorporable nucleotides or their analogs. For example, at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the nucleotides or their analogs in the reaction mixture may be unmodified deoxyribonucleotides. In any of the foregoing embodiments, the non-incorporable nucleotides or their analogs may be monophosphate nucleotides. In any of the foregoing embodiments, non-incorporable nucleotide analogs may be dissociated from the polymerase. In any of the foregoing embodiments, the average polymerization rate of the polymerase in rolling circle amplification may be inversely proportional to the concentration of one or more nucleotides or nucleotide analogs that may or may not be incorporated in the reaction mixture. In any of the foregoing embodiments, the average polymerization rate of the polymerase in rolling circle amplification may be less than 2280nt/min, less than 2000nt/min, less than 1500nt/min, less than 1250nt/min, less than 1000nt/min, less than 750nt/min, less than 500nt/min, or less than 250nt/min. In any of the foregoing embodiments, rolling circle amplification may be performed at a temperature between 18°C and 30°C. In some embodiments, rolling circle amplification is performed at 30°C.

In some aspects, a method for analyzing a biological sample is provided herein, the method comprising: (a) contacting the biological sample with a reaction mixture comprising one or more nucleotide analogs, and (b) performing rolling circle amplification (RCA) on a circular nucleic acid template in the biological sample using a polymerase, thereby generating an RCA product incorporating one or more nucleotide analogs, wherein the one or more nucleotide analogs comprise nucleotide analogs containing hydrophobic modifications. In some embodiments, the hydrophobic modification may be a base modification. In some embodiments, the hydrophobic modification may include a carbon chain. In some embodiments, the hydrophobic modification may include a triple bond. In some embodiments, the hydrophobic modification may include a vinyl or ethynyl group. In some embodiments, the nucleotide analog comprising the hydrophobic modification may be ethynyl-dUTP or vinyl-dUTP. In some such embodiments, the nucleotide analog comprising the hydrophobic modification is 5-ethynyl-dUTP or 5-vinyl-dUTP. In some embodiments, the diameter of the RCA product generated using one or more nucleotide analogs may be smaller than the reference RCA product generated using the same template without including one or more nucleotide analogs in the reaction mixture. In some embodiments, the nucleotide analogs comprising hydrophobic modifications may be added to the sample at a concentration of at least 1 μM, at least 1.25 μM, at least 2.5 μM, at least 5 μM, at least 10 μM, at least 40 μM, at least 80 μM, or at least 100 μM. In some embodiments, the nucleotide analogs may be modified dUTPs, and the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture may be between about 80:20 and about 1:99, optionally wherein the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture may be between about 80:20 and about 40:60. In some embodiments, the nucleotide analogs comprising hydrophobic modifications may be added to the sample at a concentration of about 50 μM to about 100 μM, optionally wherein the nucleotide analogs comprising hydrophobic modifications may be added to the sample at a concentration of about 80 μM to about 100 μM. In some embodiments, the median diameter of the RCA product generated using one or more nucleotide analogs may be less than the median diameter of the reference RCA product generated using the same template without including one or more nucleotide analogs in the reaction mixture. In some such embodiments, the median diameter of the RCA product generated using one or more nucleotide analogs may be less than 500nm. In some embodiments, the RCA product may be in the form of a nanosphere having a diameter between about 0.1 μm and about 3 μm, optionally wherein the diameter is between about 0.1 μm and about 0.5 μm, between about 0.5 μm and about 1 μm, between about 0.8 μm and about 1.3 μm, or between about 1 μm and about 1.5 μm. In some embodiments, the median diameter of the RCA product generated using one or more nucleotide analogs may be no more than 90% or no more than 80% of the median diameter of the reference RCA product generated using the same template without including one or more nucleotide analogs in the reaction mixture. In some embodiments, the average intensity, average signal-to-noise ratio and/or density of the RCA product incorporating one or more nucleotide analogs may be not significantly different from the average intensity, average signal-to-noise ratio and/or density of the reference RCA product produced using the same template without including one or more nucleotide analogs in the reaction mixture. In some embodiments, the diameter of the RCA product is less than the diameter of the reference RCA product. In some embodiments, the diameter of the RCA product is the average diameter, and the diameter of the reference RCA product is the average diameter. In some embodiments, the diameter of the RCA product is no more than 90%, no more than 80% or no more than 70% of the diameter of the reference RCA product. In some embodiments, the diameter of the RCA product is less than 700nm, less than 600nm or less than 500nm. In some embodiments, the method includes detecting an RCA product signal associated with the RCA product, and determining the size of the RCA product based on the RCA product signal. In some embodiments, the method does not include crosslinking the RCA product with itself, crosslinking with one or more other molecules in the biological sample and/or crosslinking with the matrix of the embedded biological sample or its molecules before determining the diameter of the RCA product. In some embodiments, the method does not include crosslinking the RCA product. In some embodiments, the average intensity, average signal to noise ratio and/or density of the RCA product signal are not significantly different from the average intensity, average signal to noise ratio and/or density of the reference RCA product signal associated with the reference RCA product. In some embodiments, compared with the reference RCA product, one or more nucleotide analogs in the RCA product increase the overall hydrophobicity of the RCA product. In some embodiments, one or more nucleotide analogs are incorporated into the RCA product to increase the overall hydrophobicity of the RCA product. In some embodiments, one or more nucleotide analogs are incorporated into the RCA product to promote the base stacking interactions between the nucleotides in the RCA product. In any of the aforementioned embodiments, linear RCA, branched RCA, dendritic RCA or any combination thereof can be used to generate the RCA product. In any of the foregoing embodiments, the RCA product can be generated using a polymerase selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and variants or derivatives thereof. In some embodiments, the polymerase is Phi29 polymerase, Vent DNA polymerase, or Bst DNA polymerase. In some embodiments, the polymerase is Phi29 polymerase. In any of the foregoing embodiments, the RCA product may be generated in situ in a biological sample or in a matrix in which the biological sample or its molecules are embedded. In any of the foregoing embodiments, the RCA product may be cross-linked with one or more other molecules in the biological sample and/or in the matrix in which the biological sample or its molecules are embedded. In any of the foregoing embodiments, the method may include detecting the RCA product in situ in a biological sample or in a matrix in which the biological sample or its molecules are embedded. In some embodiments, detecting the RCA product may include contacting the biological sample with one or more detectably labeled probes, which directly or indirectly hybridize with one or more barcode sequences contained in the RCA product. In some embodiments, the signal associated with the RCA product may be amplified in situ in a biological sample or in a matrix in which the biological sample or its molecules are embedded. In some embodiments, signal amplification may include RCA of a probe directly or indirectly bound to a rolling circle amplification (RCA) product; a hybridization chain reaction (HCR) directly or indirectly on an RCA product; a linear oligonucleotide hybridization chain reaction (LO-HCR) directly or indirectly on an RCA product; a primer exchange reaction (PER) directly or indirectly on an RCA product; assembling a branched structure directly or indirectly on an RCA product; hybridizing multiple detectable probes directly or indirectly on an RCA product, or any combination thereof. In any of the foregoing embodiments, the method may further include terminating the rolling circle amplification by thermal denaturation or by contacting the biological sample with a polymerase inhibitor. In some embodiments, terminating the rolling circle amplification may include contacting the biological sample with a polymerase inhibitor, the polymerase inhibitor being selected from the group consisting of a pyrophosphate analog, a polymerase allosteric inhibitor, a non-catalytic ion bound to a polymerase, and a chain terminating nucleotide. In any of the foregoing embodiments, the circular nucleic acid template may be a circular probe or a circularized probe generated by a circularizable probe or probe set, wherein the circular probe or the circularizable probe or probe set is hybridized with a target nucleic acid in a biological sample. In some embodiments, the target nucleic acid can be used as a connection template to generate a circularized probe by a circularizable probe or a probe group. In some embodiments, the circularizable probe group can include two, three or more probes. In some embodiments, enzymatic ligation and/or chemical ligation can be used to generate a circularized probe. In some embodiments, template-dependent ligation and/or non-template-dependent ligation can be used to generate a circularized probe. In any of the aforementioned embodiments, a ligase with RNA-templated DNA ligase activity and/or RNA-templated RNA ligase activity can be used to generate a circularized probe. In any of the aforementioned embodiments, a ligase selected from the group consisting of Chlorella virus DNA ligase (PBCV DNA ligase), T4 RNA ligase, T4 DNA ligase and single-stranded DNA (ssDNA) ligase can be used to generate a circularized probe. In some embodiments, PBCV-1 DNA ligase or its variant or derivative and/or T4 RNA ligase 2 (T4 Rnl2) or its variant or derivative is used to generate a circularized probe. In any of the aforementioned embodiments, the RCA product can include one or more barcode sequences or their complementary sequences. In some embodiments, one or more barcode sequences or their complements correspond to a target nucleic acid or a portion thereof. In any of the foregoing embodiments, the RCA product may comprise between about 10 and about 100, between about 100 and about 1,000, between about 1,000 and about 5,000, between about 5,000 and about 10,000, or more than 10,000 copies of a circular nucleic acid template or a circular or circularized probe. In any of the foregoing embodiments, the RCA product may be in the form of a nanosphere having a diameter between about 0.1 μm and about 3 μm, optionally wherein the diameter is between about 0.1 μm and about 0.5 μm, between about 0.5 μm and about 1 μm, between about 0.8 μm and about 1.3 μm, or between about 1 μm and about 1.5 μm. In any of the foregoing embodiments, the length of the RCA product can be between about 1 kilobase and about 15 kilobases, between about 15 kilobases and about 25 kilobases, between about 25 kilobases and about 35 kilobases, between about 35 kilobases and about 45 kilobases, between about 45 kilobases and about 55 kilobases, between about 55 kilobases and about 65 kilobases, between about 65 kilobases and about 75 kilobases, or more than 75 kilobases. In any of the foregoing embodiments, the target nucleic acid can include DNA and/or RNA. In some embodiments, the target nucleic acid can be a reporter oligonucleotide of a marker that is directly or indirectly bound to an analyte in a biological sample, such as genomic DNA/RNA, mRNA, cDNA, or a marker. In any of the foregoing embodiments, the method can also include crosslinking the RCA product with itself, crosslinking with one or more other molecules in the biological sample, and/or crosslinking with a matrix that embeds the biological sample or its molecules. In some embodiments, the crosslinking reduces the mobility of the amplified product in the biological sample and/or in the matrix. In any of the foregoing embodiments, the biological sample can be a fixed and/or permeabilized biological sample. In any of the foregoing embodiments, the biological sample can be a non-homogenized tissue sample or tissue section. In some embodiments, the biological sample can be a formalin-fixed paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample. In some embodiments, the biological sample can be a tissue section with a thickness between about 1 μm and about 50 μm, optionally wherein the thickness of the tissue section is between about 5 μm and about 35 μm. In some embodiments, the biological sample can be cross-linked. In some embodiments, the biological sample can be embedded in a matrix, optionally wherein the matrix is a hydrogel. In some embodiments, the biological sample can be transparentized.

In some aspects, provided herein is a method for analyzing a biological sample, the method comprising: (a) contacting the biological sample with: (i) a first circular probe or a circularizable probe or a probe set comprising one or more modified nucleotide or nucleotide analog residues, wherein the first circular probe or the circularizable probe or the probe set hybridizes with a first target nucleic acid in the biological sample, and (ii) a second circular probe or a circularizable probe or a probe set, wherein the second circular probe or the circularizable probe or the probe set hybridizes with a second target nucleic acid in the biological sample, and (b) (i) performing rolling circle amplification (RCA) on the first circular probe or the first circularized probe generated by the first circularizable probe or the probe set using a polymerase to generate a first RCA product, and (ii) performing rolling circle amplification (RCA) on the second circular probe or the second circularized probe generated by the second circularizable probe or the probe set using a polymerase to generate a second RCA product. In some embodiments, the presence of one or more modified nucleotide or nucleotide analog residues reduces the polymerization rate of the polymerase on the first circular or circularized probe compared to a reference circular template without the one or more modified nucleotide or nucleotide analog residues. In some embodiments, the polymerization rate of the polymerase on the first circular or circularized probe is slower than the polymerization rate of the polymerase on the second circular or circularized probe.

In some embodiments, the second circular probe or circularizable probe or probe set does not contain modified nucleotides or nucleotide analog residues. In some embodiments, the second circular probe or circularizable probe or probe set contains less modified nucleotides or nucleotide analog residues than the first circular probe or circularizable probe or probe set. In some embodiments, the first target nucleic acid is more abundant in the biological sample than the second target nucleic acid.

In some aspects, a kit for performing rolling circle amplification is provided herein, the kit comprising: (a) a circular probe or a circularizable probe or a probe set comprising one or more modified nucleotides or nucleotide analog residues, wherein the circular probe or the circularizable probe or the probe set comprises a target hybridization region complementary to a target sequence of a target nucleic acid; and (b) a polymerase; wherein the one or more modified nucleotides or nucleotide analog residues reduce the polymerization rate of the polymerase in a rolling circle amplification reaction on the circular probe or the circularized probe generated by the circularizable probe or the probe set compared to a reference circular template without the one or more modified nucleotides or nucleotide analog residues. In some embodiments, the kit may also include a ligase for generating a circularized probe from the circularizable probe or the probe set. In some embodiments, the one or more modified nucleotides or nucleotide analog residues may include a nucleotide residue modified with a sugar and/or a nucleotide residue modified with a backbone. In some embodiments, one or more modified nucleotides or nucleotide analog residues can be selected from the group consisting of: 2'-O-methyl ribonucleic acid (2'-OMeRNA), locked nucleic acid (LNA) nucleotides, 2'-fluoro ribonucleic acid (2'-F RNA), phosphorothioate backbone nucleotides, phosphorothioate backbone nucleotides, triazole modified nucleotides, and combinations thereof. In any of the foregoing embodiments, the kit may also include: one or more non-incorporable nucleotides or nucleotide analogs, the one or more non-incorporable nucleotides or nucleotide analogs being configured to transiently bind to the polymerase but not be incorporated by the polymerase; and/or one or more incorporable nucleotides or nucleotide analogs, the one or more incorporable nucleotides or nucleotide analogs being configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate, optionally wherein the one or more non-incorporable nucleotides. In some aspects, a kit for rolling circle amplification is provided herein, the kit comprising: (a) a polymerase; (b) one or more non-incorporable nucleotides or nucleotide analogs, the one or more non-incorporable nucleotides or nucleotide analogs being configured to bind transiently to the polymerase but not incorporated by the polymerase; and/or one or more incorporable nucleotides or nucleotide analogs, the one or more incorporable nucleotides or nucleotide analogs being configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate; and (c) one or more circular probes or circularizable probes or probe sets for generating circularized probes as rolling circle amplification templates, or one or more reagents for generating circularized templates for rolling circle amplification. In any of the foregoing embodiments, the one or more non-incorporable nucleotides or nucleotide analogs and/or the one or more incorporable nucleotides or nucleotide analogs may comprise α-thiol nucleotides, diphosphate nucleotides and/or monophosphate nucleotides. In any of the foregoing embodiments, the kit may also comprise unmodified deoxyribonucleotide triphosphates. In any of the foregoing embodiments of the kit, a combination of one or more non-incorporable nucleotides or nucleotide analogs, one or more incorporable nucleotides or nucleotide analogs, and/or unmodified deoxyribonucleotide triphosphates can be included in a reaction mixture for rolling circle amplification. In some aspects, a kit for rolling circle amplification is provided herein, the kit comprising: (a) a polymerase; (b) one or more nucleotide analogs comprising one or more hydrophobic modifications and configured to be incorporated by a polymerase; and (c) one or more circular probes or circularizable probes or probe sets for generating circularized probes as templates for rolling circle amplification, or one or more reagents for generating circularized templates for rolling circle amplification. In some embodiments, one or more nucleotide analogs may comprise a modified dUTP selected from ethynyl-dUTP and/or vinyl dUTP. In some embodiments, one or more nucleotide analogs may be provided as a reaction mixture comprising one or more nucleotide analogs and unmodified deoxyribonucleotide triphosphates. In some embodiments, the ratio of modified dUTP to unmodified dTTP in the reaction mixture may be at least about 80:20.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

Figures 1A to 1B show schematic diagrams illustrating exemplary circular or circularized probes with (Figure 1A) or without (Figure 1B) one or more modified nucleotide or nucleotide analog residues and rolling circle amplification (RCA) of the probe. One or more modified nucleotide or nucleotide analog residues can reduce the polymerization rate during RCA, thereby producing smaller RCA products compared to probes with unmodified nucleotide residues (e.g., residues with A, T, C or G bases and unmodified/natural sugar-phosphate backbones) at corresponding positions in the probe under otherwise identical RCA conditions (e.g., reaction temperature, time, etc.). Figure 1C shows the structure of an unmodified DNA residue and an exemplary modified RNA residue (2'-OMeRNA) with a modified sugar moiety. Figure 1D shows the structure of an exemplary modified nucleotide or nucleotide analog residue or nucleotide analog, which comprises a modified backbone that forms a triazole bond with another residue.

Fig. 2A shows a schematic diagram illustrating an exemplary method for analyzing a biological sample using RCA with a reaction mixture comprising (i) non-incorporable nucleotides (e.g., dNMPs or modified nucleotides) or nucleotide analogs that are not incorporated (e.g., the non-incorporable nucleotides or nucleotide analogs are configured to be only transiently bound to a polymerase and not incorporated into an RCA product), and/or (ii) incorporable nucleotides or nucleotide analogs that are configured to be incorporated by a polymerase at a slower rate than a corresponding nucleoside triphosphate (e.g., dNTPs having an A, T/U, C or G base and an unmodified/natural sugar-phosphate backbone). As shown, the reaction mixture may comprise unmodified dNTPs. FIG2B shows exemplary structures of (i) exemplary non-incorporable nucleotides (e.g., deoxyribonucleoside monophosphates, dNMPs) that can transiently bind to a polymerase and compete with other incorporable nucleotides for binding to the polymerase, and (ii) exemplary incorporable nucleotides or nucleotide analogs (e.g., deoxyribonucleoside diphosphates (dNDPs) and alpha-thiol nucleotides) that are incorporated by a polymerase at a slower rate than the corresponding dNTP.

Figure 3 shows a graph indicating the density (detected object counts per unit nuclear area) of in situ RCA products detected under the indicated experimental conditions. The concentrations of modified dNTPs (5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP) are shown as a percentage of total dNTPs. Points represent the density of experimental replicates.

Figure 4 shows a graph indicating the average size of in situ RCA product signals detected under the indicated experimental conditions. The concentrations of modified dNTPs (5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP) are shown as a percentage of total dNTPs. Points represent the average size of experimental replicates.

Figure 5 shows a graph indicating the average signal intensity of the in situ RCA product signal above the local background under the indicated experimental conditions. The concentration of modified dNTPs (5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP) is shown as a percentage of total dNTPs. Points represent the average intensity of experimental replicates.

Figure 6A shows the molecular structures of modified dNTPs 5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP used in the in situ RCA and quantitative RCA experiments described herein. Figure 6B shows the results of quantitative rolling circle amplification (qRCA) when 0%, 50% or 100% of the indicated dNTPs (5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP alone or in combination) and negative controls were added. The y-axis corresponds to relative fluorescence units (RFU) and the x-axis corresponds to imaging cycles over time.

Figure 7 shows the molecular structures of modified dNTPs 5-ethynyl-dUTP (5-EdUTP) and 5-vinyl-dUTP used in the in situ RCA experiments described herein. Dashed circles indicate increasing hydrophobic modifications (eg, hydrophobic groups).

Figures 8A to 8D show the results of RCA experiments when modified dNTPs: 5-ethynyl-dUTP (5-EdUTP) or 5-vinyl-dUTP are added at indicated concentrations. The control (Ctrl) condition does not include modified dNTPs. Figure 8A shows a bar graph indicating the density of RCA products detected under the indicated experimental conditions (RCA products detected per unit area of imaged cell nucleus). There is no data for the 40μM 5-EdUTP condition. Figure 8B shows a bar graph indicating the signal intensity (average signal intensity above local background) of RCA products detected under the indicated experimental conditions. Figure 8C shows a bar graph indicating the signal-to-noise ratio (average local signal-to-noise average) of RCA products detected under the indicated experimental conditions. Figure 8D shows a bar graph indicating the signal intensity (average signal-to-background ratio average) of RCA products detected under the indicated experimental conditions.

Figures 9A to 9B show the results of RCA experiments when the indicated concentrations of modified dNTPs were added: 5-ethynyl-dUTP (5-EdUTP, Figure 9A) or 5-vinyl-dUTP (Figure 9B). The control (Ctrl) conditions did not include modified dNTPs. The violin plots indicate the size of the RCA products detected under the indicated experimental conditions. The points represent the size of a single detected RCA product and the shapes represent the size distribution.

Figures 10A-10B show the results of RCA experiments when the indicated concentrations of modified dNTPs were added: 5-ethynyl-dUTP (5-EdUTP, Figure 10A) or 5-vinyl-dUTP (Figure 10B). The control (Ctrl) condition did not include the modified dNTPs. The graphs show the size distribution of RCA products detected under the indicated conditions in the form of probability (upper graph) and cumulative probability (middle graph). The table (lower graph) shows the summary statistics of the replicates for each condition. The results show that modified dNTPs containing hydrophobic modifications reduce the size of RCA products.

Detailed ways

All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or contradictory to a definition set forth in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. Overview

In assays involving in situ rolling circle amplification (RCA), uneven size and intensity distribution of rolling circle amplification products (RCPs) can lead to sensitivity loss because weak signals associated with certain RCPs may not exceed the detection threshold for detection of RCP signals (e.g., "spots") for image analysis, while strong signals associated with certain RCPs in close proximity can lead to optical crowding and failure to detect nearby weaker signal spots. Both undetected weak RCPs and overcrowded large RCPs can lead to sensitivity loss. In some aspects, the ability to adjust the polymerization rate will allow more control over the RCA reaction and the resulting diameter of the RCP signal spots. For example, increasing control over the RCA rate can make it easier to terminate the RCA reaction at a time point when the RCA product reaches an optimal size for detection (e.g., as small as the detection limit allows).

For a variety of reasons, controlling the polymerization rate of an RCA reaction (e.g., an RCA reaction performed in situ in a biological sample such as a tissue sample) may be challenging. The polymerization rate may depend on temperature. For example, the rate of Phi29 polymerase at 30°C is about 2280nt/min, at 15°C it is about 1490nt/min, and at 4°C it is about 290nt/min (Soengas et al., 1995 J. Mol. Biol. 253 (4): 517-529, the contents of which are incorporated herein by reference in their entirety). Therefore, lowering the reaction temperature of RCA theoretically has a significant effect on the polymerization rate. However, for some applications, it may be desirable to control the polymerization rate of RCA without changing the temperature, for example, to reduce the complexity of the instrument required to control the temperature, and/or to adjust the polymerization rate of RCA performed on multiple samples at the same temperature. In addition, performing RCA at some lower temperatures may not be feasible, for example because buffer or sample preservative components (e.g., tissue preservatives or transparentizing agents) will precipitate.

In some aspects, the present disclosure provides methods and compositions for changing the rate of rolling circle amplification using an RCA template (e.g., a circular or circularized probe) comprising one or more modified nucleotides or nucleotide analog residues, wherein the one or more modified nucleotides slow down the polymerization rate of a polymerase on the template. In some embodiments, provided herein is a method for analyzing a biological sample, the method comprising: (a) contacting the biological sample with a circular probe or a circularizable probe or a probe set comprising one or more modified nucleotides or nucleotide analog residues, wherein the circular probe or the circularizable probe or the probe set hybridizes with a target nucleic acid in the biological sample, and (b) performing rolling circle amplification (RCA) on the circular probe or the circularized probe generated by the circularizable probe or the probe set using a polymerase, thereby generating an RCA product, wherein the presence of one or more modified nucleotides or nucleotide analog residues in the circular probe or the circularized probe reduces the polymerization rate of the polymerase using the circular probe or the circularized probe as a template compared to a reference circular template without the one or more modified nucleotides or nucleotide analog residues. In some embodiments, the reference circular template is a template that does not contain one or more modified nucleotides or nucleotide analog residues, but is otherwise identical to the circular or circularized probe. For example, the reference circular template can have the same sequence as the circular or circularized probe, but contains unmodified nucleotides or nucleotide analog residues to replace one or more modified nucleotides or nucleotide analog residues of the circular or circularized probe.

One or more modified nucleotides or nucleotide analog residues may include, for example, sugar-modified nucleotides and/or nucleotide residues with triazole or thiol bonds. In some embodiments, one or more modified nucleotides may include C2' modified nucleotides. In some embodiments, one or more modified nucleotides or nucleotide analog residues may have a C3' endofolding conformation. One or more modified nucleotides or nucleotide analog residues may include one or more modified ribonucleotides or deoxyribonucleotide residues. Examples of sugar-modified nucleotides or nucleotide analog residues include, but are not limited to, 2'-OMeRNA, locked nucleic acid (LNA) nucleotides, 2'-F RNA, and combinations thereof. In some embodiments, a circular probe or a circularizable probe or probe set includes one or more thiol bonds, such as thiophosphate or phosphorothioate bonds, between residues. In some embodiments, a circular probe or a circularizable probe or probe set includes two or more triazole bonds and/or thiol bonds. In some embodiments, two or more triazole bonds and/or thiol bonds are separated by less than 100 nucleotides.

In some embodiments, one or more modified nucleotide or nucleotide analog residues include 2'-OMeRNA (e.g., 1, 2, 3, 4 or more 2'-OMeRNA residues). A single 2'-OMeRNA residue included in an exemplary circular template has been shown to reduce the rolling circle amplification rate by about 90% for Phi29 polymerase, by about 40% for Vent DNA polymerase, and by about 30% for Bst DNA polymerase. Therefore, it is recommended that 2'-OMeRNA should be avoided in RCA applications. See, e.g., Tang et al. (2016) Bioscience, Biotechnology, and Biochemistry, 80:8, 1555-1561, the contents of which are incorporated herein by reference in their entirety. Therefore, some aspects of the present disclosure are based in part on the recognition that strong inhibition of polymerization rate using a template comprising 2'-OMeRNA may be advantageous in certain applications, such as in situ detection of analytes by RCA. In some embodiments, a circular probe or circularizable probe or probe set comprises 2′-OMeRNA residues equal to or approximately equal to between any of: between 1 and 10, between 1 and 5, between 1 and 4, between 1 and 3, between 1 and 2, between 2 and 10, between 2 and 5, between 2 and 4, or between 5 and 10. In some embodiments, a circular probe or circularizable probe or probe set comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2′-OMeRNA residues.

In some aspects, the present disclosure provides a method for facilitating differential adjustment of polymerization rate in RCA reaction between different circular probes or circularizable probes or probe groups, because the polymerization rate in RCA reaction can be associated with modified nucleotides or nucleotide analog residues (such as any modified nucleotides or nucleotide analog residues described herein, for example, comprising modified sugars, thiol bonds and/or triazole bonds) in the RCA template itself. In some embodiments, circular probes or circularizable probes or probe groups comprising different modified nucleotides or nucleotide analog residues and/or different numbers of modified nucleotides or nucleotide analog residues can be designed to hybridize with different target nucleic acids. For example, a circular or circularizable probe or probe group hybridized with a first target nucleic acid in a sample can be designed to provide a circular or circularized probe amplified at a slower rate than a circular or circularized probe hybridized with a second target nucleic acid. In such an example, the first target nucleic acid may be more abundant than the second target nucleic acid. In some embodiments, the method provided herein includes: contacting a biological sample with a first circular probe or a circularizable probe or a probe group, which is hybridized with a first target nucleic acid in the sample; and contacting the biological sample with a second circular or circularizable probe or a probe group, wherein the second circular or circularizable probe or a probe group is hybridized with a second target nucleic acid in the biological sample; and using a polymerase to (i) a first circular probe or a first circularized probe generated by a first circularizable probe or a probe group and (ii) a second circularized probe or a second circularized probe generated by a second circularizable probe or a probe group to perform rolling circle amplification (RCA), thereby generating a first RCA product and a second RCA product. In some embodiments, the polymerization rate of the polymerase on the first circular or circularized probe is slower than the polymerization rate of the polymerase on the second circular or circularized probe. In some embodiments, the first RCA product is smaller than the second RCA product. In some embodiments, the first circular probe or circularized probe or probe group comprises one or more modified nucleotides, and the second circular probe or circularized probe or probe group does not comprise modified nucleotides or nucleotide analog residues. In other embodiments, the first and second circular probes or circularized probes or probe groups can all comprise one or more modified nucleotides. In some embodiments, the second circular probe or circularized probe comprises less modified nucleotides or nucleotide analog residues than the first circular probe or circularized probe. In some embodiments, the second circular probe or circularized probe comprises modified nucleotides or nucleotide analog residues different from the first circular probe or circularized probe.

In some aspects, the present disclosure provides a method for adjusting the polymerization rate of a polymerase in an RCA reaction by including one or more molecules that reduce the polymerization rate in a reaction mixture. In some embodiments, a method for analyzing a biological sample is provided herein, the method comprising: (a) contacting the biological sample with a reaction mixture comprising one or more nucleotides or their analogs, and (b) performing rolling circle amplification (RCA) on a circular nucleic acid template in the biological sample using a polymerase, thereby generating an RCA product, wherein the one or more nucleotides or their analogs include: (i) non-incorporable nucleotides or their analogs, which are transiently bound to the polymerase but not incorporated by the polymerase, and/or (ii) incorporable nucleotides or their analogs, which are configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate. The circular template can be any suitable circular template for rolling circle amplification, such as a circular probe, a circularized probe generated by a circularizable probe or a probe set, or a circularized cDNA molecule. The circularized cDNA molecules are used as circular nucleic acid templates for RCA in methods such as FISSEQ, for example as described in Lee et al., Science. 2014; 343(6177): 1360-1363, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the rate of RCA can be modified using any combination of the methods and compositions described herein. The provided method for analyzing a biological sample can include (a) using a circular or circularized probe as an RCA template comprising one or more modified nucleotide or nucleotide analog residues, wherein the inclusion of the modified nucleotide in the template reduces the polymerization rate of a polymerase using the circular or circularized probe as a template compared to a reference circular template; (b) contacting the biological sample with a reaction mixture for rolling circle amplification, the reaction mixture comprising one or more non-incorporable nucleotides or analogs thereof (e.g., non-incorporable analogs themselves), wherein the non-incorporable nucleotides or analogs thereof are transiently bound to the polymerase but not incorporated by the polymerase; and/or (c) contacting the biological sample with a reaction mixture for rolling circle amplification, the reaction mixture comprising incorporable nucleotides or analogs thereof (e.g., non-incorporable analogs themselves), wherein the incorporable nucleotides or analogs thereof are configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate, the method either comprising (a), (b) and (c) alone, or comprising any combination of (a), (b) and (c).

In some embodiments, the disclosure provides a method for adjusting the size of the RCA product by including one or more nucleotide analogs comprising hydrophobic modifications in the reaction mixture of RCA. In some embodiments, the RCA product incorporates one or more nucleotide analogs comprising hydrophobic modifications, and for example, compared to the reference RCA product generated in the absence of nucleotide analogs, the hydrophobic modification in the RCA product causes the overall size of the RCA product to be reduced. In some embodiments, incorporating one or more nucleotide analogs into the RCA product can promote base stacking interactions between nucleotides in the RCA product, thereby reducing the overall size of the RCA product. In some embodiments, there is provided herein a method for analyzing a biological sample, the method comprising: (a) contacting the biological sample with a reaction mixture comprising one or more nucleotide analogs, and (b) using a polymerase to perform rolling circle amplification (RCA) on a circular nucleic acid template in the biological sample, thereby generating an RCA product incorporating one or more nucleotide analogs, wherein the one or more nucleotide analogs comprise nucleotide analogs containing hydrophobic modifications. In some embodiments, the hydrophobic modification is a base modification. In some embodiments, the hydrophobic modification comprises a carbon chain and/or a hydrocarbon ring, a triple bond and/or a vinyl or ethynyl group. In some embodiments, the nucleotide analog comprising a hydrophobic modification is ethynyl-dUTP or vinyl-dUTP, such as 5-ethynyl-dUTP or 5-vinyl-dUTP, respectively.

In the following sections, additional descriptions of various aspects of the methods, compositions and kits disclosed herein are provided. Section II describes exemplary biological samples and analytes (e.g., products of endogenous analytes, labeling agents or endogenous nucleic acid analytes), which can be detected using the method for analyzing biological samples as described herein (e.g., by detecting a target nucleic acid as an analyte or associated with an analyte). Section III describes exemplary circular probes or circularizable probes or probe groups, which can be circularized according to the method provided herein to be used as RCA templates. Section IV describes RCA reactions and reaction mixtures. Section V describes nucleotides, nucleotide analogs and nucleotide modifications related to the methods and compositions described herein. Section VI describes a method for detecting the RCA product in a sample. Section VII provides a kit according to the present disclosure. Section VIII describes exemplary applications of the present method, compositions and kits. As described above, the section titles used herein are only used for organizational purposes and should not be construed as limiting the described subject matter.

II. Samples, Analytes, and Target Sequences

A. Sample

Samples disclosed herein can be or are derived from any biological sample. Methods and compositions disclosed herein can be used to analyze biological samples, which can be obtained from a subject using any of a variety of techniques (including but not limited to biopsy, surgery, and laser capture microscopy (LCM)), and generally include cells and/or other biological materials from a subject. In addition to the subject described above, biological samples can be obtained from prokaryotes (such as bacteria, archaea, viruses, or viroids). Biological samples can also be obtained from non-mammalian organisms (e.g., plants, insects, arthropods, nematodes, fungi, or amphibians). Biological samples can also be obtained from eukaryotic organisms, such as tissue samples, patient-derived organoids (PDO) or patient-derived xenografts (PDX). Biological samples from organisms can include one or more other organisms or components thereof. For example, in addition to mammalian cells and non-cellular tissue components, mammalian tissue sections can include prions, viroids, viruses, bacteria, fungi, or components from other organisms. The subject from whom a biological sample can be obtained can be a healthy or asymptomatic individual, an individual having or suspected of having a disease (e.g., a patient having a disease such as cancer) or susceptible to a disease, and/or an individual in need of therapy or suspected of being in need of therapy.

Biological samples can include any number of macromolecules, for example, cellular macromolecules and organelles (for example, mitochondria and nuclei). Biological samples can include nucleic acids (such as DNA or RNA), proteins/polypeptides, carbohydrates and/or lipids. Biological samples can be obtained as tissue samples (such as tissue sections, biopsy samples, core needle biopsy samples, needle aspirates or fine needle aspirates). The sample can be a fluid sample, such as a blood sample, a urine sample or a saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histological sample, a histopathological sample, a plasma or serum sample, a tumor sample, a living cell, a cultured cell, a clinical sample (for example, whole blood or blood-derived products, hemocytes or cultured tissues or cells, including cell suspensions). In some embodiments, the biological sample can include cells deposited on the surface.

A cell-free biological sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from body samples such as blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, and tears.

The biological sample may be derived from a homogenous culture or population of the subjects or organisms mentioned herein, or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

Biological samples can include one or more diseased cells. Diseased cells may have altered metabolic properties, gene expression, protein expression and/or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

The biological sample may include an analyte (e.g., protein, RNA, and/or DNA) embedded in a 3D matrix. In some embodiments, an amplicon (e.g., rolling circle amplification product) derived from an analyte (e.g., protein, RNA, and/or DNA) or associated therewith may be embedded in a 3D matrix. In some embodiments, the 3D matrix may include natural molecules and/or synthetic molecular networks chemically and/or enzymatically connected (e.g., by crosslinking). In some embodiments, the 3D matrix may include a synthetic polymer. In some embodiments, the 3D matrix includes a hydrogel.

In some embodiments, the substrate herein can be any support that is insoluble in aqueous liquids, and allows positioning biological samples, analytes, features and/or reagents (e.g., probes) on the support. In some embodiments, biological samples can be attached to substrates. The attachment of biological samples can be irreversible or reversible, depending on the subsequent steps in the properties of the sample and the analytical method. In certain embodiments, by applying a suitable polymer coating to substrates and contacting the sample with the polymer coating, the sample can be reversibly attached to the substrate. Then, for example, using an organic solvent that at least partially dissolves the polymer coating, the sample can be separated from the substrate. Hydrogels are examples of polymers suitable for this purpose.

In some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

Various steps may be performed to prepare or process a biological sample for and/or during an assay. Unless otherwise indicated, the preparation or processing steps described below may generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and/or analysis.

(i) Tissue sections

Biological samples can be obtained from subjects (e.g., by surgical biopsy, whole subject sectioning) or grown in vitro as cell populations on growth substrates or culture dishes and prepared for analysis as tissue slices or tissue sections. The grown samples can be thin enough to be analyzed without further processing steps. Alternatively, the grown samples and samples obtained via biopsy or sectioning can be prepared into thin tissue sections using mechanical cutting devices such as vibrating blade microtomes. As another alternative, in some embodiments, thin tissue sections can be prepared by applying a touch impression of the biological sample to a suitable substrate material.

The thickness of the tissue section can be a fraction of the maximum cross-sectional dimension of the cells (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1). However, tissue sections having a thickness greater than the maximum cross-sectional cell dimension can also be used. For example, cryostat sections can be used, which can be, for example, 10-20 μm thick.

More generally, the thickness of the tissue section usually depends on the method for preparing the section and the physical properties of the tissue, so sections with various thicknesses can be prepared and used. For example, the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40 or 50 μm. If necessary or convenient, thicker sections can also be used, such as at least 70, 80, 90 or 100 μm or thicker. Typically, the thickness of the tissue section is between 1-100 μm, 1-50 μm, 1-30 μm, 1-25 μm, 1-20 μm, 1-15 μm, 1-10 μm, 2-8 μm, 3-7 μm or 4-6 μm, but as mentioned above, sections with thickness greater than or less than these ranges can also be analyzed.

Multiple slices can also be obtained from a single biological sample. For example, multiple tissue slices can be obtained from a surgical biopsy sample by serially slicing the biopsy sample using a microtome blade. Spatial information between serial slices can be preserved in this way, and the slices can be analyzed continuously to obtain three-dimensional information about the biological sample.

(ii) Freezing

In some embodiments, biological samples (e.g., tissue sections as described above) can be prepared by deep freezing at a temperature suitable for maintaining or preserving the integrity (e.g., physical properties) of the tissue structure. Any number of suitable methods can be used to slice (e.g., thin slices) frozen tissue samples onto the substrate surface. For example, a freezing microtome (e.g., a cryostat) can be used to prepare tissue samples at a temperature suitable for maintaining the structural integrity of the tissue sample and the chemical properties of the nucleic acid in the sample. Such a temperature can be, for example, below -15 ° C, below -20 ° C, or below -25 ° C.

(iii) Fixation and post-fixation

In some embodiments, established method formalin fixation and paraffin embedding (FFPE) can be used to prepare biological samples. In some embodiments, formalin fixation and paraffin embedding can be used to prepare cell suspensions and other non-tissue samples. After fixing the sample and being embedded in paraffin or resin blocks, the sample can be sliced as described above. Before analysis, it is possible to remove paraffin embedded materials (e.g., dewaxing) from tissue sections by incubating tissue sections in appropriate solvents (e.g., xylene), then rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).

As an alternative to formalin fixation described above, biological samples can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, the sample can be fixed by immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

In some embodiments, acetone fixation is used for fresh frozen samples, which may include but are not limited to cortical tissue, mouse olfactory bulb, human brain tumor, human postmortem brain and breast cancer samples. When acetone fixation is performed, a pre-permeabilization step (as described below) may not be performed. Alternatively, acetone fixation may be performed in combination with a permeabilization step.

In some embodiments, the method provided herein includes one or more post-fixing (post-fixing) (also referred to as post-fixation) steps. In some embodiments, one or more post-fixing steps are performed after contacting the sample with a polynucleotide disclosed herein (e.g., one or more probes, such as circular or circularizable probes, such as padlock probes). In some embodiments, one or more post-fixing steps are performed after the hybridization complex comprising the probe and the target is formed in the sample. In some embodiments, one or more post-fixing steps are performed before the ligation reaction disclosed herein (such as the connection that circularizable probes (such as, padlock probes) are circularized).

In some embodiments, one or more post-fixation steps are performed after contacting the sample with a binding agent or labeling agent (e.g., an antibody or its antigen binding fragment) for a non-nucleic acid analyte such as a protein analyte. The labeling agent may include a nucleic acid molecule (e.g., a reporter oligonucleotide) comprising a sequence corresponding to the labeling agent and therefore corresponding to the analyte (e.g., uniquely identified). In some embodiments, the labeling agent may include a reporter oligonucleotide containing one or more barcode sequences.

The post-fixation step can be performed using any suitable fixation reagent disclosed herein (eg, 3% (w/v) paraformaldehyde in DEPC-PBS).

(iv) Embedding

As an alternative to paraffin embedding described above, biological samples can be embedded in any of a variety of other embedding materials to provide a structural substrate for the sample before sectioning and other processing steps. In some cases, the embedding material can be removed, for example, before analyzing the tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, wax, resin (e.g., methacrylic resin), epoxy resin, and agar.

In some embodiments, the biological sample can be embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this way generally involves contacting the biological sample with a hydrogel so that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material and activating the polymer material to form a hydrogel. In some embodiments, forming a hydrogel allows the hydrogel to be internalized in the biological sample.

In some embodiments, the biological sample is immobilized in the hydrogel by cross-linking of the hydrogel-forming polymeric material. The cross-linking may be performed chemically and/or photochemically, or alternatively by any other suitable hydrogel-forming method.

The composition of the hydrogel-matrix and the application to the biological sample generally depend on the nature and preparation (e.g., sliced, non-sliced, fixed type) of the biological sample. As an example, when the biological sample is a tissue slice, the hydrogel-matrix may include a monomer solution and an ammonium persulfate (APS) initiator/tetramethylethylenediamine (TEMED) accelerator solution. As another example, when the biological sample is composed of cells (e.g., cultured cells or cells dissociated from a tissue sample), the cells may be incubated with a monomer solution and an APS/TEMED solution. For cells, the hydrogel-matrix gel is formed in a compartment, which includes, but is not limited to, a device for culturing, maintaining or transporting cells. For example, a hydrogel-matrix may be formed to a depth range of about 0.1 μm to about 2 mm with a monomer solution added to the compartment plus APS/TEMED.

Additional methods and aspects of hydrogel embedding of biological samples are described, for example, in Chen et al., Science 347(6221):543–548, 2015, the entire contents of which are incorporated herein by reference.

(v) Staining and immunohistochemistry (IHC)

For the convenience of visualization, various staining agents and staining techniques can be used to stain biological samples. For example, in some embodiments, any number of staining agents and/or immunohistochemical reagents can be used to stain samples. One or more staining steps can be performed to prepare or process biological samples for determinations described herein, or can be performed during and/or after determination. In some embodiments, samples can be contacted with one or more nucleic acid stains, membrane stains (for example, cell membranes or nuclear membranes), cytological stains, or combinations thereof. In some instances, staining can be specific to the compartment of proteins, phospholipids, DNA (for example, dsDNA, ssDNA), RNA, organelles, or cells. Samples can be contacted with one or more labeled antibodies (for example, first antibodies specific to an analyte of interest and second antibodies specific to a labeling of the first antibody). In some embodiments, one or more images taken of the stained sample can be used to segment the cells in the sample.

In some embodiments, lipophilic dyes are used for staining. In some instances, lipophilic carbocyanine or aminostyrene dyes or their analogs (e.g., DiI, DiO, DiR, DiD) are used for staining. Other cell membrane stains may include FM and RH dyes or immunohistochemical reagents specific to cell membrane proteins. In some instances, stains may include but are not limited to acridine orange, acid fuchsin, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hoechst stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (e.g., rhodamine B) or safranin or their derivatives. In some embodiments, hematoxylin and eosin (H&E) may be used to stain the sample.

Samples can be stained using hematoxylin and eosin (H&E) staining techniques, using Papanicolaou staining techniques, Masson's trichrome staining techniques, silver staining techniques, Sudan staining techniques, and/or using periodic acid Schiff (PAS) staining techniques. PAS staining is usually performed after formalin or acetone fixation. In some embodiments, Romanovsky stains (including Wright's stain, Jenner's stain, Can-Grunwaldstain, Leishman stain, and Giemsa stain) can be used to stain samples.

In some embodiments, the biological sample can be decolorized. Any suitable method for destaining or decolorizing the biological sample can be used, and these methods generally depend on the nature of the stain applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample by antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide bonds by washing with a reducing agent and a detergent, treatment with a chaotropic salt, treatment with an antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiple staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444; Lin et al., Nat Commun. 2015; 6: 8390; Pirici et al., J. Histochem. Cytochem. 2009; 57: 567–75; and Glass et al., J. Histochem. Cytochem. 2009; 57: 899–905, the entire contents of each of which are incorporated herein by reference.

(vi) Isometric Expansion

In some embodiments, the biological sample embedded in the matrix (e.g., hydrogel) can be expanded isovolumetrically. The isovolumetric expansion method that can be used includes hydration, which is a preparation step in expansion microscopy, such as (e.g.) Chen et al., Science 347 (6221): 543–548, 2015 and U.S. Patent No. 10,059,990, each of which is incorporated herein by reference in its entirety.

Isovolumetric expansion can be performed by anchoring one or more components of the biological sample to a gel, followed by gel formation, protein hydrolysis, and swelling. In some embodiments, the analyte in the sample, the product of the analyte, and/or the probe associated with the analyte in the sample can be anchored to a matrix (e.g., a hydrogel). Isovolumetric expansion of the biological sample can occur before the biological sample is fixed to a substrate or after the biological sample is fixed to a substrate. In some embodiments, the isochoric expanded biological sample can be removed from the substrate before contacting the substrate with the probe disclosed herein.

In general, the steps used to perform isochoric expansion of a biological sample may depend on the characteristics of the sample (e.g., thickness of tissue sections, fixation, cross-linking) and/or the analytes of interest (e.g., different conditions for anchoring RNA, DNA, and proteins to a gel).

In some embodiments, the protein in the biological sample is anchored to a swellable gel (such as a polyelectrolyte gel). Antibodies can be directed to proteins before, after, or in conjunction with anchoring to a swellable gel. DNA and/or RNA in a biological sample can also be anchored to a swellable gel through a suitable joint. Examples of such joints include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X SE) (available from ThermoFisher, Waltham, MA), Label-IT amine (available from MirusBio, Madison, WI) and Label X (e.g., described in Chen et al., Nat. Methods 13: 679-684, 2016 and U.S. Patent No. 10,059,990, the entire contents of each document are incorporated herein by reference).

Isovolumetric expansion of a sample can increase the spatial resolution of subsequent analysis of the sample. The increase in resolution in the spatial distribution can be determined by comparing an isochoric expanded sample with a sample that has not been isochoric expanded.

In some embodiments, the biological sample is isovolumetrically expanded to a size of at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x of its unexpanded size. In some embodiments, the sample is isovolumetrically expanded to at least 2x and less than 20x of its unexpanded size.

(vii) Crosslinking and decrosslinking

In some embodiments, before or during the original position determination, biological sample is reversibly cross-linked. In some respects, the amplified product (for example, amplicon) of analyte, polynucleotide and/or analyte or the probe combined therewith can be anchored on the polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more polynucleotide probes and/or its amplified product (for example, amplicon) can be modified to contain a functional group, and this functional group can be used as the anchoring point that polynucleotide probe and/or amplified product are connected to the polymer matrix. In some embodiments, the modified probe comprising few dT can be used for combining interested mRNA molecules, followed by reversible or irreversible cross-linking of mRNA molecules.

In some embodiments, the biological sample is fixed in the hydrogel by crosslinking of the polymer material forming the hydrogel. Crosslinking can be carried out by chemical and/or photochemical means, or alternatively by any other suitable hydrogel formation method. The hydrogel can include a macromolecular polymer gel containing a network. In a network system, some polymer chains can be optionally crosslinked, but crosslinking does not always occur.

In some embodiments, the hydrogel can include hydrogel subunits such as, but not limited to, acrylamide, bisacrylamide, polyacrylamide and its derivatives, poly(ethylene glycol) and its derivatives (e.g., PEG-acrylate (PEG-DA), PEG-RGD), methacrylated gelatin (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate) and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, etc., and combinations thereof.

In some embodiments, the hydrogel comprises a hybrid material, for example, the hydrogel material comprises elements of a synthetic polymer and a natural polymer. Examples of suitable hydrogels are described in, for example, U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422 and U.S. Patent Application Publication Nos. 2017/0253918, 2018/0052081, and 2010/0055733, the entire contents of each document being incorporated herein by reference.

In some embodiments, the hydrogel can form a substrate. In some embodiments, the substrate includes a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on the top of the one or more second materials. For example, the hydrogel can be preformed and then placed on the top, bottom or any other configuration with the one or more second materials. In some embodiments, the hydrogel forms after contacting the one or more second materials during the formation of the substrate. The hydrogel formation can also occur in the structure (e.g., hole, ridge, protrusion and/or texture) located on the substrate.

In some embodiments, hydrogel formation on the substrate occurs before, simultaneously with, or after providing the probe to the sample. For example, hydrogel formation can be performed on a substrate that already contains the probe.

In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., a tissue section) is embedded in a hydrogel. In some embodiments, a hydrogel subunit is injected into a biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.

In the embodiment of forming hydrogel in biological sample, functionalization chemistry can be used.In some embodiments, functionalization chemistry includes hydrogel-tissue chemistry (HTC).Any hydrogel-tissue skeleton (for example, synthetic or natural) suitable for HTC can be used for anchoring biomacromolecules and regulating functionalization.Non-limiting examples of methods using HTC skeleton variants include CLARITY, PACT, ExM, SWITCH and ePACT.In some embodiments, the hydrogel formation in biological sample is permanent.For example, biomacromolecules can be permanently adhered to hydrogel, allowing multiple rounds of inquiry.In some embodiments, the hydrogel formation in biological sample is reversible.

In some embodiments, additional reagents are added to the hydrogel subunits before, simultaneously and/or after polymerization. For example, additional reagents may include, but are not limited to, oligonucleotides (e.g., probes), endonucleases for fragmenting DNA, fragmentation buffers for DNA, DNA polymerases, dNTPs for amplifying nucleic acids and connecting barcodes to amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, ligases, proteinase K, and DNA enzymes. Additional reagents may also include reverse transcriptases (including enzymes with terminal transferase activity), primers, and switch oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, simultaneously and/or after polymerization.

In some embodiments, the HTC agent is added to the hydrogel before, simultaneously with, and/or after polymerization. In some embodiments, the cell labeling agent is added to the hydrogel before, simultaneously with, and/or after polymerization. In some embodiments, the cell permeation agent is added to the hydrogel before, simultaneously with, and/or after polymerization.

Any suitable method can be used to make the hydrogel embedded in the biological sample transparent. For example, electrophoretic tissue clearing methods can be used to remove biomacromolecules from hydrogel-embedded samples. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixing medium, methylcellulose or other semi-solid medium) before or after the hydrogel is cleared.

In some embodiments, the methods disclosed herein include decrosslinking a reversibly crosslinked biological sample. Decrosslinking does not need to be complete. In some embodiments, only a portion of the crosslinked molecules in the reversibly crosslinked biological sample are decrosslinked and allowed to migrate.

(viii) Tissue permeabilization and processing

In some embodiments, biological samples can be permeabilized to facilitate the transfer of species (such as probes) into the sample. If the sample is not fully permeabilized, the amount of species (such as probes) transferred into the sample may be too low to be fully analyzed. On the contrary, if the tissue sample permeability is too strong, the relative spatial relationship of the analyte in the tissue sample may be lost. Therefore, it is ideal to fully permeabilize the tissue sample to obtain good signal intensity while still maintaining a balance between the spatial resolution of the analyte distribution in the sample.

Typically, biological samples can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100 ^TM or Tween-20 ^TM ) and enzymes (e.g., trypsin, protease). In some embodiments, biological samples can be incubated with cell permeabilizing agents to promote the permeabilization of samples. Other methods for sample permeabilization are described in, for example, Jamur et al., Method Mol.Biol.588:63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable sample permeabilization method can generally be used in combination with samples as described herein.

In some embodiments, the biological sample can be permeabilized by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive agents such as lysing enzymes for lysing different cell types (e.g., Gram-positive or negative bacteria, plants, yeasts, mammals), such as lysozymes, colorless peptidases, lysostaphin, labiase, kitalase, lysomucin, and various other commercially available lysing enzymes.

Other lysing agents can be added to the biological sample additionally or alternatively to promote permeabilization. For example, a lysing solution based on a surfactant can be used for lysing sample cells. The lysing solution can include an ionic surfactant, such as sodium lauryl sarcosinate and sodium lauryl sulfate (SDS). More generally, chemical lysing agents can include but are not limited to organic solvents, chelating agents, detergents, surfactants and chaotropic agents.

In some embodiments, biological samples can be permeabilized by non-chemical permeabilization methods. For example, non-chemical permeabilization methods that can be used include, but are not limited to, physical lysis techniques such as electroporation, mechanical permeabilization methods (e.g., bead beating using a homogenizer and grinding balls to mechanically disrupt sample tissue structure), acoustic permeabilization (e.g., ultrasonic treatment), and thermal lysis techniques (such as heating to induce thermal permeabilization of the sample).

Before analyzing the sample, additional reagents can be added to the biological sample to perform various functions. In some embodiments, DNA enzyme and RNA enzyme inactivators or inhibitors (such as proteinase K) and/or chelating agents (such as EDTA) can be added to the sample. For example, the method disclosed herein can include a step of increasing the accessibility of the nucleic acid for binding, for example, opening the DNA in the cell for the denaturation step of probe hybridization. For example, proteinase K treatment can be used to release DNA bound to proteins.

(ix) Selective enrichment of RNA species

In some embodiments, when RNA is an analyte, one or more RNA analyte species of interest can be selectively enriched. For example, one or more RNA species of interest can be selected by adding one or more oligonucleotides to a sample. In some embodiments, other oligonucleotides are sequences for initiating reactions by an enzyme (e.g., a polymerase). For example, one or more RNAs of interest can be amplified using one or more primer sequences having sequence complementarity with one or more RNAs of interest, for example, to generate cDNA, thereby selectively enriching these RNAs.

In some aspects, when analyzing two or more analytes, a first probe and a second probe having specificity (e.g., specific hybridization) for each RNA or cDNA analyte can be used. For example, in some embodiments of the method provided herein, templated connection is used to detect gene expression in a biological sample. Targeted analysis can be performed by an analyte of interest (such as a protein) bound by a labeling agent or a binding agent (e.g., an antibody or its epitope binding fragment), wherein the binding agent is conjugated or otherwise associated with a reporter oligonucleotide containing a reporter sequence for identifying the binding agent. The probe can be hybridized with the reporter oligonucleotide and connected in a templated ligation reaction to generate a product for analysis. In some embodiments, before connection, the gap between the probe oligonucleotides can be filled using, for example, Mu polymerase, DNA polymerase, RNA polymerase, reverse transcriptase, VENT polymerase, Taq polymerase and/or any combination thereof, derivatives and variants (e.g., engineered mutants). In some embodiments, the assay can also include an extension or amplification of the templated ligation product (e.g., a rolling circle amplification of a circular product generated in a templated ligation reaction).

The biological sample may contain one or more analytes of interest. Methods are provided for performing multiplex assays to analyze two or more different analytes in a single biological sample.

B. Analytes

A variety of different analytes can be detected and analyzed using methods and compositions disclosed herein. In some aspects, the analyte can include any biological substance, structure, part or component to be analyzed. In some aspects, the target disclosed herein can similarly include any analyte of interest. In some instances, the target or analyte can be detected directly or indirectly.

The analyte can be derived from a particular type of cell and/or a particular subcellular region. For example, the analyte can be derived from the cytosol, from the nucleus, from the mitochondria, from the microsomes, and more generally, from any other compartment, organelle or part of the cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release the analyte from the cell for analysis, and/or allow one or more reagents (e.g., probes for analyte detection) to approach the analyte in the cell or cell compartment or organelle.

Analyte can comprise any biomolecule or chemical compound, including macromolecule (such as protein or peptide, lipid or nucleic acid molecule) or small molecule (including organic or inorganic molecule). Analyte can be cell or microorganism, including virus or its fragment or product. Analyte can be any substance or entity, can develop specific binding partner (such as affinity binding partner) for it. Such specific binding partner can be nucleic acid probe (for nucleic acid analyte) and can directly lead to the generation of RCA template (such as padlock or other circularizable probe). Alternatively, specific binding partner can be coupled to nucleic acid, and described nucleic acid can be detected using RCA strategy, for example, in the determination of circular nucleic acid molecule that can be used as RCA template using or generating.

Analytes of particular interest may include nucleic acid molecules such as DNA (e.g., genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g., mRNA, microRNA, rRNA, snRNA, viral RNA, etc.); and synthetic and/or modified nucleic acid molecules (e.g., including nucleic acid domains comprising synthetic or modified nucleotides such as LNA, PNA, morpholino, etc. or consisting of these substances); protein molecules such as peptides, polypeptides, proteins or prions, or any molecule comprising a protein or polypeptide component, etc., or a fragment thereof; or lipid or carbohydrate molecules or any molecule comprising a lipid or carbohydrate component. The analyte may be a single molecule or a complex containing two or more molecular subunits, for example, including but not limited to protein-DNA complexes, which may or may not be covalently bound to each other and which may be the same or different. Therefore, in addition to cells or microorganisms, such a complex analyte may also be a protein complex or a protein interactor. Therefore, such a complex or interaction may be a homopolymer or a heteropolymer. Aggregates of molecules (e.g., proteins) can also be target analytes, such as aggregates of the same protein or different proteins. Analytes can also be complexes between proteins or peptides and nucleic acid molecules (such as DNA or RNA), such as interactors between proteins and nucleic acids (e.g., regulatory factors (such as transcription factors) and DNA or RNA).

(i) Endogenous analytes

In some embodiments, the analyte herein is endogenous to the biological sample, and may include nucleic acid analytes and non-nucleic acid analytes. The methods and compositions disclosed herein may be used in any suitable combination to analyze nucleic acid analytes (e.g., using nucleic acid probes or probe groups that are directly or indirectly hybridized with nucleic acid analytes) and/or non-nucleic acid analytes (e.g., using a labeling agent that comprises a reporter oligonucleotide and is directly or indirectly combined with a non-nucleic acid analyte).

Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-connected or O-connected), lipoproteins, phosphoproteins, specific phosphorylation or acetylation variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitination variants of proteins, sulfated variants of proteins, viral capsid proteins, extracellular proteins and intracellular proteins, antibodies and antigen binding fragments. In some embodiments, the analyte is inside the cell or on the cell surface, such as a transmembrane analyte or an analyte attached to the cell membrane. In some embodiments, the analyte can be an organelle (e.g., a nucleus or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted analyte. Exemplary analytes include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, clusters of differentiation proteins, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presentation complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, the post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation) state of cell surface proteins, nick junctions, or adherens junctions.

Examples of nucleic acid analytes include DNA analytes, such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, PCR products synthesized in situ, and RNA/DNA hybrids. A DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA (such as mRNA)) present in a tissue sample.

Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Examples of different types of RNA analytes include messenger RNA (mRNA), including nascent RNA, pre-mRNA, primary transcription RNA, and processed RNA (such as capped mRNA (e.g., with a 5'7-methylguanosine cap), polyadenylated mRNA (poly A tail at the 3' end), and spliced mRNA with one or more introns removed). Uncapped mRNA, unpolyadenylated mRNA, and unspliced mRNA are also included in the analytes disclosed herein. RNA analytes can be transcripts of another nucleic acid molecule (e.g., DNA or RNA (such as viral RNA)) present in a tissue sample. Examples of non-coding RNA (ncRNA) that are not translated into proteins include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNAs, such as microRNA (miRNA), small interfering RNA (siRNA), Piwi interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body specific RNA (scaRNA) and long ncRNA (such as Xist and HOTAIR). RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA with a length greater than 200 nucleic acid bases). Examples of small RNA include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNA, piRNA, tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). RNA can be double-stranded RNA or single-stranded RNA. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16srRNA or 23s rRNA).

In some embodiments described herein, the analyte can be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single-stranded. The nucleic acid can be denatured, for example, optionally denatured using formamide, heat, or both formamide and heat. In some embodiments, the nucleic acid is not denatured and is used in the methods disclosed herein.

In certain embodiments, analytes can be extracted from living cells. Processing conditions can be adjusted to ensure that the biological sample remains alive during analysis and that analytes are extracted (or released) from living cells of the sample. Analytes derived from living cells can be obtained only once from a sample, or can be obtained at certain intervals from a sample that is continuously kept alive.

The methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in one region of a sample or in a separate feature of a substrate.

In any of the embodiments described herein, the analyte comprises a target sequence. In some embodiments, the target sequence can be an endogenous sequence of the sample, a sequence generated in the sample, a sequence added to the sample, or a sequence associated with the analyte in the sample. In some embodiments, the target sequence is a single-stranded target sequence (e.g., a sequence in a rolling circle amplification product). In some embodiments, the analyte comprises one or more single-stranded target sequences. In one aspect, the first single-stranded target sequence is different from the second single-stranded target sequence. In another aspect, the first single-stranded target sequence is the same as the one or more second single-stranded target sequences. In some embodiments, one or more second single-stranded target sequences are contained in the same analyte (e.g., nucleic acid) as the first single-stranded target sequence. Alternatively, one or more second single-stranded target sequences are contained in an analyte (e.g., nucleic acid) different from the first single-stranded target sequence.

(ii) Labeling agent

In some embodiments, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, and cell surface or intracellular proteins and/or metabolites) in samples using one or more markers. In some embodiments, an analyte marker may include a reagent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the marker may include a reporter oligonucleotide indicating an analyte or a portion thereof interacting with the marker. For example, the reporter oligonucleotide may include a barcode sequence that allows identification of the marker. In some cases, the sample contacted by the marker may be further contacted with a probe (e.g., a single-stranded probe sequence), which is hybridized with the reporter oligonucleotide of the marker to identify the analyte associated with the marker. In some embodiments, the analyte marker includes an analyte binding moiety and a marker barcode domain, and the marker barcode domain includes one or more barcode sequences, such as a barcode sequence corresponding to an analyte binding moiety and/or an analyte. The analyte binding moiety barcode includes a barcode that associates with the analyte binding moiety or otherwise identifies the analyte binding moiety. In some embodiments, the analyte binding moiety is identified by identifying its associated analyte binding moiety barcode, and the analyte to which the analyte binding moiety binds can also be identified. The analyte binding moiety barcode can be a nucleic acid sequence of a given length and/or a sequence associated with the analyte binding moiety. The analyte binding moiety barcode can generally include any of the aspects of the barcode described herein.

In some embodiments, the methods include one or more post-fixation steps after contacting the sample with one or more labeling agents.

In the methods and systems described herein, one or more labeling agents that can be combined with or otherwise coupled to one or more features can be used to characterize analytes, cells and/or cell features. In some cases, cell features include cell surface features. Analytes can include but are not limited to proteins, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presentation complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, gap connections, adhesion connections or any combination thereof. In some cases, cell features can include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nucleoproteins, nuclear membrane proteins or any combination thereof.

In some embodiments, the analyte binding moiety may include any molecule or part capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular component). Labeling agents may include, but are not limited to, proteins, peptides, antibodies (or their epitope binding fragments), lipophilic moieties (such as cholesterol), cell surface receptor binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T cell adapters, T cell receptor adapters, B cell receptor adapters, antibody prodrugs, aptamers, monoclonal antibodies, affimers, darpins, and protein scaffolds or any combination thereof. Labeling agents may include (e.g., connected to) a reporter oligonucleotide that indicates the cell surface features to which the binding group is combined. For example, a reporter oligonucleotide may include a barcode sequence that allows identification of a labeling agent. For example, a labeling agent specific to a type of cell feature (e.g., a first cell surface feature) may have a first reporter oligonucleotide coupled thereto, and a labeling agent specific to different cell features (e.g., a second cell surface feature) may have different reporter oligonucleotides coupled thereto. For a description of exemplary labeling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Patent 10,550,429; U.S. Patent Publication 20190177800; and U.S. Patent Publication 20190367969, each of which is incorporated herein by reference in its entirety.

In some embodiments, the analyte binding moiety includes one or more antibodies or antigen binding fragments thereof. Antibodies or antigen binding fragments including analyte binding moieties can specifically bind to target analytes. In some embodiments, the analyte is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein). In some embodiments, multiple analyte labeling agents comprising multiple analyte binding moieties bind to multiple analytes present in a biological sample. In some embodiments, multiple analytes include a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which multiple analytes include a single species of analyte, the analyte binding moieties of multiple analyte labeling agents are the same. In some embodiments in which multiple analytes include a single species of analyte, the analyte binding moieties of multiple analyte labeling agents are different (e.g., members of multiple analyte labeling agents can have two or more species of analyte binding moieties, wherein each species in the two or more species of analyte binding moieties binds to a single species of analyte, such as in different binding sites). In some embodiments, multiple analytes include multiple different species of analytes (e.g., multiple different species of polypeptides).

In other cases, for example to facilitate sample multiplexing, labeling agents specific for particular cellular features can have a first plurality of labeling agents (e.g., antibodies or lipophilic moieties) coupled to a first reporter oligonucleotide and a second plurality of labeling agents coupled to a second reporter oligonucleotide.

In some aspects, these reporter oligonucleotides can include certain nucleic acid barcode sequences that allow identification of the labeling agent to which the reporter oligonucleotide is coupled. Selecting an oligonucleotide as a reporter can provide the following advantages: it can generate significant diversity in sequence, while also being easily linked to most biomolecules (e.g., antibodies, etc.), and being easy to detect (e.g., using sequencing or array technology).

The reporter oligonucleotide can be linked (coupled) to the labeling agent by any of a variety of direct or indirect, covalent or non-covalent associations or linkages. For example, the oligonucleotide can be linked (coupled) using chemical conjugation techniques (e.g., Lightning-Tran available from Innova Biosciences). Antibody labeling kits) are covalently attached to a portion of a labeling agent (such as a protein, such as an antibody or antibody fragment), as well as attached using other non-covalent attachment mechanisms, such as using a biotinylated antibody and an oligonucleotide with an avidin or streptavidin linker (or beads comprising one or more biotinylated linkers coupled to an oligonucleotide). Antibody and oligonucleotide biotinylation techniques are available. See, for example, Fang et al., "Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labelling and Affinity Purification of Synthetic Oligonucleotides," Nucleic Acids Res. 2003 Jan 15; 31(2):708-715, which is incorporated herein by reference in its entirety for all purposes. Similarly, protein and peptide biotinylation techniques have been developed and are readily available. See, for example, U.S. Pat. No. 6,265,552, which is incorporated herein by reference for all purposes. In addition, click reaction chemistry can be used to couple the reporter oligonucleotide to a labeling agent.Commercially available kits (such as those from Thunderlink and Abcam) and other suitable techniques can be used to couple the reporter oligonucleotide to a labeling agent when appropriate.In another example, the labeling agent is indirectly (e.g., via hybridization) coupled to the reporter oligonucleotide, and the reporter oligonucleotide comprises a barcode sequence for identifying the labeling agent.For example, the labeling agent can be directly coupled (e.g., covalently bound) to a hybrid oligonucleotide, and the hybrid oligonucleotide comprises a sequence hybridized with the sequence of the reporter oligonucleotide.The hybridization of the hybrid oligonucleotide and the reporter oligonucleotide couples the labeling agent to the reporter oligonucleotide.In some embodiments, such as when applying stimulation, the reporter oligonucleotide can be released from the labeling agent.For example, the reporter oligonucleotide can be connected to the labeling agent by an unstable bond (e.g., chemically unstable, light unstable, thermally unstable, etc.), as described herein elsewhere for releasing molecules from a support.In some cases, the reporter oligonucleotide described herein can include one or more functional sequences that can be used for subsequent processing.

In some cases, the labeling agent can include a reporter oligonucleotide and a label. The label can be a fluorophore, a radioisotope, a molecule capable of colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be directly or indirectly conjugated to a labeling agent (or reporter oligonucleotide) (for example, the label can be conjugated to a molecule that can be combined with a labeling agent or a reporter oligonucleotide). In some cases, the label is conjugated to a first oligonucleotide complementary to the sequence of the reporter oligonucleotide (for example, hybridization).

In some embodiments, a variety of different species of analytes (e.g., polypeptides) from biological samples can be subsequently associated with one or more physical properties of the biological sample. For example, a variety of different species of analytes can be associated with the position of the analyte in the biological sample. Such information (e.g., proteome information when the analyte binding moiety recognizes the polypeptide) can be used in combination with other spatial information (e.g., genetic information from biological samples, such as DNA sequence information, transcriptome information (e.g., transcript sequence) or both). For example, the cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., the shape, size, activity or type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte labeling agent, the labeling agent comprising an analyte binding moiety that binds to a cell surface protein and an analyte binding moiety barcode that identifies the analyte binding moiety. The protein analysis results in a sample (e.g., a tissue sample or a cell) can be associated with DNA and/or RNA analysis in the sample.

(iii) Products of endogenous analytes and/or labeling agents

In some embodiments, provided herein are methods and compositions for analyzing one or more products of endogenous analytes and/or labeling agents in biological samples. In some embodiments, endogenous analytes (e.g., viral or cellular DNA or RNA) or their products (e.g., hybridization products, connection products, extension products (e.g., by DNA or RNA polymerase), replication products, transcription/reverse transcription products and/or amplification products (such as rolling circle amplification (RCA) products)) are analyzed. In some embodiments, the labeling agent directly or indirectly combined with the analyte in the biological sample is analyzed. In some embodiments, the product of the labeling agent directly or indirectly combined with the analyte in the biological sample is analyzed (e.g., hybridization products, connection products, extension products (e.g., by DNA or RNA polymerase), replication products, transcription/reverse transcription products and/or amplification products (such as rolling circle amplification (RCA) products)).

C. Target sequence

The target sequence of the probe disclosed herein (e.g., a circular probe or a circularizable probe or a probe set) can be contained in any analyte disclosed herein, including an endogenous analyte (e.g., a viral or cellular nucleic acid), a labeling agent, or a product of an endogenous analyte and/or a labeling agent. In some embodiments, the target sequence of the probe disclosed herein (e.g., a circular probe or a circularizable probe or a probe set or a circularized probe) is contained by a target nucleic acid.

In some aspects, one or more of the target sequences comprises one or more barcodes, such as at least two, three, four, five, six, seven, eight, nine, ten or more barcodes. The barcodes can spatially resolve molecular components found in biological samples, such as molecular components within a cell or tissue sample. The barcodes can be attached to an analyte or another part or structure in a reversible or irreversible manner. The barcodes can be added to fragments of, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples before or during sample sequencing. The barcodes can allow identification and/or quantification of individual sequencing reads (e.g., the barcodes can be or can include unique molecular identifiers or "UMIs"). In some aspects, the barcode comprises about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides.

In some embodiments, the barcode includes two or more sub-barcodes that together act as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) separated by one or more non-barcode sequences. In some embodiments, one or more barcodes can also provide a platform for targeting functions, such as oligonucleotides, oligonucleotide-antibody conjugates, oligonucleotide-streptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties, enzymes, enzymes for detection assays or other functions, and/or enzymes for detection and identification of polynucleotides.

In some embodiments, a barcode or its complementary sequence (e.g., a barcode sequence or its complementary sequence contained in an RCA product herein) can be analyzed (e.g., detected or sequenced) using any suitable method or technique, including those described herein, such as RNA sequential target detection (RNA SPOT), sequential fluorescence in situ hybridization (seqFISH), single molecule fluorescence in situ hybridization (smFISH), multiple error robust fluorescence in situ hybridization (MERFISH), in situ sequencing, hybridization-based in situ sequencing (HybISS), targeted in situ sequencing, fluorescence in situ sequencing (FISSEQ), sequencing by synthesis (SBS), sequencing by ligation (SBL), sequencing by hybridization (SBH), or spatially-resolved transcript amplicon readout mapping (STARmap). In any of the foregoing embodiments, the methods provided herein can include analyzing the barcode by sequential hybridization and detection with a plurality of labeled probes (e.g., detection oligos).

In some embodiments, in a barcode sequencing method, a barcode sequence is detected to identify other molecules containing nucleic acid molecules (DNA or RNA) that are longer than the barcode sequence itself, as opposed to directly sequencing longer nucleic acid molecules. In some embodiments, given a sequencing read of N bases, an N-mer barcode sequence contains a complexity of ^4N , and molecular identification may require much shorter sequencing reads compared to non-barcode sequencing methods such as direct sequencing. For example, a barcode sequence of 5 nucleotides can be used to identify 1024 molecular species (4 ⁵ = 1024), while a barcode of 8 nucleotides can be used to identify up to 65,536 molecular species, a number greater than the total number of different genes in the human genome. In some embodiments, a barcode sequence contained in a probe or RCP is detected, rather than an endogenous sequence, which can be an effective readout of the information aspect of each sequencing cycle. Because the barcode sequences are predetermined, they can also be designed with error detection and correction mechanisms, see, for example, US2019/0055594 and US2021/0164039, which are hereby incorporated by reference in their entirety.

III. Circular or Circularizable Probes or Probe Sets

In some aspects, the methods provided herein include rolling circle amplification (RCA) of a circular probe or circularized probe generated by a circularizable probe or probe set. In some aspects, the circular probe or circularized probe comprises one or more modified nucleotides or nucleotide analog residues, which, compared to a reference circular template without one or more modified nucleotides or nucleotide analog residues, reduce the polymerization rate of a polymerase using the circular probe or circularized probe as an RCA template. In other embodiments, the circular probe or circularized probe does not comprise a modified nucleotide or nucleotide analog residue.

In some embodiments, the method provided herein includes contacting a biological sample with a circular probe or a circularizable probe or a probe group that can hybridize with a target nucleic acid in a sample. In some embodiments, a circular probe or a circularizable probe or a probe group comprises one or more modified nucleotides or nucleotide analog residues. In some embodiments, the method includes generating a circularized probe by a circularizable probe or a probe group. In some embodiments, a circularized probe comprises one or more modified nucleotides or nucleotide analog residues. In some embodiments, a circular or circularized probe is a template for rolling circle amplification. FIG. 1A shows an exemplary first circular probe or circularized probe comprising one or more modified nucleotides or nucleotide analog residues. In some aspects, compared to a reference circular template without one or more modified nucleotides or nucleotide analog residues, the presence of one or more modified nucleotides or nucleotide analog residues in a circular or circularized probe reduces the polymerization rate of a polymerase using a circular or circularized probe as a template. In some embodiments, a reference circular template is otherwise identical to a circular or circularized probe comprising one or more modified nucleotides or nucleotide analog residues. For example, a reference circular template can have the same nucleotide sequence as a circular or circularizing probe, but contain unmodified nucleotide residues in place of one or more modified nucleotide or nucleotide analog residues of the circular or circularizing probe.

The circularizable probe or probe group that can be used to generate a circularized probe (such as the exemplary probe shown in Figure 1A) can be any linear probe or linear probe group that can be circularized by connection, such as any of the following circularizable probes or probe groups. In some embodiments, the method also includes contacting the sample with a primer, which hybridizes with the circular probe, the circularizable probe or the probe group or the circularized probe to perform RCA (for example, as shown in Figure 1A). Optionally, the primer can also include a region that hybridizes with the target nucleic acid, for example, as shown by the dotted line in Figure 1A. In other embodiments, the target nucleic acid itself can trigger the rolling circle amplification of the circular probe or the circularized probe (for example, as in RollFISH. See, for example, Wu et al., Commun Biol 1, 209 (2018), which is hereby incorporated by reference in its entirety).

In some embodiments, the methods provided herein include performing RCA on a circular or circularized probe that does not comprise a modified nucleotide or nucleotide analog residue. Figure 1B shows an exemplary second circular or circularized probe that does not comprise any modified nucleotide or nucleotide analog residue. In some aspects, the circular or circularized probe shown in Figure 1B can be a reference circular template, wherein the polymerization rate of the polymerase on the corresponding circular or circularized template comprising one or more modified nucleotides or nucleotide analog residues is slower than the rate on the reference template.

In some embodiments, a method is provided herein, the method comprising performing RCA on a first circular or circularized probe that hybridizes to a first target nucleic acid and comprises one or more modified nucleotides or nucleotide analog residues (e.g., as shown in FIG. 1A ) and a second circular or circularized probe that hybridizes to a second target nucleic acid and does not comprise a modified nucleotide or nucleotide analog residue (e.g., as shown in FIG. 1B ). In some embodiments, the polymerization rate of the polymerase on the first circular or circularized probe is slower than the polymerization rate of the polymerase on the second circular or circularized probe (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% slower). In some embodiments, the polymerization rate of the polymerase on the first circular or circularized probe is slower than the polymerization rate of the polymerase on the second circular or circularized probe by no more than 95%, 50%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%. In some embodiments, the polymerization rate of the polymerase on the first circular or circularized probe is 5%-15%, 10%-15%, 10%-20%, 15%-20%, 10%-30%, 15%-30%, 10%-50%, 10%-70%, 10%-90%, 30%-50%, 30%-90%, 50%-70%, 70%-90% or 50%-90% slower than the polymerization rate of the polymerase on the second circular or circularized probe. In some embodiments, the polymerization rate of the polymerase is the number of nucleotides (nt/min) incorporated into, for example, the RCA product per minute. In some embodiments, the polymerization rate is the average polymerization rate. For example, a polymerase using a circular probe comprising modified and unmodified nucleotide residues as a template may be slower than using unmodified residues as a template when incorporating nucleotides. In such an example, the average polymerization rate using a template having modified and unmodified residues overall will be reduced, for example, compared to the average polymerization rate using a reference template lacking the modified residues. In some embodiments, a slower or reduced polymerization rate (such as a reduced average rate) results in a smaller RCA product.

In some aspects, the method results in a first RCA product produced by a first circular or circularized probe being smaller (e.g., shorter) than a second RCA product produced by a second circular or circularized probe. In any of the foregoing embodiments, the first RCA product and/or the second RCA product may comprise between about 10 and about 100, between about 100 and about 1,000, between about 1,000 and about 5,000, between about 5,000 and about 10,000, or more than 10,000 copies of a circular or circularized probe. In some embodiments, the first RCA product comprises fewer copies (e.g., at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% fewer copies) of a circular or circularized probe than the second RCA product. In some embodiments, the first RCA product comprises no more than 95%, 50%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% fewer copies of the circular or circularization probe than the second RCA product. In some embodiments, the first RCA product comprises 5%-15%, 10%-15%, 10%-20%, 15%-20%, 10%-30%, 15%-30%, 10%-50%, 10%-70%, 10%-90%, 30%-50%, 30%-90%, 50%-70%, 70%-90% or 50%-90% fewer copies of the circular or circularization probe than the second RCA product.

In any of the foregoing embodiments, the first RCA product and/or the second RCA product may be in the form of a nanosphere having a diameter between about 0.1 μm and about 3 μm, optionally wherein the diameter is between about 0.1 μm and about 0.5 μm, between about 0.5 μm and about 1 μm, between about 0.8 μm and about 1.3 μm, or between about 1 μm and about 1.5 μm. In some embodiments, the diameter of the first RCA product is smaller than the diameter of the second RCA product (e.g., at least any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% smaller than the diameter of the second RCA product). In some embodiments, the diameter of the first RCA product is no more than 95%, 50%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% smaller than the diameter of the second RCA product. In some embodiments, the diameter of the first RCA product is 5%-15%, 10%-15%, 10%-20%, 15%-20%, 10%-30%, 15%-30%, 10%-50%, 10%-70%, 10%-90%, 30%-50%, 30%-90%, 50%-70%, 70%-90% or 50%-90% smaller than the diameter of the second RCA product. In some aspects, the determined diameter of the RCA product can vary according to the measurement method of the three-dimensional RCA product. In some aspects, the determined diameter of the RCA product is represented by the maximum measured diameter, the minimum measured diameter, the average measured diameter or any other suitable method for measuring the diameter of the RCA product of the RCA product. In some aspects, the size (e.g., diameter) of the RCA product is measured based on the signal (e.g., fluorescent signal) generated by the RCA product.

In any of the foregoing embodiments, the length of the RCA product may be between about 1 kilobase and about 15 kilobases, between about 15 kilobases and about 25 kilobases, between about 25 kilobases and about 35 kilobases, between about 35 kilobases and about 45 kilobases, between about 45 kilobases and about 55 kilobases, between about 55 kilobases and about 65 kilobases, between about 65 kilobases and about 75 kilobases, or more than 75 kilobases. In some embodiments, the length of the first RCA product is shorter than the length of the second RCA product (e.g., at least any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% shorter). In some embodiments, the length of the first RCA product is no more than any of 95%, 50%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% shorter than the length of the second RCA product. In some embodiments, the length of the first RCA product is 5%-15%, 10%-15%, 10%-20%, 15%-20%, 10%-30%, 15%-30%, 10%-50%, 10%-70%, 10%-90%, 30%-50%, 30%-90%, 50%-70%, 70%-90%, or 50%-90% shorter than the length of the second RCA product.

In some embodiments, the circular probe or circularizable probe or probe set comprises 1, 2, 3, 4, 5, 6 or more modified nucleotides or nucleotide analog residues. Examples of suitable modified nucleotides or nucleotide analog residues include sugar-modified nucleotides, such as 2'-O-methyl ribonucleic acid (2'-OMeRNA), locked nucleic acid (LNA) nucleotides, 2'-fluoro ribonucleic acid (2'-FRNA) or any combination thereof. In some embodiments, the modified nucleotides or nucleotide analog residues include one or more of the following: sugar-modified nucleotides, C2' modified nucleotides, nucleotides with C3' endofolding conformation, modified ribonucleotide residues, modified deoxyribonucleotide residues, modified nucleotides or nucleotide analog residues comprising triazole or thiol bonds instead of phosphodiester bonds, and/or modified nucleotides comprising thiophosphate bonds. In some embodiments, the modified nucleotide or nucleotide analog residue comprises any suitable modification (e.g., sugar, base and/or bond modifications), such as any modification described herein or in Ochoa and Milam, Molecules, 25(20):4659 (2020); and McKenzie et al., Chem Soc Rev., 50(8):5126-5164 (2021), the entire contents of each of which are incorporated herein by reference.

In some embodiments, the circular probe, circularizable probe or probe set or circularized probe consists mainly of deoxyribonucleotide residues. In some embodiments, the probe consists mainly of deoxyribonucleotide residues, with more than 50% of the nucleotide residues being deoxyribonucleotide residues.

Figure 1C depicts the structure of exemplary modified nucleotides or nucleotide analog residues that can reduce the polymerization rate of a polymerase on a circular or circularized probe comprising the modified nucleotides or nucleotide analog residues (2'-OMeRNA). In some embodiments, the inclusion of a single 2'-OMeRNA residue in a template (e.g., in a circular or circularized probe) reduces the polymerization rate by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the polymerization rate on a corresponding reference template.

In some embodiments, the circular probe or circularization probe may contain no more than 20%, 15%, 10%, 5%, 2% or 1% of the total number of residues in the circular probe or circularization probe. Modified nucleotides or nucleotide analog residues and/or modified or unmodified ribonucleotide residues. In some embodiments, the circular probe or circularization probe may contain at least 1%, 2%, 3%, 4%, 5% or 10% of the total number of residues in the circular probe or circularization probe. Modified nucleotides or nucleotide analog residues and/or modified or unmodified ribonucleotide residues. In some embodiments, the circular probe or circularization probe may contain 1%-20%, 1%-15%, 1%-10%, 1%-5%, 5%-10%, 5%-20% or 10%-20% of the total number of residues in the circular probe or circularization probe.

In some embodiments, one or more modified nucleotides or nucleotide analog residues may include one or more backbone modified nucleotides or nucleotide analog residues. For example, one or more modified nucleotides or nucleotide analog residues may include one or more thiol bonds and/or one or more triazole bonds. The exemplary structure of triazole bonds is shown in Figure 1D. In some embodiments, thiol and/or triazole bonds are internal bonds (for example, not formed at the connection point where a cyclized probe or probe group generates a cyclized probe). In some embodiments, the method includes contacting a biological sample with a circular probe or a cyclized probe or a probe group comprising one, two or more thiol and/or triazole bonds. In some embodiments, a circular probe or a cyclized probe or a probe group comprises two or more thiol and/or triazole bonds. In some embodiments, at least two of two or more thiol and/or triazole bonds are separated by less than 100 nucleotide (nt) residues. In some embodiments, at least two of the two or more thiol and/or triazole bonds are separated by less than 90nt residues, less than 80nt residues, less than 70nt residues, less than 60nt residues, less than 50nt residues, less than 40nt residues, less than 30nt residues, less than 20nt residues, less than 10nt residues, or less than 5nt residues. In some embodiments, one or more thiol bonds comprise one or more phosphorothioate and/or phosphorothioate bonds. In some embodiments, one or more backbone-modified nucleotides or nucleotide analog residues comprise any bond and/or modification described herein or in the following documents: Ochoa and Milam, Molecules, 25(20):4659(2020); and McKenzie et al., Chem Soc Rev., 50(8):5126-5164(2021), the entire contents of each document are incorporated herein by reference.

In some embodiments, one or more modified nucleotides or nucleotide analog residues may include one or more nucleotide residues comprising a base other than A, T, G, C or U. In some embodiments, the modified nucleotides or nucleotide analog residues comprise universal or semi-universal bases. In some embodiments, the universal base is 5-nitroindole. In some embodiments, 5-nitroindole guides the random incorporation of nucleotides or incorporable nucleotide analogs in the RCA product. In some embodiments, the semi-universal base is deoxyinosine. Deoxyinosine can guide the partial random incorporation of nucleotides or incorporable nucleotide analogs (e.g., some are biased towards incorporation of cytosine nucleotides or nucleotide analogs) in the RCA product. In some embodiments, the universal base and/or semi-universal base is located outside the barcode sequence or target hybridization sequence contained in the circular or circularizable probe or probe set.

In some embodiments, one or more modified nucleotides or nucleotide analog residues (e.g., 5-nitroindole) reduce the processivity of the polymerase. Processivity is the ability of a DNA polymerase to perform continuous DNA synthesis on a template DNA without frequent dissociation. Processivity can be measured by the average number of nucleotides incorporated by the DNA polymerase in a single association/dissociation event. Therefore, in some aspects, the polymerization rate of a polymerase using a circular or cyclized probe comprising one or more modified nucleotides or nucleotide analog residues as a template is reduced by increasing the number or frequency of association/dissociation events. Modified nucleotides or nucleotide analog residues can act as "speed bumps" to slow down the polymerization of the polymerase on the RCA template.

In some embodiments, the circular probe or circularizable probe or probe group is hybridized with a target nucleic acid in a biological sample. In some embodiments, the target nucleic acid is an endogenous analyte or is associated with an endogenous analyte (e.g., a labeling agent or a component thereof), as described in Section II above. In some embodiments, hybridization with a target nucleic acid in a biological sample includes pairing of substantially complementary or complementary nucleic acid sequences in two different molecules or groups of molecules (one of which is an endogenous analyte or a labeling agent (e.g., a reporter oligonucleotide connected thereto), and the other is a circular probe or circularizable probe or probe group). Pairing can be achieved by any method, wherein the nucleic acid sequence is combined with a substantially or completely complementary sequence by base pairing to form a hybridization complex. For the purpose of hybridization, if at least 60% (e.g., at least 70%, at least 80%, at least 90% or at least 95%) of the bases of the two nucleic acid sequences are each complementary to each other, the two nucleic acid sequences are "substantially complementary".

In some embodiments, the circularizable probe or probe set provided herein can be connected to DNA templates (such as from cDNA molecules). See, for example, U.S. Patent 8,551,710, which is hereby incorporated by reference in its entirety. In some embodiments, circularizable probes or probe sets capable of RNA template connection are provided herein. See, for example, U.S. Patent Publication 2020/0224244, which is hereby incorporated by reference in its entirety. In some embodiments, the circularizable probe set is a SNAIL probe set. See, for example, U.S. Patent Publication 20190055594, which is hereby incorporated by reference in its entirety. In some embodiments, multiple proximity connection assays are provided herein. See, for example, U.S. Patent Publication 20140194311, which is hereby incorporated by reference in its entirety. In some embodiments, circularizable probes or probe sets capable of proximity connection are provided herein, such as RNA proximity connection assays (e.g., PLAYR) probe sets. See, for example, U.S. Patent Publication 20160108458, which is hereby incorporated by reference in its entirety. In some embodiments, the circular probe can be indirectly hybridized with the target nucleic acid. In some embodiments, the circular construct is formed by a circularizable probe group (e.g., a neighboring connection in situ hybridization (PLISH) probe group) capable of adjacent connection. See, for example, U.S. Patent Publication 2020/0224243, which is hereby incorporated by reference in its entirety. In some embodiments, the target nucleic acid of the circularizable probe or probe group provided herein is a probe (such as an L-shaped probe) hybridized with an analyte (such as an mRNA molecule) in a sample, such as in a RollFISH assay. See, for example, Wu et al., Commun Biol 1, 209 (2018), which is hereby incorporated by reference in its entirety.

Any suitable circularizable probe or probe set or in fact more generally circularizable reporter molecules can be used to generate an RCA template for generating an RCA product. Any circularizable probe or probe set described herein can be modified to include one or more modified nucleotides or nucleotide analog residues described herein, so as to reduce the polymerization rate of the polymerase using the circularized probe generated therefrom as a template. The so-called "circularizable" means that the probe or reporter (RCA template) is a linear molecule with a connectable end, which can be circularized by connecting the ends (for example) directly or indirectly to each other or connecting to the corresponding ends of the inserted ("gap") oligonucleotide or connecting to the extended 3' end of the circularizable RCA template. The circularizable probe or template can also be provided in two or more parts, i.e., two or more molecules (such as oligonucleotides) that can be connected together to form a ring. When the probe or RCA template is circularizable, it is circularized by connecting before RCA. The connection template can be used to template the connection, and in the case of circularizable probes (such as padlock probes and molecular inversion probes), the target analyte can provide the connection template, or the connection template can be provided separately. The circularisable probe or RCA template (or part or portion thereof) will contain complementarity at its respective 3' and 5' terminal regions to corresponding homologous complementary regions (or binding sites) in the ligation template, which complementary regions may be adjacent to where the ends are directly ligated to each other, or not adjacent, with an intervening "gap" sequence where indirect ligation will occur.

In the case of a circularizable probe (such as a padlock probe), in one embodiment, the ends of the probe can be adjacent to each other by hybridizing with adjacent sequences on a target nucleic acid molecule (such as a target analyte), and the target nucleic acid molecule serves as a connection template, so that the ends are allowed to be connected together to form a circular nucleic acid molecule, thereby allowing the circularized probe to serve as a template for RCA reactions. In such an example, the end sequence of the probe hybridized with the target nucleic acid molecule will be specific to the target analyte in question, and will be replicated repeatedly in the RCA product. Therefore, they can serve as a marker sequence indicating the target analyte. Therefore, it can be seen that the marker sequence in the RCA product can be equivalent to the sequence present in the target analyte itself. Alternatively, a marker sequence (for example, a label or a barcode sequence) can be provided in the non-target complementary portion of the probe. In a further embodiment, the marker sequence can be present in a gap oligonucleotide hybridized between the corresponding hybridization ends of the probe, where they hybridize with non-adjacent sequences in the target molecule. Such gap filling probes are similar to molecular inversion probes.

In some embodiments, a similar circularization probe suitable as an RCA template molecule can be generated using a molecular inversion probe (a kind of circularizable probe). Like padlock probes, these are also generally linear nucleic acid molecules that can hybridize with target nucleic acid molecules (such as target analytes) and be circularized. The two ends of the molecular inversion probe can hybridize with the target nucleic acid molecule at sites close to each other but not directly adjacent, thereby generating a gap between the two ends. In some embodiments, the size of the gap can be in the range of a larger gap of 100 to 500 nucleotides or longer in other embodiments from only a single nucleotide in some embodiments. Therefore, it is necessary to provide a polymerase and a nucleotide (or nucleotide analog) source or an additional gap filling oligonucleotide to fill the gap between the two ends of the molecular inversion probe so that it can be circularized. Like padlock probes, the end sequence of the molecular inversion probe hybridized with the target nucleic acid molecule and the sequence therebetween will be specific for the target analyte in question, and will be replicated repeatedly in the RCA product. Therefore, they can serve as a marker sequence indicating the target analyte. Alternatively, a marker sequence (eg, a tag or barcode sequence) may be provided in the non-target complementary portion of the molecular inversion probe.

In some embodiments, circularized probes comprising modified nucleotides and/or nucleotide analogs can be generated by incorporating modified nucleotides and/or nucleotide analogs during gap filling of a circularizable probe.

In some embodiments, probe disclosed herein can be an invasion probe, for example, to generate circular nucleic acid such as cyclization probe. Such probe is particularly useful in the detection of single nucleotide polymorphism. Therefore, the method for analyzing biological samples provided by the present disclosure can be used to detect single nucleotide polymorphisms or any variant bases in target nucleic acid sequences. The probe for this method can be designed so that the 3' ligatable end of the cyclization probe or probe part of the probe group is complementary to the nucleotide (variant nucleotide) in the target molecule of interest and can hybridize, and the nucleotide at the 3' end of the 5' additional sequence at the end of the 5' end of the cyclization probe 5' end or another different probe part 5' end of the cyclization probe group is complementary to the same described nucleotide, but is prevented from hybridizing with it (for example, it is a substituted nucleotide) by the 3' ligatable end. Cutting the probe to remove the additional sequence will provide a 5' ligatable end, if the 3' ligatable end is correctly hybridized with the target nucleic acid molecule (for example, complementary to the target nucleic acid molecule), then the 5' ligatable end can be connected to the 3' ligatable end of the probe or probe part. Probes designed according to this principle provide a high degree of discrimination between different variants at the position of interest, because only probes in which the 3' ligatable end is complementary to the nucleotide at the position of interest can participate in the ligation reaction. In one embodiment, the probe is provided in a single portion, and the 3' and 5' ligatable ends are provided by the same probe. In some embodiments, the intrusion probe is a padlock probe (intrusion padlock or "iLock"), such as described in Krzywkowski et al., Nucleic Acids Research 45, e161, 2017 and US2020/0224244, which are incorporated herein by reference in their entirety.

In some embodiments, the ligation involves chemical ligation. In some embodiments, the ligation involves template-dependent ligation. In some embodiments, the ligation involves template-independent ligation. In some embodiments, the ligation involves enzymatic ligation.

In some embodiments, enzymatic ligation involves the use of a ligase. In some aspects, the ligase used herein includes enzymes that are commonly used to connect polynucleotides together or connect single polynucleotide ends. RNA ligase, DNA ligase or another ligase can be used to connect two nucleotide sequences together. Ligase includes ATP-dependent double-stranded polynucleotide ligase, NAD-i-dependent double-stranded DNA or RNA ligase and single-stranded polynucleotide ligase, such as any ligase described in EC 6.5.1.1 (ATP-dependent ligase), EC 6.5.1.2 (NAD+-dependent ligase), EC 6.5.1.3 (RNA ligase). Specific examples of ligases include bacterial ligases (such as E. coli DNA ligase), Tth DNA ligase, Thermococcus species (strain 9°N) DNA ligase (9°N ^TM DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase ^TM (Epicentre Biotechnologies), and bacteriophage ligases (such as T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase), and mutants thereof. In some embodiments, the ligase is T4 RNA ligase. In some embodiments, the ligase is splintR ligase. In some embodiments, the ligase is a single-stranded DNA ligase. In some embodiments, the ligase is T4 DNA ligase. In some embodiments, the ligase is a ligase with DNA splinting DNA ligase activity. In some embodiments, the ligase is a ligase with RNA splinting DNA ligase activity. In some embodiments, the ligase has RNA templated DNA ligase activity and/or RNA templated RNA ligase activity. In some embodiments, the ligase is Chlorella virus DNA ligase (also known as PBCV-1 DNA ligase), T4 RNA ligase, T4 DNA ligase, or single-stranded DNA (ssDNA) ligase. In some embodiments, the ligase is PBCV-1 DNA ligase or a variant or derivative thereof and/or T4 RNA ligase 2 (also known as T4 Rn12) or a variant or derivative thereof.

In some embodiments, the connection herein is directly connected. In some embodiments, the connection herein is indirectly connected. "Direct connection" means that the ends of polynucleotides are hybridized in close proximity to each other to form a substrate for a ligase, thereby causing them to be connected to each other (intramolecular connection). Alternatively, "indirect" means that the ends of polynucleotides are hybridized non-adjacently to each other, for example, separated by one or more insertion nucleotides or "gaps". In some embodiments, the ends are not directly connected to each other, but by one or more insertions (so-called "gaps" or "filling gaps" (oligo) nucleotides) of intermediates or by extending the 3' end of the probe to "fill" the "gap" corresponding to the insertion nucleotide and occur (intermolecular connection). In some cases, the gap of one or more nucleotides between the polynucleotide hybridization ends can be "filled" with one or more "gap" (oligo) nucleotides complementary to a splint, a circularizable probe (such as a padlock probe) or a target nucleic acid. The gap can be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In a specific embodiment, the gap can be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, a gap of any integer (or integer range) of nucleotides between the values shown. In some embodiments, the gap between the terminal regions can be filled by a gap oligonucleotide or by extending the 3' end of the polynucleotide. In some cases, the connection involves connecting the end of the probe to at least one gap (oligo) nucleotide so that the gap (oligo) nucleotide is incorporated into the resulting polynucleotide. In some embodiments, gap filling is performed before the connection herein. In other embodiments, the connection herein does not require gap filling.

In some embodiments, the probes disclosed herein (e.g., circularizable probes or probe sets) may include 5' flanks that can be recognized by structure-specific cutting enzymes, such as enzymes that can recognize the junction between a single-stranded 5' overhang and a DNA duplex and cut the single-stranded overhang. It should be understood that the branched three-stranded structure that is the substrate of the structure-specific cutting enzyme can be formed by the 5' end of one probe portion and the 3' end of another probe portion (when both have hybridized with the target nucleic acid molecule) as well as by the 5' and 3' ends of a single-part probe. Enzymes suitable for such cutting include flanking nucleases (FENS), which are enzymes that have endonuclease activity and can catalyze the hydrolytic cutting of phosphodiester bonds at the junction of single-stranded and double-stranded DNA. Therefore, in some embodiments, the cutting of the additional sequence at 5' of the first target-specific binding site is performed by a structure-specific cutting enzyme (e.g., a flanking nuclease). Suitable flanking endonucleases are described in the following documents: for example, Ma et al., 2000. JBC 275, 24693-24700 and US2020/0224244, the entire contents of each document are incorporated herein by reference, and may include P. furiosus (Pfu), A. fulgidus (Afu), M. jannaschii (Mja) or M. thermoautotrophicum (Mth). In other embodiments, enzymes capable of recognizing and degrading single-stranded oligonucleotides with free 5' ends can be used to cut additional sequences (5' flanks) from structures as described above. Therefore, enzymes with 5' nuclease activity can be used to cut 5' additional sequences. Such 5' nuclease activity can be 5' exonuclease activity and/or 5' endonuclease activity. 5' nucleases can recognize the free 5' ends of single-stranded oligonucleotides and degrade the single-stranded oligonucleotides. 5' exonucleases degrade single-stranded oligonucleotides with free 5' ends by degrading the oligonucleotides from their 5' ends into constituent mononucleotides. 5' endonuclease activity can internally cut 5' flanking sequences at one or more nucleotides. Once the enzyme recognizes free 5' ends, the enzyme will pass through the single-stranded oligonucleotides to reach the duplex region, and cut the single-stranded region into larger constituent nucleotides (e.g., dinucleotides or trinucleotides), or cut the entire 5' single-stranded region, so that 5' nuclease activity occurs, such as Lyamichev et al., 1999.PNAS 96, 6143-6148 for Taq DNA polymerase and its 5' nuclease described, the content of the document is incorporated herein by reference in its entirety. Preferred enzymes with 5' nuclease activity include exonucleases VIII, or natural or recombinant DNA polymerases from aquatic Thermus (Taq), Thermus thermophilus or Thermus flavus, or nuclease domains thereof.

In some embodiments, the melting temperature of the polynucleotide generated by polynucleotide ligation is higher than the melting temperature of the unligated polynucleotide.Thus, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotide prior to subsequent steps (including amplification and detection).

In some aspects, a high-fidelity ligase is used, such as a thermostable DNA ligase (e.g., Taq DNA ligase). The thermostable DNA ligase is active at elevated temperatures, allowing further differentiation by incubating the ligation at a temperature close to the melting temperature ( _Tm ) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates compared to annealed fully base-paired substrates (expected to have a slightly lower _Tm around mismatches). Therefore, high-fidelity ligation can be achieved by a combination of the inherent selectivity of the ligase active site and equilibrium conditions to reduce the incidence of annealed mismatched dsDNA.

In some embodiments, the RCA template may include a target analyte or a portion thereof, wherein the target analyte is a nucleic acid, or the RCA template may be provided or generated as a substitute or marker for an analyte. As described above, any suitable assay may be used to detect a variety of different analytes, which use an RCA-based detection system, for example, wherein a signal is provided by generating an RCP from a circular RCA template provided or generated in the assay, and the RCP is detected to detect the analyte. Therefore, the RCP may be considered a reporter molecule, which is used to detect the target analyte. However, the RCA template may also be considered a reporter molecule for the target analyte; the RCP is generated based on the RCA template and comprises a complementary copy of the RCA template. The RCA template determines the detected signal, and therefore indicates the target analyte. As will be described in more detail below, the RCA template may be a probe or a part or component of a probe, or may be generated by a probe, or may be a component of a detection assay (e.g., a reagent in a detection assay), which is used as a reporter molecule, or a part of a reporter molecule, or a signal generating system for the assay. Therefore, the RCA template for generating RCP can be a circular (e.g., cyclized) reporter nucleic acid molecule, i.e., from any RCA-based detection assay that uses or generates a circular nucleic acid molecule as a reporter molecule for the assay. Because the RCA template generates the RCP reporter molecule, it can be considered as part of the reporter system for the assay. In some embodiments, the RCA template may include one or more modified nucleotides or nucleotide analog residues as described above. In other embodiments, the RCA template does not include modified nucleotides or nucleotide analog residues.

IV. Rolling Circle Amplification

In some embodiments, the probes disclosed herein are amplified by rolling circle amplification (RCA).In some embodiments, a circular or circularizable probe or probe set (such as a padlock probe or a probe set comprising a padlock probe) comprises one or more barcodes or their complements.

In some embodiments, RCA primers are added to trigger RCA, for example as shown in Figure 1A. In some embodiments, RCA primers are added after probe hybridization and/or cyclization. In some embodiments, amplification primers are added together with circular or cyclizable probes or probe groups. In some embodiments, RCA primers are complementary and hybridized to circular or cyclized probes. In some embodiments, RCA primers can also be complementary to target nucleic acid, such as the target nucleic acid portion adjacent to the target nucleic acid portion hybridized with the circular or cyclized probe, for example as shown in Figure 1A. In some cases, RCA primers can be complementary to target nucleic acid and cyclizable probe (for example, SNAIL probe). In some cases, target nucleic acid is a probe (such as an L-shaped probe) that can also be used as a primer (for example, as in RollFISH). In some embodiments, washing steps are carried out to remove any unbound probes, primers, etc. In some embodiments, washing is a strict washing. Washing steps can be carried out at any time point during the methods described herein to remove any component of the reaction as required. For example, washing steps can be used to remove unhybridized probe, non-specifically bound probe, probe that has not yet ligated, or other components present in or in contact with the reaction mixture and/or biological sample.

In some cases, after adding DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, RCA primers are extended by duplication of multiple copies of circular templates. The amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after forming a hybridization complex and any subsequent cyclization (such as connecting, for example, a padlock probe), a circularization probe undergoes rolling circle amplification to generate RCA products (for example, amplicons) of multiple copies of the complementary sequence comprising a circular template (for example, a circular probe or a circularization probe). In some embodiments, RCA includes linear RCA. In some embodiments, RCA includes branched RCA. In some embodiments, RCA includes dendritic RCA. In some embodiments, RCA includes any combination of the foregoing.

Suitable examples of DNA polymerases that can be used include, but are not limited to, E. coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT ^™ DNA polymerase, DEEPVENT ^™ DNA polymerase, Taq DNA polymerase, Hot Start Taq DNA Polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase, DNA polymerase, Quick- DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-Fidelity DNA Polymerase, Platinum Pfx DNA Polymerase, AccuPrime Pfx DNA Polymerase, Phi29 DNA Polymerase, Klenow fragment, Pwo DNA Polymerase, Pfu DNA Polymerase, T4 DNA Polymerase, and T7 DNA Polymerase.

In some embodiments, the rolling circle amplification product is generated using a polymerase selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and variants or derivatives of any of the foregoing.

In some embodiments, amplification is performed at a temperature equal to or approximately equal to between 20°C and 60°C. In some embodiments, amplification is performed at a temperature equal to or approximately equal to between 30°C and 40°C. In some aspects, an amplification step (such as rolling circle amplification (RCA)) is performed at a temperature equal to or approximately equal to between 25°C and equal to or approximately equal to 50°C (such as equal to or approximately equal to 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C or 49°C). In some embodiments, RCA is performed at a temperature between 18°C and 30°C. In some embodiments, RCA is performed at 30°C.

In some aspects, polynucleotide and/or amplification product (for example, amplicon) can be anchored to a polymer matrix.For example, a polymer matrix can be a hydrogel.In some embodiments, one or more polynucleotide probes can be modified to contain a functional group, which can be used as an anchoring point for attaching polynucleotide probes and/or amplification products to a polymer matrix.Exemplary modifications and polymer matrices that can be used according to the embodiment provided include, for example, those described in US 2016/0024555, US2018/0251833 and US2017/0219465, and the entire contents of each document are incorporated herein by reference.In some instances, support also includes modifications or functional groups that can react with probe groups or amplification products or incorporate modifications or functional groups of probe groups or amplification products.In some instances, support can include oligonucleotides, polymers or chemical groups, to provide matrix and/or supporting structure.

In some embodiments, after adding DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, primers are extended to produce multiple copies of circular templates. This amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after hybridization complex formation and amplification probe combination, hybridization complex rolling circle amplification occurs and generates cDNA nanospheres (e.g., amplicon) comprising multiple copies of cDNA. The technology for rolling circle amplification (RCA) includes linear RCA, branched RCA, tree-like RCA and their combination. See, e.g., Baner et al., Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al., Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 Nov 15;49(11):2540–2550; Schweitzer et al., Proc. Natl Acad. Sci. USA 97:101 13-1 19, 2000; Faruqi et al., BMC Genomics 2:4, 2000; Nallur et al., Nucl. Acids Res. 29:el 18, 2001; Dean et al., Genome Res. 1 1:l095-1099, 2001; Schweitzer et al., Nature Biotech. 20:359-365, 2002; and U.S. Pat. Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329, and 6,368,801, the entire contents of each of which are incorporated herein by reference. Exemplary polymerases for RCA include DNA polymerases such as phi29 Polymerase, Klenow fragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase or DNA polymerase I. In some aspects, a DNA polymerase that has been engineered or mutated to have the desired characteristics can be used. In some embodiments, the polymerase is phi29 DNA polymerase.

The amplified product can be fixed in the matrix, usually in the position where the nucleic acid is amplified, thereby producing a local colony of the amplicon. The amplified product can be fixed in the matrix by spatial factors. The amplified product can also be fixed in the matrix by covalent bonds or non-covalent bonds. In this way, it can be considered that the amplified product is connected to the matrix. By being fixed on the matrix, such as by covalent bonds or crosslinking, the size and spatial relationship of the original amplicon are maintained. By being fixed on the matrix, such as by covalent bonds or crosslinking, the amplified product is resistant to moving or spreading under mechanical stress.

In some aspects, the amplification products are copolymerized and/or covalently linked to the surrounding matrix, thereby retaining their spatial relationship and any inherent information thereof. For example, if the amplification products are those generated by DNA or RNA in cells embedded in the matrix, the amplification products can also be functionalized to form covalent connections with the matrix, retaining their spatial information in the cell, thereby providing a subcellular localization distribution pattern. In some embodiments, the provided method involves embedding one or more polynucleotide probe groups and/or amplification products in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, the described hydrogel-histochemistry includes covalently linking nucleic acids to in situ synthetic hydrogels for tissue clearing, enzyme diffusion, and multi-cycle sequencing, while existing hydrogel-histochemistry methods cannot. In some embodiments, in order to enable the amplification products to be embedded in the tissue-hydrogel setting, amine-modified nucleotides are included in the amplification step (e.g., RCA), functionalized to acrylamide moieties using acrylic acid N-hydroxysuccinimide esters, and copolymerized with acrylamide monomers to form a hydrogel.

After amplification, the sequence of the amplicon (e.g., RCA product) or a portion thereof is determined or otherwise analyzed, for example by using detectably labeled probes and imaging, as described in Section VI. Sequencing or analysis of the amplification product may include sequencing by hybridization, sequencing by ligation, and/or fluorescence in situ sequencing, and/or wherein the in situ hybridization includes sequential fluorescence in situ hybridization.

A. Reaction mixture used to slow down RCA

As discussed in Section III above, in some aspects, the RCA rate can be reduced by including one or more modified nucleotides or nucleotide analog residues in the RCA template (e.g., in a circular probe or a circularizable probe or probe set for generating a circularized probe). One or more modified nucleotides in a circular or circularized probe can effectively act as a "speed bump" to slow down the polymerase and/or reduce the continuous synthesis capacity of the polymerase. Additionally or alternatively, the method provided herein may include contacting the sample with a reaction mixture designed to reduce the polymerase polymerization rate. In some embodiments, the reaction mixture comprises one or more nucleotides or their analogs. In some embodiments, the nucleotide or its analog comprises a non-incorporable nucleotide or its analog, which is transiently bound to the polymerase but not incorporated by the polymerase. In some embodiments, the nucleotide or its analog comprises an incorporable nucleotide or its analog, which is configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate. In some embodiments, the nucleotide or analog may comprise any nucleotide or analog thereof or any nucleotide modification described herein or in the following documents: Ochoa and Milam, Molecules, 25(20):4659 (2020); and McKenzie et al., Chem Soc Rev., 50(8):5126-5164 (2021), the entire contents of each document are incorporated herein by reference.

As disclosed herein, reducing the polymerization rate in RCA (e.g., by contacting a sample with a reaction mixture comprising nucleotides or their analogs) can provide several advantages. For example, in some embodiments, the RCA rate can be altered so that there are few large signal spots (e.g., even for highly expressed genes) overlapping each other and/or masking adjacent smaller signal spots, thereby improving optical crowding problems and allowing adjacent spots to be distinguished from each other and/or better resolved, for example, when the size and brightness of the RCA products become more uniform in the same microscope field of view. In some embodiments, the RCA rate can be adjusted so that there are few very bright signal spots, thereby allowing relatively dark spots to be detected simultaneously with bright spots. In some embodiments, adjusting the polymerization rate in RCA (e.g., by contacting a sample with a reaction mixture) can allow adjacent reactions to proceed at different rates while being performed at the same temperature and/or within the same amount of time, thereby, for example, reducing the complexity of the instrument and/or experimental setup. In such an example, in parallel reactions, the first reaction may include only unmodified dNTPs, and the second reaction may also include nucleotides or analogs thereof (e.g., non-incorporable nucleotides and/or incorporable nucleotides or analogs thereof, which are configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphates), which results in a reduced polymerization rate in RCA. In some embodiments, the polymerization rate may be controlled based on the concentration of one or more nucleotides or analogs thereof to slow down polymerization.

As shown in Figure 2A, in some embodiments, a method for analyzing a biological sample may include contacting the biological sample with a reaction mixture comprising (i) a non-incorporable nucleotide or an analog thereof that is transiently bound to a polymerase but not incorporated by the polymerase, and/or (ii) an incorporable nucleotide or an analog thereof that is configured to be incorporated by a polymerase at a slower rate than a corresponding nucleoside triphosphate. Optionally, the reaction mixture additionally comprises unmodified dNTPs, as shown.

In some embodiments, the nucleotide or its analog that cannot be incorporated is a nucleotide or nucleotide analog that cannot be incorporated into a polymerase or cannot be incorporated substantially. In some embodiments, the nucleotide or its analog that cannot be incorporated is combined with a polymerase, but is not incorporated into the RCA product being generated by the polymerase. In some embodiments, the nucleotide or its analog that cannot be incorporated cannot be incorporated by a polymerase. In some embodiments, the nucleotide or its analog that cannot be incorporated is temporarily combined with a polymerase, which means that it can be dissociated from the polymerase. Therefore, the combination of the nucleotide or its analog that cannot be incorporated with a polymerase will not terminate the RCA reaction. In some embodiments, the nucleotide or its analog that cannot be incorporated is a nucleotide monophosphate (e.g., deoxyribonucleoside monophosphate (dNMP)) or its analog, such as shown in Figure 2B. In some embodiments, one or more nucleotides or its analogs that cannot be incorporated include one or more of AMP, GMP, CMP, UMP, dAMP, dGMP, dCMP and/or dTMP. In some embodiments, the non-incorporable nucleotide or analog thereof competes with one or more NTPs and/or dNTPs for binding to the polymerase, thereby reducing the rate of polymerization.

In some embodiments, the nucleotide or its analogue that can be incorporated that is configured to be incorporated by polymerase at a slower speed than corresponding nucleoside triphosphate is a nucleotide or nucleotide analogue other than unmodified NTP/dNTP. The change of nucleotide configuration can affect the incorporation rate of polymerase. For example, the rate-limiting step (such as NTP or dNTP recognition) may be sensitive to both core base and backbone modification. In some embodiments, the nucleotide or its analogue that can be incorporated is configured to be incorporated by polymerase at a speed lower than any one of 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the corresponding nucleoside triphosphate incorporation rate. In some embodiments, the nucleotide or its analogue that can be incorporated is configured to be incorporated by polymerase at a rate of at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 65% or 70% of the corresponding nucleoside triphosphate incorporation rate. In some embodiments, the nucleotide or its analogue that can be incorporated is configured to be incorporated by polymerase at a rate of 5%-10%, 5%-20%, 5%-30%, 5%-80%, 10%-80%, 10%-60%, 10%-50%, 10%-20%, 20%-30%, 20%-80%, 40%-80%, 50%-80%, 40%-50%, 50%-60%, 60%-70% or 70%-80% of the corresponding nucleoside triphosphate incorporation rate.

In some embodiments, the incorporable nucleotide or its analogue configured to be incorporated by a polymerase at a slower rate than the corresponding nucleoside triphosphate corresponds to any one of the following nucleoside triphosphates: dATP, dCTP, dGTP, dTTP, ATP, GTP, CTP and UTP. Generally, the incorporable nucleotide or its analogue corresponds to the nucleoside triphosphate with the most similar structure and/or the same or similar nucleobase. For example, in some embodiments, α-thio-dATP (α-thio derivative of dATP) corresponds to dATP. Similarly, in some embodiments, α-thio-dTTP corresponds to dTTP, α-thio-dCTP corresponds to dCTP, α-thio-GTP corresponds to dGTP, and so on. It can be seen that in some aspects, for a given incorporable nucleotide or its analogue described herein, the corresponding nucleoside triphosphate will be easily identified. In some embodiments, the corresponding nucleoside triphosphate is naturally occurring.

In some embodiments, the reaction mixture has a ratio of one or more incorporable nucleotides or analogs thereof (e.g., α-thio-dNTPs) to unmodified dNTPs of at least 1:1000, 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 100:1, or 1000:1. In some embodiments, the reaction mixture comprises one or more incorporable nucleotides or analogs thereof and does not comprise any unmodified dNTPs. In some embodiments, the reaction mixture has a ratio of one or more incorporable nucleotides or analogs thereof (e.g., α-thio-dNTPs) to unmodified dNTPs of no more than 1:1000, 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 100:1, or 1000:1. In some embodiments, the reaction mixture has a ratio of one or more incorporable nucleotides or analogs thereof (e.g., α-thio-dNTPs) to unmodified dNTPs of no more than any of 1:1000-1:100, 1:1000-1:50, 1:1000-1:20, 1:1000-1:1, 1:2-100:1, 1:2-1000:1, or 1:10-10:1.

In some embodiments, the reaction mixture has a ratio of one or more non-incorporable nucleotides or analogs thereof (e.g., NMP/dNMP) to unmodified dNTPs of at least any of 1:1000, 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, or 100: 1. In some embodiments, the reaction mixture has a ratio of one or more non-incorporable nucleotides or analogs thereof (e.g., NMP/dNMP) to unmodified dNTPs of no more than any of 1:1000, 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 100:1, or 1000:1. In some embodiments, the reaction mixture has a ratio of one or more non-incorporable nucleotides or analogs thereof (e.g., NMP/dNMP) to unmodified dNTPs of no more than any of 1:1000-1:100, 1:1000-1:50, 1:1000-1:20, 1:1000-1:1, 1:2-100:1, 1:2-1000:1, or 1:10-10:1.

In some embodiments, nucleoside diphosphates (e.g., ADP, GDP, CDP, UDP, dADP, dGDP, dCDP and/or dUDP) are included in the reaction mixture. In some embodiments, the methods provided herein include contacting the sample with a reaction mixture, the reaction mixture comprising one or more incorporable nucleotides other than unmodified dNTPs, the one or more incorporable nucleotides being configured to be incorporated by a polymerase at a slower rate than unmodified dNTPs. In some embodiments, the incorporable nucleotides may be NDP/dNDP. In some embodiments, NDP/dNDP may be incorporated by a polymerase at a slower rate than unmodified NDP/dNDP (e.g., as Cassandra et al., PNAS 2018, 115 (5) 980-985, Varela et al., Biochemistry 2021, 60 (5) 373-380, Garforth et al., PLoS One. 2008, 3 (4): e2074, the contents of each document are incorporated herein by reference in their entirety). For example, the incorporation rate of NDP/dNDP can be at least 10 times, at least 15 times, at least 20 times or at least 30 times slower than the incorporation rate of the corresponding unmodified dNTP. However, it is not necessary to incorporate NDP/dNDP into the RCA product by including NDP/dNDP in the reaction mixture to slow down the polymerization rate of the polymerase. In some cases, NDP/dNDP is temporarily combined with the polymerase and dissociates when not incorporated. Therefore, NDP/dNDP can slow down the polymerization rate by competing with other nucleotides (such as dNTP) to bind to the polymerase. In some embodiments, the reaction mixture includes one or more dNDPs selected from dADP, dGDP, dCDP, dTDP and a combination thereof. In some embodiments, the reaction mixture includes one or more NDPs selected from ADP, GDP, CDP, UDP and a combination thereof. In some embodiments, the reaction mixture includes NDP/dNDP and dNTP. In some embodiments, the polymerization rate of the polymerase is inversely proportional to the concentration of NDP and/or dNDP in the reaction mixture and/or the ratio of dNDP to dNTP in the reaction mixture.

In another example of an incorporable nucleotide or its analogue configured to be incorporated by a polymerase at a slower rate than a nucleoside triphosphate (e.g., dATP, dTTP, dUTP, dCTP or dUTP), an incorporable α-thiol nucleotide analogue can be included in the reaction mixture. The structure of an α-thiol nucleotide is shown in FIG. 2B, which contains sulfur to replace oxygen at the α-phosphate position. The effect of α-thiol dNTP on the polymerization rate of Klenow fragments has been described. See, e.g., Pugliese et al., J Am Chem Soc. 2015; 137(30): 9587-9594, the contents of which are incorporated herein by reference in their entirety. In some embodiments, an α-thiol nucleotide is configured to be incorporated by a polymerase at a rate lower than any one of 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the incorporation rate of the corresponding nucleoside triphosphate. In some embodiments, the alpha thiol nucleotide is configured to be incorporated by the polymerase at a rate of at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, or 70% of the incorporation rate of the corresponding nucleoside triphosphate. In some embodiments, the alpha thiol nucleotide is configured to be incorporated by the polymerase at a rate of 5%-10%, 5%-20%, 5%-30%, 5%-80%, 10%-80%, 10%-60%, 10%-50%, 10%-20%, 20%-30%, 20%-80%, 40%-80%, 50%-80%, 40%-50%, 50%-60%, 60%-70%, or 70%-80% of the incorporation rate of the corresponding nucleoside triphosphate. The corresponding nucleoside triphosphate is the dNTP with the same base as the alpha thiol (eg, α-thio-dATP corresponds to dATP, α-thio-dGTP corresponds to dGTP, and so on).

In some embodiments, the alpha thiol nucleotide increases the amount of time (τ _open ) that the polymerase is in an open conformation when processing the alpha thiol nucleotide compared to the corresponding unmodified nucleoside triphosphate. In some embodiments, the alpha thiol nucleotide is configured to increase τ _open by at least 1.2 times, 1.5 times, 2 times, 2.5 times, or 3 times compared to the τ _open of the unmodified nucleoside triphosphate. In some embodiments, the average open time for the polymerase to incorporate an incorporable nucleotide (e.g., an alpha thiol nucleotide) during polymerization is at least 125%, 150%, 200%, 225%, or 250% of the average open time for incorporating the corresponding nucleoside triphosphate.

In some embodiments, one or more incorporable nucleotides or analogs thereof comprise a γ-thiol modified nucleotide. In some embodiments, the γ-thiol modified nucleotide is selected from γ-thio-dATP, γ-thio-dGTP, γ-thio-dTTP, and γ-thio-dCTP. In some embodiments, the γ-thiol modified nucleotide is γ-thio-dATP.

In some embodiments, one or more incorporable nucleotides or their analogs that are configured to be incorporated by polymerase at a slower rate than nucleoside triphosphates include one or more nucleotides with modified bases. In some instances, one or more unmodified dNTPs selected from dATP, dGTP, dTTP and dCTP are replaced by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the corresponding modified nucleotides or their analogs. In some embodiments, one or more unmodified dNTPs selected from dATP, dGTP, dTTP and dCTP are replaced by no more than 90%, no more than 80%, no more than 75%, no more than 60%, no more than 50%, no more than 40% or no more than 30% of the corresponding modified nucleotides or their analogs. Any combination of these range endpoints can also be used, such as, for example, between 25% and 75%, between 30% and 60%, or between 70% and 90% replacement. In some embodiments, a plurality of unmodified dNTPs selected from dATP, dGTP, dTTP and dCTP are partially or completely replaced by modified nucleotides or analogs thereof in the reaction mixture. In some embodiments, the effect of replacing a plurality of different nucleotides with modified nucleotides is additive.

In some embodiments, one or more incorporable nucleotides or their analogs include one or more azide-modified nucleotides and/or one or more alkyne-modified nucleotides. In some embodiments, the reaction mixture includes at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% azide-modified dNTPs that replace the corresponding unmodified dNTPs. In some embodiments, the reaction mixture includes no more than 90%, no more than 80%, no more than 75%, no more than 60%, no more than 50%, no more than 40% or no more than 30% azide-modified dNTPs that replace the corresponding unmodified dNTPs. Exemplary azide-modified dNTPs include, but are not limited to, 5-azidomethyl-dUTP, azide-PEG ₄ -aminoallyl-dUTP and 5-azido-PEG ₄ -dCTP. In some embodiments, the reaction mixture comprises at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of alkyne-modified dNTPs, which replace the corresponding unmodified dNTPs. In some embodiments, the reaction mixture comprises no more than 90%, no more than 80%, no more than 75%, no more than 60%, no more than 50%, no more than 40% or no more than 30% of alkyne-modified dNTPs, which replace the corresponding unmodified dNTPs. Exemplary alkyne-modified nucleotides include, but are not limited to, 5-ethynyl-dUTP and C8-alkyne-dCTP. In some embodiments, the method described herein comprises incorporating one or more azide and/or alkyne-modified dNTPs into the RCA product, wherein the method does not include performing a click reaction or a copper-catalyzed reaction.

In some embodiments, one or more incorporable nucleotides or analogs thereof comprise one or more modified nucleotides having base modifications. In addition to the above exemplary azide and/or alkyne base modifications, exemplary base modifications may also include dibenzylcyclooctyl (DBCO) modifications, vinyl modifications, trans-cyclooctene (TCO), and the like. In some embodiments where the nucleotide modification comprises a functional group suitable for performing a click reaction, the method does not include performing a click reaction on the biological sample prior to imaging to detect the RCA product.

In some aspects, the reaction mixture does not include amine-modified nucleotides. In some aspects, the reaction mixture does not include nucleotides modified with fluorophores. In some aspects, the reaction mixture does not include amine-modified nucleotides or fluorescently modified nucleotides. In some aspects, the amplification product (e.g., RCA product) does not include amine-modified nucleotides or fluorescently modified nucleotides or nucleotide analog residues. In other embodiments, one or more nucleotides or its analogs that can be incorporated can include one or more amine-modified nucleotides. In some embodiments, one or more nucleotides or its analogs that can be incorporated can be functionalized, for example, for anchoring or cross-linking the generated amplification product (e.g., nanosphere) to a support, a cell structure and/or other amplification products (e.g., other nanospheres). In some aspects, the amplification product includes modified nucleotides, such as amine-modified nucleotides. Examples of other amine-modified nucleotides include but are not limited to 5-aminoallyl-dUTP partial modifications, 5-propargylamino-dCTP partial modifications, N ⁶ -6-aminohexyl-dATP partial modifications or 7-deaza-7-propargylamino-dATP partial modifications. In some embodiments, the modified nucleotide comprises an acrylic acid or N-hydroxysuccinimide (NHS) moiety modification.

In some embodiments, the average polymerization rate of the polymerase in rolling circle amplification is less than 2280 nt/min, less than 2000 nt/min, less than 1500 nt/min, less than 1250 nt/min, less than 1000 nt/min, less than 750 nt/min, less than 500 nt/min, or less than 250 nt/min, optionally wherein the polymerization rate is measured at 30° C. In some embodiments, the average polymerization rate of the polymerase in rolling circle amplification is at least any one of 100, 200, 250, 500, 750, 1000, 1250, 1500, or 2000 nt/min, optionally wherein the polymerization rate is measured at 30° C. In some embodiments, the average polymerization rate of the polymerase is within any of 100nt/min-2280nt/min, 100nt/min-2000nt/min, 100nt/min-1500nt/min, 200nt/min-2280nt/min, 200nt/min-2000nt/min, 200nt/min-1500nt/min, 200nt/min-1000nt/min, 500nt/min-1000nt/min, 1000nt/min-1500nt/min, 1250nt/min-2000nt/min, and 1500nt/min-2000nt/min.

In some embodiments, the RCA reaction of multiple circular templates in the sample can be terminated simultaneously to provide multiple rolling circle amplification products. In some embodiments, the RCA reaction can be terminated by contacting the sample with a buffer comprising EDTA (eg, Tris-EDTA or TE buffer).

B. Reaction mixture for reducing the size of RCA products

As discussed in Section III and Section IV.A. above, various methods are provided herein to slow down or reduce the speed of polymerase polymerization RCA products. In some aspects, slowing down the polymerization of RCA products reduces the size of RCA products. In some aspects, there is provided herein a method for reducing the size of RCA products independently of the slowing effect on polymerization (e.g., by promoting the compression of RCA products and/or base stacking interactions within the RCA products). In some aspects, reducing the size (e.g., average diameter) of RCA products can reduce optical crowding. In some aspects, there is provided herein a method for reducing the size (e.g., average diameter) of RCA products without significantly reducing the average intensity, average signal-to-noise ratio and/or average density of RCA products in samples.

In some aspects, a method for analyzing a biological sample is provided herein, the method comprising: (a) contacting the biological sample with a reaction mixture comprising one or more nucleotide analogs, and (b) performing rolling circle amplification (RCA) on a circular nucleic acid template in the biological sample using a polymerase, thereby generating an RCA product incorporating one or more nucleotide analogs, wherein the one or more nucleotide analogs comprise nucleotide analogs containing hydrophobic modifications. In some embodiments, the hydrophobic modification is a base modification. In some embodiments, the hydrophobic modification comprises a carbon chain and/or a hydrocarbon ring. In some embodiments, the hydrophobic modification comprises a triple bond. In some embodiments, the hydrophobic modification comprises a vinyl or ethynyl group. Exemplary nucleotide analogs comprising hydrophobic modifications include, but are not limited to, ethynyl-dUTP or vinyl-dUTP. As shown in, for example, Figure 7, 5-ethynyl-dUTP (5-EdUTP) and 5-vinyl-dUTP comprise hydrophobic modifications (dashed circles in the figure). In some embodiments, the nucleotide analogs comprising hydrophobic modifications are 5-ethynyl-dUTP or 5-vinyl-dUTP. In some aspects, incorporation of one or more nucleotide analogues into the RCA product increases the overall hydrophobicity of the RCA product.Example 5 provides data demonstrating the compaction effect of exemplary nucleotide analogues 5-ethynyl-dUTP or 5-vinyl-dUTP incorporated into the RCA product.

Without being bound by theory, in some aspects, incorporating one or more nucleotide analogs into the RCA product promotes base stacking interactions between nucleotides in the RCA product, thereby promoting the folding of the RCA product into a smaller structure. For example, in an aqueous environment, the hydrophobic portion of the RCA product (RCP) will favorably locate inwardly toward the center of the RCP, forming a hydrophobic core, while charged groups (e.g., phosphate groups) will tend to be located on the outside of the RCP.

In some embodiments, the diameter of the RCA product generated using one or more nucleotide analogs is smaller than the reference RCA product generated using the same template without including one or more nucleotide analogs in the reaction mixture. In some embodiments, the median diameter of the RCA product generated using one or more nucleotide analogs is smaller than the median diameter of the reference RCA product generated using the same template without including one or more nucleotide analogs in the reaction mixture. In some embodiments, the median diameter of the RCA product generated using one or more nucleotide analogs is less than 500nm. In some embodiments, the median diameter of the RCA product generated under the same conditions without nucleotide analogs (e.g., only containing unmodified dNTPs) is at least 600nm, at least 700nm, or at least 800nm. In some embodiments, the median diameter of the RCA product generated using one or more nucleotide analogs is no more than 90% or no more than 80% of the median diameter of the reference RCA product generated using the same template without including one or more nucleotide analogs in the reaction mixture. In some aspects, the average diameter of the RCA product incorporating one or more nucleotide analogs is reduced, but the average intensity, average signal-to-noise ratio and/or density of the RCA product incorporating one or more nucleotide analogs are not significantly different from the average intensity, average signal-to-noise ratio and/or density of the reference RCA product produced using the same template without including one or more nucleotide analogs in the reaction mixture. Any suitable method for determining the significance of numerical differences can be used. For example, in some embodiments, using a two-tailed or one-tailed t-test, a significant difference can be defined as a p-value less than or equal to 0.05. In some embodiments, the size (e.g., diameter) of one or more RCA products is determined by any suitable means, such as those described in Section III.

In some embodiments, the nucleotide analogs comprising a hydrophobic modification are added to the sample at a concentration of at least 1 μM, at least 1.25 μM, at least 2.5 μM, at least 5 μM, at least 10 μM, at least 20 μM, at least 40 μM, at least 80 μM, or at least 100 μM. In some embodiments, the nucleotide analogs comprising a hydrophobic modification are added to the sample at a concentration of no more than 1 mM, no more than 750 μM, no more than 500 μM, 200 μM, no more than 150 μM, no more than 100 μM, no more than 80 μM, no more than 60 μM, or no more than 40 μM. In some embodiments, a nucleotide analog comprising a hydrophobic modification is added to the sample at a concentration equal to or approximately equal to any of: between 1 μM and 200 μM, between 1 μM and 150 μM, between 1 μM and 100 μM, between 1 μM and 80 μM, between 10 μM and 200 μM, between 10 μM and 150 μM, between 10 μM and 100 μM, between 10 μM and 80 μM, between 50 μM and 200 μM, between 50 μM and 100 μM, between 50 μM and 80 μM, between 60 μM and 200 μM, between 60 μM and 100 μM, between 60 μM and 80 μM, or between 80 μM and 100 μM. In some embodiments, a nucleotide analog comprising a hydrophobic modification is added to a sample at a concentration between about 60 μM and about 100 μM or between about 80 μM and about 100 μM. In some embodiments, a nucleotide analog comprising a hydrophobic modification is added to a sample at a concentration of about 80 μM or at least about 80 μM. In some embodiments, a nucleotide analog comprising a hydrophobic modification is added to a sample at a concentration equal to or approximately equal to 1 μM, equal to or approximately equal to 1.25 μM, equal to or approximately equal to 2.5 μM, equal to or approximately equal to 5 μM, equal to or approximately equal to 10 μM, equal to or approximately equal to 20 μM, equal to or approximately equal to 40 μM, equal to or approximately equal to 80 μM, or equal to or approximately equal to 100 μM. In some embodiments, a nucleotide analog comprising a hydrophobic modification is added to a sample at a concentration of 100 μM.

In some embodiments, the nucleotide analog is a modified dUTP and the ratio of modified dUTP to unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 1:99, optionally wherein the ratio of modified dUTP to unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 40:60.

In some embodiments according to any of the methods described herein for slowing down RCA and/or reducing the size of RCA products, the plurality of RCA products may have an average or median diameter of about 0.05 μm, about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1.0 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, or about 1.5 μm, or any of the foregoing values. In some embodiments, the plurality of rolling circle amplification products may have an average or median diameter of less than 0.25 μm.

In some embodiments, the plurality of RCA products may have an average or median length of about 1 kb, about 2 kb, about 5 kb, about 10 kb, about 20 kb, about 30 kb, about 40 kb, about 50 kb, about 60 kb, or about 70 kb, or any of the foregoing values. In some embodiments, the plurality of rolling circle amplification products may have an average or median length of less than 20 kb or less than 10 kb.

In some embodiments, the average or median copy number of the unit sequence complementary to the circular nucleic acid in the plurality of RCA products may be about 10, about 50, about 100, about 500, about 1,000, about 5,000, or about 10,000 or more.

In some embodiments, the average or median copy number of the unit sequence complementary to the circular nucleic acid in the plurality of rolling circle amplification products may be less than 100 or less than 1,000.

In some embodiments, polymerase extension (e.g., RCA) can be carried out for no more than 3 hours. In some embodiments, polymerase extension can be carried out for no more than 2 hours. In some embodiments, polymerase extension can be carried out for no more than 1 hour. In some embodiments, polymerase extension can be carried out for no more than 30 minutes. In any of the embodiments herein, polymerase extension (e.g., RCA) can be performed at between about 20°C to about 40°C, such as at about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C or about 37°C for less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 minutes, less than about 60 minutes, less than about 65 minutes, less than about 70 minutes, less than about 75 minutes, less than about 80 minutes, less than about 85 minutes, less than about 90 minutes, less than about 95 minutes, less than about 100 minutes, less than about 105 minutes, less than about 110 minutes, less than about 115 minutes, less than about 120 minutes. In some embodiments, polymerase extension can be carried out for less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, less than about 23 hours, less than about 24 hours, less than about 30 hours, less than about 35 hours, or less than about 40 hours. In a specific embodiment, polymerase extension can be carried out for between about 10 and 24 hours.

V. Nucleotides, Nucleotide Analogs, and Modifications

In some aspects, provided herein are nucleotides, nucleotide analogs, and modified nucleotides for use in conjunction with any of the methods and compositions described herein.

In some aspects, nucleotides, such as naturally occurring/unmodified nucleotides, comprise (i) pentose (e.g., sugar moieties or sugar rings), such as ribose of ribonucleotides or deoxyribose of deoxyribonucleotides, (ii) nucleobases (e.g., adenine, cytosine, guanine, thymine or uracil), and (iii) one or more phosphate groups. Nucleotides can exist as monomers not included by larger nucleic acid molecules, such as nucleoside triphosphates. In some aspects, nucleotides can be nucleotide residues in nucleic acid molecules (e.g., polynucleotides or oligonucleotides). In some aspects, in nucleic acid molecules, multiple nucleotides (e.g., nucleotide residues) are covalently linked by a sugar-phosphate backbone, and each nucleotide is connected to the next nucleotide by a phosphodiester bond. The structure of unmodified nucleotides, nucleotide residues and nucleic acids has been described in detail, for example, in Alberts et al., Molecular Biology of the Cell, Seventh Edition (W.W.Norton & Company, 2022).

In some embodiments, a modified nucleotide (e.g., a modified nucleotide or nucleotide analog residue, or a nucleotide analog) is, for example, any suitable nucleotide comprising a modification compared to an unmodified nucleotide (such as an unmodified deoxyribonucleotide or ribonucleotide). In some aspects, a modified nucleotide is a nucleotide analog. In some embodiments, the modification is a chemical and/or structural difference between a modified nucleotide and an unmodified nucleotide (e.g., a corresponding unmodified nucleotide). In some embodiments, a modified nucleotide comprises a modification to a nucleobase, a sugar moiety, one or more phosphate groups, and/or a phosphodiester bond (in the case of a nucleotide residue). In some embodiments, a modified nucleotide comprises a modification present in nature. In some embodiments, a modified nucleotide comprises a modification not present in nature. In some embodiments, a modified nucleotide comprising a naturally non-existent modification is incorporated less efficiently and/or at a slower rate by a polymerase (which may be naturally occurring or modified by protein engineering) than a corresponding unmodified nucleotide (e.g., unmodified dATP, dTTP, dUTP, dCTP, or dGTP) that does not comprise the modification in the modified nucleotide. In some embodiments, the modified nucleotide may comprise any suitable modification or combination thereof, such as any modification described herein or, for example, any modification described in Ochoa and Milam, Molecules, 25(20):4659(2020); and McKenzie et al., Chem Soc Rev., 50(8):5126-5164(2021), the entire contents of each of which are incorporated herein by reference.

In some embodiments, the nucleoside triphosphate is deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), or deoxyuridine triphosphate (dUTP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP) and uridine triphosphate (UTP). In some embodiments, the unmodified nucleotide residue is a nucleotide residue generated by polymerase incorporating nucleoside triphosphate into nucleic acid, or a nucleotide residue having the same structure as a nucleotide residue generated by polymerase incorporating nucleoside triphosphate into nucleic acid.

In some embodiments, the modified nucleotide comprises a modification to the sugar-phosphate backbone. In some embodiments, a backbone-modified nucleotide or a backbone-modified nucleotide is a nucleotide, such as a nucleotide residue, comprising a sugar and/or phosphate modification (eg, a modification to the sugar-phosphate backbone).

In some embodiments, the modified nucleotide is a sugar-modified nucleotide or a sugar-modified nucleotide. In some embodiments, the sugar-modified nucleotide comprises a modification of the sugar moiety. In some aspects, the sugar-modified nucleotide comprises a modification on a specific carbon atom of the sugar moiety and is named accordingly. In some aspects, the carbon atoms of the sugar moiety of the nucleotide are numbered 1 to 5 (e.g., C1', C2', C3', C4', C5'). Therefore, in some embodiments, a C2'-modified nucleotide is a sugar-modified nucleotide with a modification on the C2' carbon of the sugar moiety; a C3'-modified nucleotide is a sugar-modified nucleotide with a modification on the C3' carbon of the sugar moiety; and so on. For example, the C2' carbon can be connected to fluorine, such as in the case of 2'-fluoro ribonucleic acid (2'-F RNA). Alternatively, the C2' carbon can be connected to a methoxy/OMe (-OCH ₃ ) group, such as in the case of 2'-O-methyl ribonucleic acid (2'-OMeRNA). Other exemplary C2' modifications include an amino (-NH ₂ ) group or an azido (-N ₃ ) group attached to the C2' carbon.

In some embodiments, the sugar-modified nucleotide is a nucleotide with a C3' inner folding conformation. In some embodiments, folding (puckering) refers to a non-planar conformation of a sugar ring, such as in a nucleotide. In folding, atoms can be displaced from the sugar ring plane. In some embodiments, the folding conformation is defined by the position of C2' and/or C3' atoms relative to the plane formed by the C1', O4' and C4' atoms of the sugar ring. In the "inner" folding, the displacement occurs on the β face of the ring, i.e., on the same side of the C4'-C5' bond and the substrate. In the outer folding, the displacement occurs on the α face of the ring, i.e., on the opposite side of the plane of the C4'-C5' bond. Therefore, in some embodiments, in a nucleotide with a C3' inner folding conformation, relative to the plane formed by the C1', O4' and C4' atoms of the sugar ring, the C3' carbon is displaced to the same side of the C4'-C5' bond. Figure 1C, for example, shows 2'-OMeRNA, an exemplary nucleotide with a 3' inner folding conformation.

In some embodiments, the nucleotide analog is a locked nucleic acid (LNA) nucleotide. In some embodiments, LNA nucleotides are also called bridged nucleic acid (BNA) nucleotides. In some embodiments, LNA nucleotides are nucleotides that contain a methylene bridge between the 2'O and 4'C of the sugar ring. In some embodiments, the methylene bridge conformationally locks the sugar into an N-folded conformation.

In some embodiments, modified nucleotides and/or probes comprising modified nucleotides (such as any circular probe, circularizable probe or probe set or circularizing probe described herein) comprise modified bonds. In some embodiments, modified bonds are any bonds that are different from unmodified (e.g., naturally occurring) phosphodiester bonds (e.g., observed in DNA and RNA). In some embodiments, modified nucleotides are nucleotides that are connected to adjacent nucleotides by modified bonds but are otherwise unmodified.

In some embodiments, the key is a triazole bond or a triazole-based bond. In some embodiments, triazole is a five-membered ring comprising three nitrogen atoms and two carbon atoms. In some embodiments, the key between two residues comprises triazole. Figure 1D shows exemplary modified nucleotides or nucleotide analog residues comprising triazole bonds. In some aspects, the positioning of nitrogen atoms in different triazole rings can be different. In some embodiments, the key is a thiol bond or a thiol-based bond. In some embodiments, the key is a thiophosphate or a thiophosphate bond. In some embodiments, a thiophosphate, a thiophosphate bond or a thiophosphate internucleotide bond is a bond in which one of the phosphate oxygens (e.g., non-connected oxygen) is replaced by a sulfur atom. In some embodiments, one or more modified nucleotides (such as nucleotide residues) comprise thiophosphate backbone nucleotides, thiophosphate backbone nucleotides and/or triazole-modified nucleotides. In some embodiments, a thiophosphate backbone nucleotide is a nucleotide residue comprising a thiophosphate bond. In some embodiments, a thiophosphate backbone nucleotide is a nucleotide residue comprising a thiophosphate bond. In some embodiments, a triazole-modified nucleotide is a nucleotide residue comprising a triazole linkage.

In some embodiments, a nucleotide or an analog thereof is any nucleotide described herein, such as an unmodified nucleotide or a modified nucleotide. In some embodiments, a nucleotide analog is a modified nucleotide. In some embodiments, an incorporable nucleotide or an analog thereof is a nucleotide or an analog thereof (such as a modified nucleotide) that can be incorporated into a nucleic acid by a polymerase (e.g., by incorporating a phi29 DNA polymerase using a circularization probe as a template at the 3' end of an RCA product). In some embodiments, a non-incorporable nucleotide or an analog thereof is a nucleotide or an analog thereof (such as a modified nucleotide) that cannot be incorporated into a nucleic acid by a polymerase (e.g., by incorporating a phi29 DNA polymerase using a circularization probe as a template at the 3' end of an RCA product). In some embodiments, a non-incorporable nucleotide or an analog thereof comprises a monophosphate nucleotide. In some embodiments, a monophosphate nucleotide is a nucleotide monomer (e.g., an unincorporated nucleotide) having a single phosphate, such as shown in Figure 2B (left), which shows the structure of a deoxyribonucleoside monophosphate. In some embodiments, an incorporable nucleotide or an analog thereof comprises a diphosphate nucleotide. In some embodiments, a diphosphate nucleotide is a nucleotide monomer having two phosphates (e.g., an unincorporated nucleotide), such as shown in FIG. 2B (middle), which shows the structure of a deoxyribonucleotide diphosphate. Incorporable and non-incorporable nucleotides or their analogs are further described, such as in section IV.A.

In some embodiments, the nucleotides provided herein (such as modified nucleotides or nucleotides or analogs thereof that can be incorporated) are α-thiol nucleotides. In some aspects, starting from the group closest to ribose, the phosphoryl groups of nucleoside triphosphates are respectively referred to as α phosphate, β phosphate and γ phosphate. In some embodiments, α-thiol nucleotides are nucleotides in which one oxygen combined with α phosphate is replaced by a sulfur atom. An exemplary structure of α-thiol nucleotides is shown in Fig. 2B (right). The "base" in the figure can be any unmodified or modified nuclear base, such as any nuclear base described herein. In some embodiments, α-thiol nucleotides can be α-thio-dATP, α-thio-dTTP, α-thio-dCTP or α-thio-dGTP.

In some embodiments, the nucleotides provided herein (such as modified nucleotides or incorporable nucleotides or their analogs) are alkyne-modified nucleotides. In some aspects, alkynes are unsaturated hydrocarbons containing at least one carbon-carbon triple bond. In some embodiments, alkyne-modified nucleotides include alkynes. In some embodiments, unmodified nucleobases (and their corresponding nucleotides) do not include alkynes. In some embodiments, alkyne-modified nucleotides include alkyne modifications on nucleobases. Examples of alkyne-modified nucleotides include 5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP (5-EdUTP) (e.g., as shown in Figure 6A).

In some embodiments, the nucleotides provided herein (such as modified nucleotides or nucleotides or analogs thereof that can be incorporated) are azide-modified nucleotides. In some aspects, azide-modified nucleotides are nucleotides comprising azides. In some aspects, azide-modified nucleotides are nucleotides comprising azides. In some aspects, azide-modified nucleotides are nucleotides comprising azide modifications. In some aspects, azide modifications are located on any suitable portion of a nucleotide. For example, in some embodiments, 8-azido-adenine is an azide-modified adenine nucleotide with azide modifications in a nucleobase. In another example, 2'-azido nucleotides include azide modifications in the sugar moiety. In another example, azide modifications (for example, azide can replace phosphate) can be performed to the phosphate of nucleoside triphosphates. In some embodiments, azide-modified nucleotides include 5-azido-PEG ₄ -dCTP (for example, as shown in Figure 6A). It can be seen that modified nucleotides can include more than one modification. For example, 5-azido- _PEG4 -dCTP comprises an azide modification and an alkyne modification.

In some embodiments, nucleotides or their analogs (such as modified nucleotides) comprise hydrophobic modifications. In some embodiments, the hydrophobic modifications are located on any suitable portion of the nucleotide, such as a base, a sugar moiety, and/or one or more phosphate groups. In some embodiments, the hydrophobic modifications are base modifications and can be at any one or more positions on the base of the nucleotide.

In some embodiments, the hydrophobic modification is a modification that increases the hydrophobicity of the nucleotide. In some embodiments, the hydrophobicity increase is due to the addition of non-polar groups or molecules, such as carbon chains and/or hydrocarbon rings. In some embodiments, the hydrophobic modification may include carbon chains and/or hydrocarbon rings. In some embodiments, the hydrophobic modification includes double bonds and/or triple bonds, such as double bonds or triple bonds between two carbon atoms. In some embodiments, the hydrophobic modification includes vinyl or ethynyl groups. In some embodiments, the vinyl group is a functional group with the formula -CH=CH _2. In some embodiments, the vinyl group is connected to the nucleobase of a nucleotide or its analog, such as any one of adenine, cytosine, guanine, thymine and uracil. In some embodiments, the nucleotide or its analog is vinyl-dNTP, such as vinyl-dUTP. In some embodiments, vinyl-dUTP is a dUTP with vinyl modification, such as a dUTP comprising a vinyl group. In some embodiments, the nucleotide or its analog is 5-vinyl-deoxyuridine triphosphate, for example as shown in Figure 7. In some embodiments, the hydrophobic modification includes an ethynyl group. In some embodiments, the ethynyl group is a functional group with the formula -C≡CH. In some embodiments, the ethynyl group is connected to the nucleobase of a nucleotide or its analog, including any one of adenine, cytosine, guanine, thymine and uracil. In some embodiments, the nucleotide or its analog is an ethynyl-dNTP, such as an ethynyl-dUTP. In some embodiments, the nucleotide or its analog is a 5-ethynyl-dUTP (5-EdUTP), for example as shown in Figure 7.

In some embodiments, for example, compared with a reference RCA product not comprising nucleotide analogs, incorporating one or more nucleotide analogs comprising hydrophobic modifications in the RCA product increases the overall hydrophobicity of the RCA product. In some embodiments, incorporating one or more nucleotide analogs comprising hydrophobic modifications in the RCA product promotes base stacking interactions between the nucleotides in the RCA product. In some aspects, base stacking refers to the arrangement of nucleobases found in the three-dimensional structure of nucleic acids, wherein two or more planar bases are closely arranged in a stacking configuration. In some aspects, base stacking causes water to be repelled, and increased base hydrophobicity (e.g., via hydrophobic modifications) can further promote base stacking. In some aspects, base stacking contributes to the shape and/or size of the RCA product. In some embodiments, promoting base stacking between the nucleotides in the RCA product (e.g., by comprising nucleotide analogs containing hydrophobic modifications) can cause the RCA product size (e.g., diameter) to decrease.

VI. Signal Amplification, Detection, and Analysis

In some aspects, the provided method relates to analyzing (e.g., detecting or determining) one or more sequences present in a circular RCA template (e.g., a circular probe or a circularized probe) and/or its RCA product. In some cases, one or more captured images are analyzed, and the one or more images may be processed and/or the observed signals may be quantified. For example, the analysis may include processing one or more cell types, one or more types of biomarkers, the number or level of biomarkers, and/or the number or level of cells detected in a specific region of the sample. In some embodiments, the analysis includes detecting a sequence, such as a barcode present in the sample. In some embodiments, the analysis includes quantification of a small point (puncta) (e.g., if an amplification product is detected). In some cases, the analysis includes determining whether there are specific cells and/or signals associated with one or more biomarkers from a specific panel. In some embodiments, the information obtained may be compared with positive and negative controls, or compared with a threshold value of a feature to determine whether the sample exhibits a certain feature or phenotype. In some cases, the information may include signals from cells, regions, and/or include readings from multiple detectable labels. In some cases, the analysis also includes displaying information from an analysis or detection step. In some embodiments, software may be used to automate the processing, analysis, and/or display of data.

In some embodiments, the RCA product in the detection biological sample includes contacting the sample with a nucleic acid probe that can hybridize with the RCA product. After contacting the nucleic acid probe with the sample, the probe can be directly detected by determining a detectable label (if present), and/or by using one or more other probes that are directly or indirectly combined with the probe or its product to detect the probe. The one or more other probes can include a detectable label. For example, the first nucleic acid probe can be combined with the RCA product in the sample, and the second nucleic acid probe can be introduced to combine with the first nucleic acid probe, wherein the second nucleic acid probe or its product can be detected using a detectably labeled probe subsequently. A higher-order probe that is directly or indirectly combined with the second nucleic acid probe or its product can also be used, and then the higher-order probe or its product can be detected using a detectably labeled probe.

In some embodiments, detection can be spatial, for example, two-dimensional or three-dimensional. In some embodiments, detection can be quantitative, for example, the amount or concentration of the RCA product (and the corresponding target nucleic acid) can be determined. In some embodiments, depending on the application, the primary probe, secondary probe, higher order probe and/or detectably labeled probe can include any of a variety of entities capable of hybridizing with nucleic acids, for example, DNA, RNA, LNA and/or PNA, etc.

The detection probe can include a recognition sequence that hybridizes with a sequence in the RCA product (for example, hybridizes with a barcode sequence or its complementary sequence included in the RCA product). The recognition sequence can have any length, and multiple recognition sequences in the same or different secondary nucleic acid probes can have the same or different lengths. If more than one recognition sequence is used, the recognition sequence can independently have the same or different lengths. For example, the length of the recognition sequence can be at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40 or at least 50 nucleotides. In some embodiments, the length of the recognition sequence can be no more than 48, no more than 40, no more than 32, no more than 24, no more than 16, no more than 12, no more than 10, no more than 8 or no more than 6 nucleotides. Combinations of any of these are also possible, for example, the recognition sequence can have a length of between 5 and 8 nucleotides, between 6 and 12 nucleotides, or between 7 and 15 nucleotides, etc. In one embodiment, the recognition sequence has the same length as the barcode sequence or its complement of the circular or circularized probe or its product. In some embodiments, the recognition sequence can be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% complementary to the barcode sequence or its complement.

In some embodiments, the RCA product generated according to the method described herein can be detected by a method including signal amplification. Exemplary signal amplification methods include targeted deposition of detectable reactive molecules around probe hybridization sites, targeted assembly of branched structures (e.g., bDNA or branching assays using locked nucleic acids (LNA)), programmed in situ growth of concatemers by enzymatic rolling circle amplification (RCA) (e.g., as described in US2019/0055594, the contents of which are incorporated herein by reference in their entirety), hybridization chain reaction, assembly of topological concatenated DNA structures using consecutive rounds of chemical ligation (clampFISH), signal amplification by hairpin-mediated concatenation (e.g., as described in US 2020/0362398, the contents of which are incorporated herein by reference in their entirety), such as primer exchange reactions, such as signal amplification by exchange reactions (SABER) or SABER with DNA exchange (exchange-SABER). In some embodiments, non-enzymatic signal amplification methods can be used.

In some embodiments, signal amplification can be realized using hybridization chain reaction (HCR).HCR is an enzyme-free nucleic acid amplification based on the hybridization trigger chain of nucleic acid molecules, starting from HCR monomers, these monomers hybridize with each other to form a nicked nucleic acid polymer.The polymer is the product of HCR reaction, which is ultimately detected to indicate the presence of target analytes.HCR is described in detail in Dirks and Pierce, 2004, PNAS, 101 (43), 15275-15278 and in US 7,632,641 and US 7,721,721, and the entire contents of each document are incorporated herein by reference. See also US 2006/00234261; Chemeris et al., 2008 Doklady Biochemistry and Biophysics, 419, 53-55; Niu et al., 2010, 46, 3089-3091; Choi et al., 2010, Nat. Biotechnol. 28 (11), 1208-1212; and Song et al., 2012, Analyst, 137, 1396-1401, the entire contents of each document are incorporated herein by reference. HCR monomers generally include hairpins or other metastable nucleic acid structures. In the simplest HCR form, when "initiator" nucleic acid molecules are introduced, two different types of stable hairpin monomers (here referred to as the first and second HCR monomers) undergo hybridization chain reaction events to form a double-stranded DNA molecule with a long nick. HCR monomers have a hairpin structure, which includes a double-stranded stem region, a loop region connecting two chains of the stem region, and a single-stranded region at one end of the double-stranded stem region. The single-stranded region that exposes (and therefore can be used for hybridization with another molecule such as initiator or other HCR monomers) when monomer is in hairpin structure can be referred to as "foothold area" (or "input domain"). The first HCR monomer each also includes a sequence complementary to the sequence in the foothold area exposed with the second HCR monomer. This complementary sequence in the first HCR monomer can be referred to as "interaction region" (or "output domain"). Similarly, the second HCR monomer each includes an interaction region (output domain), such as a sequence complementary to the foothold area (input domain) exposed with the first HCR monomer. In the absence of HCR initiators, these interaction regions are protected by secondary structure (for example, they are not exposed), so hairpin monomers are stable or kinetics are trapped (also referred to as "metastable state"), and remain as monomers (for example, to prevent system rapid equilibrium), because the first and second groups of HCR monomers can not hybridize to each other. However, once an initiator is introduced, it can hybridize with the foothold area exposed with the first HCR monomer, and invade it, causing it to open. This will expose the interaction zone (for example, sequence complementary to the foothold region of the second HCR monomer) of the first HCR monomer, thereby allowing it to hybridize with the second HCR monomer at the foothold region and invade the second HCR monomer. This hybridization and invasion and then open the second HCR monomer, expose its interaction zone (it is complementary to the foothold region of the first HCR monomer), and allow it to hybridize with another first HCR monomer and invade another first HCR monomer. Reaction continues in this way, until all HCR monomers are exhausted (for example, all HCR monomers are incorporated into polymer chain). Finally, the chain reaction causes the notched chain of the alternating unit forming the first and second monomer species. Therefore, the presence of HCR initiator is needed to trigger HCR reaction by hybridizing with the first HCR monomer and invading the first HCR monomer. The first and second HCR monomers are designed to hybridize each other and therefore can be defined as homologous to each other. They are also homologous to a given HCR initiator sequence. The HCR monomers interacting with each other (hybridization) can be described as a group of HCR monomers or HCR monomers or hairpin systems.

The HCR reaction can be carried out with more than two kinds or types of HCR monomers.For example, a system involving three HCR monomers can be used.In such a system, each first HCR monomer can include an interaction zone combined with the foothold area of the second HCR monomer; Each second HCR can include an interaction zone combined with the foothold area of the 3rd HCR monomer; and each 3rd HCR monomer can include an interaction zone combined with the foothold area of the first HCR monomer.Then, the HCR polymerization reaction will be carried out as described above, except that the product therefrom will be a polymer with a continuous first, second and third monomer repeating unit.It is easy to think of a corresponding system with a large amount of HCR monomer groups.

In some embodiments, similar to the HCR reaction using hairpin monomers, linear oligonucleotide hybridization chain reaction (LO-HCR) can also be used for signal amplification. In some embodiments, a method for detecting an analyte in a sample is provided herein, comprising: (i) performing a linear oligonucleotide hybridization chain reaction (LO-HCR), wherein an initiator is contacted with a plurality of LO-HCR monomers of at least the first and second species to generate a polymerized LO-HCR product hybridized with a target nucleic acid molecule, wherein the first species comprises a first hybridization region complementary to the initiator and a second hybridization region complementary to the second species, wherein the first species and the second species are linear single-stranded nucleic acid molecules; wherein the initiator is provided in one or more parts and is directly or indirectly hybridized with the target nucleic acid molecule or is contained in the target nucleic acid molecule; and (ii) detecting the polymer product, thereby detecting the analyte. In some embodiments, the first species and/or the second species may not contain a hairpin structure. In some embodiments, the plurality of LO-HCR monomers may not contain a metastable secondary structure. In some embodiments, the LO-HCR polymer may not contain a branched structure. In some embodiments, performing a linear oligonucleotide hybridization chain reaction comprises contacting the target nucleic acid molecule with an initiator to provide the initiator to hybridize to the target nucleic acid molecule.In any of the embodiments herein, the target nucleic acid molecule and/or the analyte may be a RCA product.

In some embodiments, the in situ detection of nucleic acid sequences includes a combination of the RCA method described herein and a component for branch signal amplification. In some embodiments, the component complex comprises an amplifier that hybridizes directly or indirectly (via one or more oligonucleotides) to the sequence of the RCA product. In some embodiments, the component includes one or more amplifiers, each of which includes an amplifier repeat sequence. In some aspects, the one or more amplifiers are labeled. This article describes a method using the aforementioned components, which includes, for example, using the component in a multiple error robust fluorescence in situ hybridization (MERFISH) application, wherein branch DNA amplification is used for signal readout. In some embodiments, the amplifier repeat sequence is about 5-30 nucleotides and is repeated N times in the amplifier. In some embodiments, the amplifier repeat sequence is about 20 nucleotides and is repeated at least twice in the amplifier. In some aspects, the one or more amplifier repeat sequences are labeled. For exemplary branched signal amplification, see, for example, U.S. Patent Publication Nos. US2020/0399689 and US 2022/0064697 and Xia et al., Multiplexed Detection of RNA using MERFISH and branched DNA amplification, Scientific Reports (2019), the entire contents of each of which are incorporated herein by reference.

In some embodiments, the RCA product can be detected by a method including signal amplification by performing a primer exchange reaction (PER). In various embodiments, a primer having a domain on its 3' end is combined with a catalytic hairpin, and a new domain is extended by a strand displacement polymerase. For example, a primer having domain 1 at its 3' end is combined with a catalytic hairpin, and a new domain 1 is extended by a strand displacement polymerase, and a concatemer of a repeating domain 1 sequence is generated by repeated cycles. In various embodiments, the strand displacement polymerase is Bst. In various embodiments, the catalytic hairpin includes a stopper for releasing a strand displacement polymerase. In various embodiments, the branch migration displaces the extended primer, and then the extended primer can be dissociated. In various embodiments, the primer undergoes repeated cycles to form a concatemer primer. In various embodiments, a variety of concatemer primers are contacted with a sample comprising an RCA product generated using the method described herein. In various embodiments, the RCA product can be contacted with a plurality of concatemer primers and a plurality of labeled probes. For exemplary molecules and PER reaction components, see, e.g., U.S. Patent Publication No. US20190106733, which is incorporated herein by reference.

In some embodiments, the RCA product can be detected by providing a detection probe such as a probe for performing a chain reaction (e.g., HCR) forming an amplified product. In some embodiments, analysis includes determining the sequence of all or part of the amplified product. In some embodiments, analysis includes detecting the sequence present in the amplified product. In some embodiments, the sequence of all or part of the amplified product indicates the identity of the region of interest in the target nucleic acid. In other embodiments, the provided method relates to analyzing (e.g., detecting or determining) one or more sequences present in a polynucleotide probe (e.g., a barcode sequence present in the overhang region of the first and/or second probe).

In some embodiments, the method includes sequencing all or part of the RCA product (such as one or more barcode sequences present in the RCA product). In some embodiments, analysis and/or sequence determination include sequencing all or part of the RCA product or probe and/or in situ hybridization of the RCA product or probe. In some embodiments, the sequencing step is related to sequencing while hybridization, sequencing while connection and/or fluorescence in situ sequencing, in situ sequencing based on hybridization and/or wherein in situ hybridization includes sequential fluorescence in situ hybridization. In some embodiments, analysis and/or sequence determination include detecting a polymer generated by hybridization chain reaction (HCR) reaction, for exemplary probes and HCR reaction components, see, for example, US2017/0009278, which is incorporated herein by reference. In some embodiments, detection or determination include hybridization with amplification product using a detection oligonucleotide labeled with a fluorophore, isotope, mass label or a combination thereof. In some embodiments, detection or determination include imaging the amplification product. In some embodiments, the target nucleic acid is an mRNA in a tissue sample, and the target nucleic acid and/or amplification product are detected or determined when they are located in situ in the tissue sample.

In some aspects, the methods provided include imaging one or more portions of an amplified product (e.g., an amplicon) and/or a polynucleotide, for example, by combining and detecting a detectable label with a detection probe. In some embodiments, the detection probe comprises a detectable label that can be measured and quantified. Labels and detectable labels include directly or indirectly detectable portions associated with (e.g., conjugated to) a molecule to be detected, such as a detectable probe, including but not limited to a fluorophore, a radioisotope, a fluorescent agent, a chemiluminescent agent, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a chromophore, a dye, a metal ion, a metal sol, a ligand (e.g., biotin or a hapten), etc.

Fluorophores include substances or a portion thereof that can display fluorescence within a detectable range. Specific examples of labels that can be used according to the provided embodiments include, but are not limited to, phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acridinium esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, β-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease.

Fluorescence detection in tissue samples may usually be hindered by the presence of strong background fluorescence. Autofluorescence is a term used to distinguish background fluorescence (which can be caused by a variety of sources (including aldehyde fixation, extracellular matrix components, erythrocytes, lipofuscin, etc.) from the desired immunofluorescence of fluorescently labeled antibodies or probes. Tissue autofluorescence may cause it to be difficult to distinguish the signal caused by fluorescent antibodies or probes from the general background. In some embodiments, the method disclosed herein utilizes one or more reagents to reduce tissue autofluorescence, such as autofluorescence eliminator (Sigma/EMD Millipore), TrueBlack lipofuscin autofluorescence quencher (Biotium), MaxBlock autofluorescence reduction kit (MaxVisionBiosciences) and/or very strong black dyes (e.g., Sudan black or suitable dark chromophores).

In some embodiments, detectable probes containing detectable labels can be used to detect one or more polynucleotides and/or amplification products (e.g., amplicons) described herein. In some embodiments, the method involves incubating a detectable probe containing a detectable label with a sample, washing unbound detectable probes, and detecting the label, e.g., by imaging.

Examples of detectable labels include, but are not limited to, various radioactive moieties, enzymes, prosthetic groups, fluorescent labels, luminescent labels, bioluminescent labels, metal particles, protein-protein binding pairs, and protein-antibody binding pairs. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, and phycoerythrin.

Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacteria, fireflies, and click beetles), luciferin, aequorin, and the like. Examples of enzyme systems with visually detectable signals include, but are not limited to, beta-galactosidase, glucuronidase, phosphatase, peroxidase, and cholinesterase. Identifiable markers also include radioactive compounds, such as ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³ H. Identifiable markers are commercially available from a variety of sources.

Examples of fluorescent labels and nucleotides and/or polynucleotides conjugated to such fluorescent labels include those described in, e.g., Hoagland, Handbook of Fluorescent Probes and Research Chemicals, 9th Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991), the entire contents of each of which are incorporated herein by reference. In some embodiments, exemplary techniques, methods and methodologies suitable for the provided embodiments include, for example, those described in US 4,757,141, US 5,151,507 and US 5,091,519, the entire contents of each document are incorporated herein by reference. In some embodiments, one or more fluorescent dyes are used as labels for labeled target sequences, for example, as described in the following documents: US 5,188,934 (4,7-dichlorofluorescein dye); US 5,366,860 (spectrally resolvable rhodamine dye); US 5,847,162 (4,7-dichlororhodamine dye); US 4,318,846 (ether-substituted fluorescein dye); US 5,800,996 (energy transfer dye); US 5,066,580 (xanthine dye); and US 5,688,648 (energy transfer dye), the entire contents of each document are incorporated herein by reference. Quantum dots can also be used for labeling, as described in US 6,322,901, US 6,576,291, US 6,423,551, US 6,251,303, US 6,319,426, US 6,426,513, US 6,444,143, US 5,990,479, US 6,207,392, US2002/0045045 and US2003/0017264, the entire contents of each document are incorporated herein by reference. In some embodiments, the fluorescent label is a signal transduction moiety that conveys information through the fluorescence absorption and/or emission characteristics of one or more molecules. Exemplary fluorescence characteristics include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer.

Examples of commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and/or polynucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED ^™ -5-dUTP, CASCADEBLUE ^™ -7-dUTP, BODIPY™FL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN ^™ -5-dUTP, OREGON GREENR ^™ 488-5-dUTP, TEXAS RED ^™ -12-dUTP, BODIPY ^™ 630/650-14-dUTP, BODIPY ^™ 650/665-14-dUTP, ALEXA FLUOR ^TM 488-5-dUTP, ALEXA FLUOR ^TM 532-5-dUTP, ALEXA FLUOR ^TM 568-5-dUTP, ALEXA FLUOR ^TM 594-5-dUTP, ALEXA FLUOR ^TM 546-14-dUTP, Fluorescein-12-UTP, Tetramethyl Rhodamine-6-UTP, TEXAS RED ^TM -5-UTP, mCherry, CASCADE BLUE ^TM -7-UTP, BODIPY ^TM FL-14-UTP, BODIPY ^TM R-14-UTP, BODIPY ^TM TR-14-UTP, RHOD AMINE GREEN ^TM -5-UTP, ALEXA FLUOR ^TM 488-5-UTP and ALEXA FLUOR ^TM 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.) Methods for custom synthesis of nucleotides with other fluorophores are described, for example, in Henegariu et al., (2000) Nature Biotechnol. 18:345, the contents of which are incorporated herein by reference in their entirety.

Other fluorophores available for post-synthetic attachment include, but are not limited to, ALEXA FLUOR ^™ 350, ALEXA FLUOR ^™ 532, ALEXA FLUOR ^™ 546, ALEXA FLUOR ^™ 568, ALEXA FLUOR ^™ 594, ALEXA FLUOR ^™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Blue, and Cascade Blue. Yellow, dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, NJ). FRET tandem fluorophores can also be used, including but not limited to PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), and APC-Alexa dyes.

In some cases, metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and/or polynucleotide sequences. See, e.g., Lakowicz et al., (2003) Bio Techniques 34:62, the contents of which are incorporated herein by reference in their entirety.

Biotin or its derivatives can also be used as labels on nucleotides and/or polynucleotide sequences, and are subsequently bound by detectably labeled avidin/streptavidin derivatives (e.g., phycoerythrin-conjugated streptavidin) or detectably labeled anti-biotin antibodies. Digoxigenin can be incorporated as a label, and is subsequently bound by detectably labeled anti-digoxigenin antibodies (e.g., fluoresceinized anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into polynucleotide sequences, and subsequently coupled to fluorescent dyes derived from N-hydroxysuccinimide (NHS). In general, any member of the conjugate pair can be incorporated into the detection polynucleotide as long as the detectably labeled conjugation partner can be combined to allow detection. In some embodiments, the antibody is an antibody molecule of any class or any subfragment thereof, such as Fab.

Other suitable labels for polynucleotide sequences can include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis) and phosphor-amino acids (e.g., P-tyr, P-ser, P-thr). In some embodiments, the following hapten/antibody pairs are used for detection, wherein each antibody is derivatized with a detectable label: biotin/a-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNP)/a-DNP, 5-carboxyfluorescein (FAM)/a-FAM.

In some embodiments, the nucleotide and/or polynucleotide sequence can be indirectly labeled, particularly with a hapten, which is then bound by a capture agent, such as disclosed in US 5,344,757, US 5,702,888, US 5,354,657, US 5,198,537, US 4,849,336, and US 5,073,562, the entire contents of each of which are incorporated herein by reference. Many different hapten-capture agent pairs are available for use. Exemplary haptens include, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, Cy5, and digoxigenin. For biotin, the capture agent can be avidin, streptavidin, or an antibody. Antibodies can be used as capture agents for other haptens (many dye-antibody pairs are commercially available, for example, Molecular Probes, Eugene, Oreg.).

In some aspects, analysis and/or sequence determination can be performed at room temperature to best preserve tissue morphology with low background noise and reduced errors. In some embodiments, analysis and/or sequence determination includes eliminating error accumulation as sequencing proceeds.

In some embodiments, analysis and/or sequence determination involves washing to remove unbound polynucleotides, after which the fluorescent product is revealed for imaging.

In some aspects, detection involves the use of detection methods such as flow cytometry; sequencing; probe binding and electrochemical detection; pH changes; catalysis induced by enzymes bound to DNA tags; quantum entanglement; Raman spectroscopy; terahertz wave technology; and/or scanning electron microscopy. In some aspects, flow cytometry is mass cytometry or fluorescence activated flow cytometry. In some aspects, detection includes performing microscopy, scanning mass spectrometry, or other imaging techniques described herein. In these aspects, detection includes determining a signal, such as a fluorescent signal.

In some aspects, detection (including imaging) is performed using any of a number of different types of microscopy, such as confocal microscopy, two-photon microscopy, light-field microscopy, intact tissue expansion microscopy, and/or CLARITY ^™ -optimized light sheet microscopy (COLM).

In some embodiments, fluorescence microscopy is used to detect and image the detection probe. In some aspects, a fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of or in addition to reflection and absorption to study the properties of organic or inorganic substances. In fluorescence microscopy, the sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluorescence is then imaged by a microscope objective, and the fluorescence wavelength is usually longer than the illumination wavelength. Two filters can be used in this technology; an illumination (or excitation) filter that ensures that the illumination is close to monochromatic and at the correct wavelength, and a second emission (or barrier) filter that ensures that no excitation light source reaches the detector. Alternatively, these functions can all be achieved by a single dichroic filter. "Fluorescence microscope" includes any microscope that uses fluorescence to generate images, whether it is a simpler device like an epifluorescence microscope, or a more complex design like a confocal microscope, which uses optical sectioning to obtain better resolution of fluorescent images.

In some embodiments, confocal microscopy is used to detect and image the detection probe. Confocal microscopy uses point illumination and a pinhole in an optical conjugate plane in front of the detector to eliminate out-of-focus signals. Since only the light generated by the fluorescence very close to the focal plane can be detected, the optical resolution of the image, especially in the sample depth direction, is much better than that of a wide-field microscope. However, since many lights from the sample fluorescence are blocked at the pinhole, this improvement in resolution is at the expense of a reduction in signal intensity--therefore a long exposure time is usually required. Since only one point in the sample is irradiated at a time, 2D or 3D imaging needs to be scanned in the sample with a regular grating (e.g., a rectangular pattern of parallel scan lines). The achievable thickness of the focal plane is mainly limited by the wavelength of the light used divided by the numerical aperture of the objective lens, but is also limited by the optical properties of the sample. Thin optical sections make these types of microscopes particularly good in 3D imaging and surface profile analysis of samples. CLARITY ^TM -Optimized Light Sheet Microscopy (COLM) provides an alternative microscopy for fast 3D imaging of large clarified samples. COLM interrogates large immunostained tissues, allowing for increased acquisition speeds and resulting in higher quality generated data.

Other types of microscopy that may be employed include brightfield microscopy, oblique illumination microscopy, darkfield microscopy, phase contrast microscopy, differential interference contrast (DIC) microscopy, interference reflection microscopy (also known as reflected interference contrast or RIC), single plane illumination microscopy (SPIM), ultra-high resolution microscopy, laser microscopy, electron microscopy (EM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), reflection electron microscopy (REM), scanning transmission electron microscopy (STEM), and low voltage electron microscopy. (LVEM), scanning probe microscopy (SPM), atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic force microscopy (C-AFM), electrochemical scanning tunneling microscopy (ECSTM), electrostatic force microscopy (EFM), fluid force microscopy (FluidFM), force modulation microscopy (FMM), feature-oriented scanning probe microscopy (FOSPM), Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM ), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM, scanning near-field optical microscopy, SNOM, piezoelectric response force microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, photothermal microspectroscopy/microscopy (PTMS), SCM, scanning capacitance microscopy (SCM), SECM, scanning electrochemical microscopy (SECM), SGM, scanning gate microscopy (SGM), SHPM, scanning Hall probe microscopy ( The invention relates to the present invention to a method for the treatment of tissue damage and the occurrence of tissue instability. The invention relates to a method for the treatment of tissue damage and the occurrence of tissue instability. The invention relates to a method for the treatment of tissue damage and the occurrence of tissue instability.

In some embodiments, nucleic acid probes such as circular probes or circularizable probes or probe sets or nucleic acid probes hybridized with RCA products can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 32 or more, 40 or more, or 50 or more barcode sequences. The barcode sequence can be located at any position in the nucleic acid probe. If there is more than one barcode sequence, the barcode sequence can be positioned adjacent to each other, and/or interspersed with other sequences. In some embodiments, two or more of the barcode sequences can also overlap at least partially. In some embodiments, two or more barcode sequences in the same probe do not overlap. In some embodiments, all barcode sequences in the same probe are separated from each other by at least one phosphodiester bond (e.g., they can be adjacent to each other but do not overlap), such as separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

If present, the barcode sequence can be of any length. If more than one barcode sequence is used, the barcode sequences can independently be of the same or different lengths, such as at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50 nucleotides in length. In some embodiments, the barcode sequence can be no more than 120, no more than 112, no more than 104, no more than 96, no more than 88, no more than 80, no more than 72, no more than 64, no more than 56, no more than 48, no more than 40, no more than 32, no more than 24, no more than 16, or no more than 8 nucleotides in length. Any combination of these is also possible, for example, the barcode sequence can be between 5 and 10 nucleotides, between 8 and 15 nucleotides, etc.

The barcode sequence can be arbitrary or random. In some cases, the barcode sequence is selected to reduce or minimize homology with other components in the sample, for example, so that the barcode sequence itself does not bind or hybridize with other nucleic acids suspected to be in cells or other samples. In some embodiments, the homology between a specific barcode sequence and another sequence (e.g., a cell nucleic acid sequence in a sample or other barcode sequences in a probe added to a sample) may be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the homology may be less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases, and in some embodiments, the bases are continuous bases.

In some embodiments, the number of different barcode sequences in a population of circularized probes or circularizable probes or probe sets is less than the number of different targets (e.g., nucleic acid analytes and/or protein analytes) of nucleic acid probes, but different targets can still be uniquely identified from each other, for example, by encoding the probes with different combinations of barcode sequences. However, it is not necessary to use all possible combinations of a given set of barcode sequences. For example, each probe can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. or more barcode sequences. In some embodiments, the populations of nucleic acid probes can each contain the same number of barcode sequences, but in other cases, different numbers of barcode sequences can be present on the various probes.

In some embodiments, the methods disclosed herein include multiplex analysis of biological samples, including probe hybridization to RCA products, continuous cycles of fluorescence imaging and signal removal, wherein signal removal includes removing fluorophores from fluorophore-labeled reactive molecules (e.g., tyramide). Exemplary detectable reactive reagents and methods are described in US 6,828,109, US2019/0376956, US 2019/0376956, US2022/0026433, US2022/0128565, and US2021/0222234, each of which is incorporated herein by reference in its entirety.

In some embodiments, detecting the RCA product in the sample may include sequencing all or a portion of the RCA product (e.g., one or more barcode sequences or their complementary sequences contained in the RCA product). In some embodiments, sequencing may be performed in situ. In situ sequencing generally involves incorporating labeled nucleotides (e.g., fluorescently labeled mononucleotides or dinucleotides) in a sequential, template-dependent manner or hybridizing labeled primers (e.g., labeled random hexamer) to a nucleic acid template so that the identity (e.g., nucleotide sequence) of the incorporated nucleotides or labeled primer extension products can be determined, and thus the nucleotide sequence of the corresponding template nucleic acid is determined. Aspects of in situ sequencing are described in, for example, Mitra et al., (2003) Anal. Biochem. 320, 55-65 and Lee et al., (2014) Science, 343 (6177), 1360-1363, the entire contents of each document are incorporated herein by reference. In addition, examples of methods and systems for performing in situ sequencing are described in US 2016/0024555, US 2019/0194709, and in US 10,138,509, US 10,494,662, and US 10,179,932, the entire contents of each of which are incorporated herein by reference. Exemplary techniques for in situ sequencing include, but are not limited to, STARmap (described, e.g., in Wang et al., (2018) Science, 361(6499)5691, the contents of which are incorporated herein by reference in their entirety), MERFISH (described, e.g., in Moffitt, (2016) Methods in Enzymology, 572, 1-49, the contents of which are incorporated herein by reference in their entirety), hybridization-based in situ sequencing (HybISS) (described, e.g., in Gyllborg et al., Nucleic Acids Res (2020) 48(19):e112, the contents of which are incorporated herein by reference in their entirety), and FISSEQ (described, e.g., in US2019/0032121, the contents of which are incorporated herein by reference in their entirety).

In some embodiments, sequencing can be performed by sequencing by synthesis (SBS). In some embodiments, a sequencing primer is complementary to a sequence at or near one or more barcodes. In such embodiments, sequencing by synthesis can include reverse transcription and/or amplification to generate a template sequence to which a primer sequence can bind. Exemplary SBS methods include, for example, but are not limited to, those described in US 2007/0166705, US 2006/0188901, US 7,057,026, US 2006/0240439, US 2006/0281109, US 2011/005986, US 2005/0100900, US 9,217,178, US 2009/0118128, US 2012/0270305, US 2013/0260372, and US 2013/0079232, the entire contents of each of which are incorporated herein by reference.

In some embodiments, sequencing can be performed by sequential fluorescent hybridization (eg, sequencing by hybridization). Sequential fluorescent hybridization can involve the sequential hybridization of detection probes comprising an oligonucleotide and a detectable label.

In some embodiments, sequencing can be performed using single molecule sequencing while ligating. Such technology utilizes DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides generally have different tags related to the identity of specific nucleotides in the sequence to which the oligonucleotides hybridize. Aspects and features involved in sequencing while ligating are described in, for example, Shendure et al., Science (2005), 309: 1728-1732, and US 5,599,675; US 5,750,341; US 6,969,488; US 6,172,218; and US 6,306,597, the entire contents of each document are incorporated herein by reference.

In some embodiments, the barcode or its complementary sequence contained in the RCA product or one or more probes hybridized therewith is targeted by a detectably labeled detection oligonucleotide (such as a fluorescently labeled oligonucleotide). In some embodiments, one or more decoding schemes are used to decode signals (such as fluorescence) for sequence determination. In any embodiment herein, any suitable method or technique can be used to analyze (e.g., detect or sequence) barcodes, the method or technique includes those described herein, such as RNA sequential target detection (RNA SPOT), sequential fluorescence in situ hybridization (seqFISH), single molecule fluorescence in situ hybridization (smFISH), multiple error robust fluorescence in situ hybridization (MERFISH), hybridization-based in situ sequencing (HybISS), in situ sequencing, targeted in situ sequencing, fluorescence in situ sequencing (FISSEQ), or spatially resolved transcript amplicon read segment mapping (spatially-resolved transcript amplicon readout mapping, STARmap). In some embodiments, the method provided herein includes analyzing barcodes by sequential hybridization and detection with multiple labeled probes (e.g., detection oligonucleotides). Exemplary decoding schemes are described in Eng et al., "Transcriptome-scale Super-Resolved Imaging in Tissues by RNA SeqFISH+," Nature 568(7751):235-239(2019); Chen et al., "Spatially resolved, highly multiplexed RNA profiling in single cells," Science; 348(6233):aaa6090(2015); Gyllborg et al., Nucleic Acids Res (2020) 48(19):e112; US10,457,980B2; US2016/0369329A1; WO 2018/026873A1; and US2017/0220733A1, each of which is incorporated by reference in its entirety. In some embodiments, these assays enable simultaneous signal amplification, combined decoding, and error correction schemes.

In some embodiments, nucleic acid hybridization can be used for sequencing. These methods utilize labeled nucleic acid decoder probes that are complementary to at least a portion of the barcode sequence. Multiplexed decoding can be performed with a pool of many different probes with distinguishable labels. Non-limiting examples of nucleic acid hybridization sequencing are described in, for example, US 8,460,865 and Gunderson et al., Genome Research 14:870--877 (2004), the entire contents of each document being incorporated herein by reference.

In some embodiments, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET), as described in, for example, Levene et al., Science (2003), 299, 682-686, Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al., Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181, the entire contents of each document are incorporated herein by reference.

In some embodiments, sequence analysis of nucleic acid (e.g., nucleic acid comprising a barcode sequence, such as a probe or RCP) can be performed by sequential hybridization (e.g., sequencing while hybridizing and/or sequential in situ fluorescent hybridization). Sequential fluorescent hybridization may involve sequential hybridization of detectable probes comprising oligonucleotides and detectable labels. In some embodiments, methods disclosed herein include sequential hybridization of detectable probes disclosed herein, including detectably labeled probes (e.g., fluorophore-conjugated oligonucleotides) and/or probes that are not detectably labeled themselves but can be combined with detectably labeled probes (e.g., via nucleic acid hybridization) and detected by detectably labeled probes. Exemplary methods for sequential fluorescent hybridization comprising detectable probes are described in US2019/0161796, US 2020/0224244, US2022/0010358, US2021/0340618, and WO 2021/138676, all of which are incorporated herein by reference. In some embodiments, the methods provided herein can include analyzing an identifier sequence (eg, an analyte sequence or a barcode sequence) by sequential hybridization and detection with a plurality of labeled probes (eg, detection oligonucleotides).

In some embodiments, sequence detection includes: contacting a biological sample with one or more intermediate probes that hybridize directly or indirectly to the RCP, wherein the one or more intermediate probes can be detected using one or more detectably labeled probes (e.g., probes comprising branched junctions, such as the probes described in Section II.B); and dehybridizing one or more intermediate probes and/or one or more detectably labeled probes from the rolling circle amplification product. In some aspects, dehybridizing the probes includes removing the probes from the RCP. In some embodiments, the one or more intermediate probes include one or more overhang regions (e.g., 5' and/or 3' ends of the probes that do not hybridize to the rolling circle amplification product). A probe comprising a single overhang region may be referred to as an "L-shaped probe", and a probe comprising two overhangs may be referred to as a "U-shaped probe". In some cases, the overhang region includes a binding region for binding to one or more detectably labeled probes. In some embodiments, detection includes contacting a biological sample with an intermediate probe pool corresponding to different barcode sequences or portions thereof and a detectably labeled probe pool corresponding to different detectable labels. In some embodiments, the biological sample is contacted with different intermediate probe pools in sequence. In some cases, a common or universal detectably labeled probe pool is used in multiple sequential hybridization steps (eg, with different intermediate probe pools).

In some embodiments, the barcode sequence is detected by sequential hybridization of a probe with a barcode sequence or its complementary sequence and detecting the complex formed by the probe and the barcode sequence or its complementary sequence. In some cases, each barcode sequence or its complementary sequence is assigned a signal code sequence (e.g., a time signal feature or code for identifying an analyte) for identifying the barcode sequence or its complementary sequence, and detecting the barcode sequence or its complementary sequence can include detecting the corresponding sequence of the signal code detected from the continuous pool of sequential hybridization, detection and removal of intermediate probes and the universal pool of detectably labeled probes to decode the barcode sequence or its complementary sequence. In some cases, the signal code sequence can be a fluorophore sequence assigned to the corresponding barcode sequence or its complementary sequence. In some embodiments, the detectably labeled probe is fluorescently labeled. In some embodiments, the barcode sequence or its complementary sequence is decoded by sequential probe hybridization, as described in US2021/0340618, the contents of which are incorporated herein by reference in their entirety.

In any embodiment herein, the detection step may include contacting the biological sample with one or more detectably labeled probes that hybridize directly or indirectly to a barcode sequence or its complement (e.g., in an amplification product generated using a probe or probe set), and dehybridizing the one or more detectably labeled probes. In any embodiment herein, the contacting and dehybridization steps may be repeated with one or more detectably labeled probes that hybridize directly or indirectly to a barcode sequence or its complement and/or one or more other detectably labeled probes. In some aspects, the method includes sequential hybridization of detectably labeled probes to generate a spatiotemporal signal signature or code for identifying an analyte.

In any embodiment herein, the detection step may include contacting the biological sample with one or more first detectably labeled probes that are directly hybridized with multiple probes or probe sets. In some cases, the detection step may include contacting the biological sample with one or more first detectably labeled probes that are indirectly hybridized with multiple probes or probe sets. In any embodiment herein, the detection step may include contacting the biological sample with one or more second detectably labeled probes that are directly or indirectly hybridized with multiple probes or probe sets.

In any embodiment herein, the detection step may include contacting the biological sample with one or more intermediate probes that hybridize directly or indirectly to a barcode sequence or its complement (e.g., a barcode sequence or its complement of a rolling circle amplification product generated using multiple probes or probe sets, wherein the one or more intermediate probes are detectable using one or more detectably labeled probes. In any embodiment herein, the detection step may also include dehybridizing one or more intermediate probes and/or one or more detectably labeled probes from a barcode sequence or its complement (e.g., a barcode sequence or its complement of a rolling circle amplification product generated using multiple probes or probe sets, or using multiple probes or probe sets). In any embodiment herein, the contact and dehybridization steps may be repeated with one or more intermediate probes, one or more detectably labeled probes, one or more other intermediate probes, and/or one or more other detectably labeled probes.

In some aspects, the present invention provides a method for detecting RCP and decoding an identifier sequence in RCP, the method comprising: a) providing an intermediate probe and a fluorescently labeled probe, and RCPs in a cell or tissue sample (e.g., a fixed sample, a FFPE sample, or a hydrogel-embedded and cleared sample), each RCP comprising multiple copies of an identifier sequence having an assigned signal code sequence from a codebook; b) in a first cycle, delivering (e.g., using a fluidics module of an instrument or system) a first plurality of intermediate probe/fluorescently labeled probe pairs to the cell or tissue sample, wherein the intermediate probe and the fluorescently labeled probe in each pair form a complex , the complex comprising an intermediate probe that hybridizes to an RCP in a plurality of RCPs and a fluorescently labeled probe that hybridizes to the intermediate probe, wherein the intermediate probe comprises (i) a recognition sequence that is complementary to an identifier sequence in the RCP and (ii) an overhang sequence, and wherein the fluorescently labeled probe comprises (i) a sequence that is complementary to the overhang sequence and (ii) a fluorescent label; c) detecting (e.g., using an optical module of an instrument or system) a first signal (or the absence thereof) associated with the fluorescent label of a first plurality of probe pairs at a plurality of locations on a solid support, wherein the first signal or the absence thereof detected at a particular location corresponds to the presence of a fluorescent label in the RCP at the particular location. d) in a second cycle, delivering (e.g., using a fluidics module of an instrument or system) a second plurality of intermediate probe/fluorescently labeled probe pairs to a cell or tissue sample, wherein the intermediate probe and the fluorescently labeled probe in each pair form a complex comprising an intermediate probe hybridized to an RCP in a plurality of RCPs and a fluorescently labeled probe hybridized to the intermediate probe, wherein the intermediate probe comprises (i) a recognition sequence complementary to an identifier sequence in the RCP and (ii) an overhang sequence, and wherein the fluorescently labeled probe comprises (i) a sequence complementary to the overhang sequence and (ii) a fluorescent label; e) in a solid The method comprises the steps of: detecting (e.g., using an optical module of an instrument or system) a second signal (or the absence thereof) associated with the fluorescent label of a second plurality of probe pairs at multiple positions on a phase support, wherein the second signal or the absence thereof detected at a specific position corresponds to a second signal code in a signal code sequence assigned to an identifier sequence in an RCP at the specific position, thereby generating a signal code sequence comprising at least a first signal code and a second signal code at each position in the multiple positions; and f) comparing (e.g., using a system controller of an instrument or system) the signal code sequence generated for the RCP at multiple positions with a signal code sequence from a code book, thereby decoding the identifier sequence in the RCP.

In some embodiments, in the first cycle, a first pool of intermediate probes and a universal pool of fluorescently labeled probes are delivered to a solid support, wherein each intermediate probe in the first pool of intermediate probes comprises (i) a recognition sequence complementary to one of the different identifier sequences in the RCP, and (ii) a hybridization sequence complementary to the fluorescently labeled probes of the universal pool. In some embodiments, in the second cycle, a second pool of intermediate probes and a universal pool of fluorescently labeled probes are delivered to a solid support, wherein each intermediate probe in the second pool of intermediate probes comprises (i) a recognition sequence complementary to one of the different identifier sequences in the RCP, and (ii) a hybridization sequence complementary to the fluorescently labeled probes of the universal pool. In some embodiments, the number of different identifier sequences in the RCP is at least 9, and the number of fluorescently labeled probes of different sequences in the universal pool is 4.

In any embodiment herein, each fluorescently labeled probe of different sequences in the universal pool can be labeled with a fluorophore of a different color. In any embodiment herein, the signal code sequence comprises a first signal code, a second signal code, a third signal code corresponding to the third cycle, and a fourth signal code corresponding to the fourth cycle. In any embodiment herein, the signal code sequence can include a dark signal code corresponding to no signal in the corresponding cycle.

VII. Kit

Also provided herein is a kit, which, for example, comprises one or more polynucleotides, such as any circular probe or circularizable probe or probe set described in Section III, and reagents for carrying out the methods provided herein, such as reagents required for one or more steps including hybridization, connection, amplification, detection, sequencing and/or sample preparation described herein. In some embodiments, the kit also comprises a target nucleic acid, such as any target nucleic acid described in Section II. In some embodiments, any or all polynucleotides are DNA molecules. In some embodiments, the target nucleic acid is a messenger RNA molecule. In some embodiments, the kit also comprises a ligase, such as for forming a circularized probe from a circularizable probe or probe set. In some embodiments, the ligase has DNA splinting DNA ligase activity. In some embodiments, the ligase has RNA splinting ligase activity. In some embodiments, the kit also comprises a polymerase, such as for example, a polymerase for rolling circle amplification of a circular or circularized probe using any method described in Section IV. In some embodiments, the kit also comprises a primer (e.g., RCA primer) for amplification.

In some embodiments, disclosed herein is a kit for analyzing biological samples using rolling circle amplification, the kit comprising: (a) a circular probe or a circularizable probe or a probe set comprising one or more modified nucleotides or nucleotide analog residues, wherein the circular probe or the circularizable probe or the probe set comprises a target hybridization region complementary to a target sequence of a target nucleic acid; and (b) a polymerase; wherein the one or more modified nucleotides or nucleotide analog residues reduce the polymerization rate of the polymerase using the circular probe or the circularized probe generated by the circularizable probe or the probe set as a template in a rolling circle amplification reaction compared to a reference circular template without the one or more modified nucleotides or nucleotide analog residues. In some embodiments, the kit may also include a ligase for generating a circularized probe from the circularizable probe or the probe set. In some embodiments, the one or more modified nucleotides or nucleotide analog residues may include a nucleotide residue modified with a sugar and/or a nucleotide residue modified with a backbone. In some embodiments, one or more modified nucleotides or nucleotide analog residues can be selected from the group consisting of: 2'-O-methyl ribonucleic acid (2'-OMeRNA), locked nucleic acid (LNA) nucleotides, 2'-fluoro ribonucleic acid (2'-F RNA), phosphorothioate backbone nucleotides, phosphorothioate backbone nucleotides, triazole modified nucleotides, and combinations thereof. In any of the foregoing embodiments, the kit may also include: one or more non-incorporable nucleotides or analogs thereof, which are configured to transiently bind to the polymerase but are not incorporated by the polymerase; and/or one or more incorporable nucleotides or analogs thereof, which are configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate.

In some aspects, a kit for performing rolling circle amplification (RCA) is provided herein, the kit comprising: (a) a polymerase; (b) one or more non-incorporable nucleotides or analogs thereof, the one or more non-incorporable nucleotides or analogs thereof being configured to bind transiently to the polymerase but not incorporated by the polymerase; and/or one or more incorporable nucleotides or analogs thereof, the one or more incorporable nucleotides or analogs thereof being configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate; and (c) one or more circular probes used as RCA templates, one or more circularizable probes or probe sets for generating circularized probes used as RCA templates, and/or one or more reagents for generating RCA circularized templates. In some embodiments, one or more non-incorporable nucleotides or analogs thereof and/or one or more incorporable nucleotides or analogs thereof may comprise α-thiol nucleotides, alkyne-modified nucleotides, azide-modified nucleotides, diphosphate nucleotides and/or monophosphate nucleotides. In any of the foregoing embodiments, the kit may also comprise unmodified deoxyribonucleotide triphosphates.

In any of the foregoing embodiments of the kit, a combination of one or more non-incorporable nucleotides or analogs thereof, one or more incorporable nucleotides or analogs thereof and/or unmodified deoxyribonucleotide triphosphates may be included in a reaction mixture for RCA, such as any reaction mixture described in Section IV.

In some aspects, a kit for performing rolling circle amplification (RCA) is provided herein; the kit comprises: (a) a polymerase; (b) one or more nucleotide analogs comprising one or more hydrophobic modifications and configured to be incorporated by the polymerase; and (c) one or more circular probes or circularizable probes or probe sets for generating circularization probes as RCA templates, or one or more reagents for generating RCA circularization templates. In some embodiments, one or more nucleotide analogs comprise a modified dUTP selected from ethynyl-dUTP and/or vinyl dUTP. In some embodiments, one or more nucleotide analogs are provided as a reaction mixture comprising one or more nucleotide analogs and unmodified deoxyribonucleotide triphosphates. In some embodiments, the ratio of modified dUTP to unmodified dTTP in the reaction mixture is at least about 80:20.

The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined into a single container. In some embodiments, the kit also contains instructions for using the kit components to perform the provided methods.

In some embodiments, the test kit may contain reagents and/or consumables required for one or more steps of the method provided. In some embodiments, the test kit contains reagents for fixing, embedding and/or permeabilizing biological samples. In some embodiments, the test kit contains reagents, such as enzymes and buffers for connection and/or amplification, such as ligases and/or polymerases. In some respects, the test kit may also include any reagent as described herein, such as washing buffer and connection buffer. In some embodiments, the test kit contains reagents for detection and/or sequencing, such as barcode detection probes or detectable labels. In some embodiments, the test kit optionally contains other components, such as nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, reagents for other determinations.

VIII. Application

In some respects, the embodiments provided can be applied to the in situ methods for analyzing nucleic acid sequences, such as in situ transcriptome analysis or in situ sequencing, such as nucleic acid sequences from intact tissues or samples in which spatial information has been retained. In some respects, the embodiments can be applied to imaging or detection methods for multiple nucleic acid analysis. In some respects, the embodiments provided can be used to identify or detect regions of interest in target nucleic acids.

In some embodiments, the region of interest comprises a single nucleotide polymorphism (SNP). In some embodiments, the region of interest is a single nucleotide variant (SNV). In some embodiments, the region of interest comprises a single nucleotide substitution. In some embodiments, the region of interest comprises a point mutation. In some embodiments, the region of interest comprises a single nucleotide insertion.

In some respects, the embodiments can be applied to research and/or diagnostic applications, for example, for characterizing or evaluating specific cells or tissues from a subject. The application of the provided method can include biomedical research and clinical diagnosis. For example, in biomedical research, applications include but are not limited to spatially resolved gene expression analysis for biological research or drug screening. In clinical diagnosis, applications include but are not limited to detecting gene markers such as disease, immune response, bacteria or viral DNA/RNA of patient samples.

In some aspects, embodiments may be applied to visualize the distribution of genetically encoded markers throughout a tissue at subcellular resolution, such as chromosomal abnormalities (inversions, duplications, translocations, etc.), loss of genetic heterozygosity, the presence of alleles indicative of disease predisposition or good health, likelihood of response to treatment, or in personalized medicine or lineage.

IX. Terminology

Specific terminology is used throughout this disclosure to explain various aspects of the described devices, systems, methods, and compositions.

Some illustrative embodiments of the present disclosure have been described, but it will be apparent to those skilled in the art that the foregoing is illustrative and non-restrictive and has been presented by way of example only. Many modifications and other illustrative embodiments are within the scope of those of ordinary skill in the art and are considered to fall within the scope of the present disclosure. Specifically, although many of the examples presented herein relate to specific combinations of method actions or system elements, it will be understood that these actions and these elements may be combined in other ways to achieve the same goal.

As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more".

As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments involving that value or parameter per se.

Throughout this disclosure, various aspects of the claimed subject matter are presented in the form of ranges. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within the range. For example, in the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range and any other stated or intermediate values in the stated range are all included in the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the claimed subject matter, but are subject to any specific exclusion limits in the stated range. In the case where the stated range includes one or two limits in these limits, the range excluding any one or two limits in those included limits is also included in the claimed subject matter. Regardless of the breadth of the range, this applies.

The use of ordinal terms (such as "first", "second", "third", etc.) in the claims to modify claim elements does not by itself imply any priority, precedence, or order of one claim element over another sequential or chronological order of the acts of performing the method, but is merely used as a marker to distinguish one claim element with a particular name from another element with the same name (but using an ordinal term) to distinguish these claim elements. Similarly, the use of a), b), etc. or i), ii), etc. in the claims does not by itself imply any priority, precedence, or order of steps. Similarly, the use of these terms in the specification does not by itself imply any required priority, precedence, or order.

(i) Barcode

A "barcode" is a label or identifier that conveys or is capable of conveying information (e.g., information about an analyte in a sample and/or bead). A barcode can be part of an analyte or can be separate from the analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes.

Barcodes can have a variety of different forms. For example, barcodes can include polynucleotide barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences. The barcode can be connected to an analyte or another part or structure in a reversible or irreversible manner. The barcode can be added to a fragment of, for example, a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sample sequencing. The barcode can allow identification and/or quantification of individual sequencing reads (e.g., the barcode can be or can include a unique molecular identifier or "UMI").

A barcode can spatially resolve molecular components present in a biological sample, for example, at single-cell resolution (e.g., a barcode can be or can include a "spatial barcode"). In some embodiments, a barcode includes both a UMI and a spatial barcode. In some embodiments, a barcode includes two or more sub-barcodes that together act as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) separated by one or more non-barcode sequences.

(ii) Nucleic acids and nucleotides

The terms "nucleic acid" and "nucleotide" are intended to comply with their use in the art and include unmodified naturally occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids can hybridize with nucleic acids in a sequence-specific manner (e.g., can hybridize with two nucleic acids so that connection can occur between the two hybridized nucleic acids) or can be used as a replication template for a specific nucleotide sequence. Unmodified nucleic acids generally have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone connections. Unmodified nucleic acids generally have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

Nucleic acid can comprise nucleotides of any of a variety of analogs with a sugar moiety. Nucleic acid can comprise natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C) or guanine (G), and ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C) or guanine (G).

(iii) Probe and target

"Probe" or "target," when used to refer to a nucleic acid or nucleic acid sequence, is intended in the context of methods or compositions as a semantic identifier of the nucleic acid or sequence, and does not limit the structure or function of the nucleic acid or sequence beyond that explicitly indicated.

(iv) Oligonucleotides and polynucleotides

The terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to single-stranded nucleotide polymers of about 2 to about 500 nucleotides in length. Oligonucleotides can be synthetic, enzymatic (e.g., by polymerization) or prepared using a "split-pool" method. Oligonucleotides can include ribonucleotide monomers (e.g., can be oligoribonucleotides) and/or deoxyribonucleotide monomers (e.g., oligodeoxyribonucleotides). In some instances, an oligonucleotide can include a combination of deoxyribonucleotide monomers and ribonucleotide monomers in an oligonucleotide (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). For example, oligonucleotide can be 4 to 10,10 to 20,21 to 30,31 to 40,41 to 50,51 to 60,61 to 70,71 to 80,80 to 100,100 to 150,150 to 200,200 to 250,250 to 300,300 to 350,350 to 400 or the length of 400-500 nucleotide.Oligonucleotide can include one or more functional parts connected with polymer structure (for example, covalently or non-covalently).For example, oligonucleotide can include one or more detectable labels (for example, radioisotope or fluorophore).

(v) Hybridizing, Annealing

The terms "hybridizing", "hybridize", "annealing", "anneal" are used interchangeably in the present disclosure and refer to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any method in which a nucleic acid sequence binds to a substantially or completely complementary sequence through base pairing to form a hybridization complex. For the purpose of hybridization, two nucleic acid sequences are "substantially complementary" if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the individual bases of each nucleic acid sequence are complementary to each other.

(vi) Primers

"Primer" is a single-stranded nucleic acid sequence with a 3' end, which can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed by RNA nucleotides and used for RNA synthesis, while DNA primers are formed by DNA nucleotides and used for DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (for example, in a random or designed pattern). Primers can also include other natural or synthetic nucleotides that can have additional functions as described herein. In some instances, a DNA primer can be used to initiate RNA synthesis, and vice versa (for example, an RNA primer can be used to initiate DNA synthesis). The length of a primer can vary. For example, a primer can be about 6 bases to about 120 bases. For example, a primer can include up to about 25 bases. In some cases, a primer can refer to a primer binding sequence.

(vii) Primer extension

"Primer extension" refers to any method by which two nucleic acid sequences are connected (e.g., hybridized) by overlapping complementary nucleic acid sequences at their respective ends (e.g., 3' ends). Such connection may be followed by nucleic acid extension (e.g., enzymatic extension) of one or both ends using another nucleic acid sequence as an extension template. Enzymatic extension may be performed by enzymes including, but not limited to, polymerases and/or reverse transcriptases.

(viii) Nucleic acid extension

"Nucleic acid extension" generally involves incorporating one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs or derivatives thereof) into a molecule (such as, but not limited to, a nucleic acid sequence) in a template-dependent manner, such that consecutive nucleic acids are incorporated by an enzyme (such as a polymerase or reverse transcriptase) to generate a newly synthesized nucleic acid molecule. For example, a primer that hybridizes to a complementary nucleic acid sequence can be used to synthesize a new nucleic acid molecule by using the complementary nucleic acid sequence as a template for nucleic acid synthesis. Similarly, the 3' polyadenylated tail of an mRNA transcript that hybridizes to a poly (dT) sequence can be used as a template for single-stranded synthesis of a corresponding cDNA molecule.

(ix) PCR amplification

"PCR amplification" refers to the use of polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. Suitable reagents and conditions for implementing PCR are described in, for example, U.S. Patents 4,683,202, 4,683,195, 4,800,159, 4,965,188 and 5,512,462, the entire contents of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes genetic material to be amplified, an enzyme, one or more primers for primer extension reactions and reagents for the reaction. Oligonucleotide primers have sufficient length to provide hybridization with complementary genetic material under annealing conditions. The length of the primer is usually dependent on the length of the amplification domain, but will usually be at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (bp), at least 11bp, at least 12bp, at least 13bp, at least 14bp, at least 15bp, at least 16bp, at least 17bp, at least 18bp, at least 19bp, at least 20bp, at least 25bp, at least 30bp, at least 35bp, and can be up to 40bp or longer, wherein the length of the primer will usually be in the range of 18 to 50bp. The genetic material can be contacted with a single primer or a set of two primers (forward primer and reverse primer), depending on whether primer extension, linear or exponential amplification of the genetic material is desired.

In some embodiments, the PCR amplification process uses a DNA polymerase. The DNA polymerase activity can be provided by one or more different DNA polymerases. In certain embodiments, the DNA polymerase is from a bacterium, for example, the DNA polymerase is a bacterial DNA polymerase. For example, the DNA polymerase can be from a bacterium of the genus Escherichia, Bacillus, Thermophilus or Pyrococcus.

Suitable examples of DNA polymerases that can be used include, but are not limited to, E. coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT ^™ DNA polymerase, DEEPVENT ^™ DNA polymerase, Taq DNA polymerase, Hot Start Taq DNA Polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase, DNA polymerase, Quick- DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-Fidelity DNA Polymerase, Platinum Pfx DNA Polymerase, AccuPrime Pfx DNA Polymerase, Phi29 DNA Polymerase, Klenow fragment, Pwo DNA Polymerase, Pfu DNA Polymerase, T4 DNA Polymerase, and T7 DNA Polymerase.

The term "DNA polymerase" includes not only naturally occurring enzymes, but also all modified derivatives thereof, and also derivatives of naturally occurring DNA polymerases. For example, in some embodiments, the DNA polymerase may have been modified to remove 5'-3' exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that can be used include, but are not limited to, mutants that retain at least some functions (e.g., DNA polymerase activity) of the wild-type sequence. Mutations can affect the activity distribution of enzymes under different reaction conditions (e.g., temperature, template concentration, primer concentration, etc.), such as increasing or decreasing polymerization rates. Mutations or sequence modifications may also affect exonuclease activity and/or the thermostability of the enzyme.

In some embodiments, PCR amplification can include reactions such as, but not limited to, strand displacement amplification reactions, rolling circle amplification reactions, ligase chain reactions, transcription-mediated amplification reactions, isothermal amplification reactions, and/or loop-mediated amplification reactions.

In some embodiments, PCR amplification uses a single primer that is complementary to the 3' tag of the target DNA fragment. In some embodiments, PCR amplification uses a first primer and a second primer, wherein at least the 3' terminal portion of the first primer is complementary to at least a portion of the 3' tag of the target nucleic acid fragment, and wherein at least the 3' terminal portion of the second primer displays the sequence of at least a portion of the 5' tag of the target nucleic acid fragment. In some embodiments, the 5' terminal portion of the first primer is not complementary to the 3' tag of the target nucleic acid fragment, and the 5' terminal portion of the second primer does not display the sequence of at least a portion of the 5' tag of the target nucleic acid fragment. In some embodiments, the first primer comprises a first universal sequence and/or the second primer comprises a second universal sequence.

In some embodiments (for example, when the captured DNA is amplified by PCR), DNA ligase can be used to connect the PCR amplification product to another sequence. DNA ligase activity can be provided by one or more different DNA ligases. In some embodiments, DNA ligase is from bacteria, for example, DNA ligase is a bacterial DNA ligase. In some embodiments, DNA ligase is from a virus (for example, a bacteriophage). For example, DNA ligase can be T4 DNA ligase. Other enzymes suitable for the ligation step include but are not limited to Tth DNA ligase, Taq DNA ligase, Thermococcus (strain 9oN) DNA ligase (9oNTM DNA ligase, available from New England Biolabs, Ipswich, MA) and Ampligase ^TM (available from Epicentre Biotechnologies, Madison, WI). Their derivatives (for example, sequence-modified derivatives) and/or mutants can also be used.

In some embodiments, the genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The required reverse transcriptase activity can be provided by one or more different reverse transcriptases, suitable examples of which include, but are not limited to, M-MLV, MuLV, AMV, HIV, ArrayScript ^™ , MultiScribe ^™ , ThermoScript ^™ , and I, II, III and IV enzymes. "Reverse transcriptase" includes not only the naturally occurring enzyme, but also all such modified derivatives thereof, and also includes derivatives of the naturally occurring reverse transcriptase.

In addition, reverse transcription can be performed using sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases, including mutants that retain at least some functional activity of the wild-type sequence (e.g., reverse transcriptase activity). The reverse transcriptase can be provided as part of a composition that includes other components, such as a stabilizing component that enhances or improves reverse transcriptase activity, such as an RNase inhibitor, a DNA-dependent DNA synthesis inhibitor, such as actinomycin D. Many sequence-modified derivatives or mutants of reverse transcriptases (e.g., M-MLV), as well as compositions comprising unmodified and modified enzymes, are commercially available, such as ArrayScript ^™ , MultiScribe ^™ , ThermoScript ^™ , and I, II, III and IV enzymes.

Certain reverse transcriptases (e.g., avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can use both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates to synthesize complementary DNA strands. Therefore, in some embodiments, the reverse transcription reaction can use an enzyme (reverse transcriptase) that can use both RNA and ssDNA as templates for extension reactions, such as AMV or MMLV reverse transcriptase.

In some embodiments, techniques well known in the art are used, such as but not limited to "TAQMAN ^™ " or or in a capillary (“ In some embodiments, the quantification of the genetic material is determined by optical absorbance and real-time PCR. In some embodiments, the quantification of the genetic material is determined by digital PCR. In some embodiments, the analyzed gene can be compared to reference nucleic acid extracts (DNA and RNA) corresponding to expression (mRNA) and quantity (DNA) in order to compare the expression level of the target nucleic acid.

(x) Antibodies

"Antibodies" are polypeptide molecules that recognize and bind to complementary target antigens. Antibodies generally have a molecular structural shape similar to a Y shape. Naturally occurring antibodies, called immunoglobulins, belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can also be produced synthetically. For example, recombinant antibodies that are monoclonal antibodies can be synthesized using synthetic genes by recovering antibody genes from source cells, amplifying them into appropriate vectors, and introducing the vectors into hosts so that the hosts express the recombinant antibodies. In general, recombinant antibodies can be cloned from antibody-producing animals of any species using suitable oligonucleotide primers and/or hybridization probes. Recombinant techniques can be used to generate antibodies and antibody fragments, including non-endogenous species.

Synthetic antibodies can be derived from non-immunoglobulin sources. For example, antibodies can be generated from nucleic acids (e.g., aptamers) and from non-immunoglobulin protein scaffolds (such as peptide aptamers) into which hypervariable loops are inserted to form antigen binding sites. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than immunoglobulin-derived antibodies, resulting in greater tissue penetration.

Antibodies can also include affimer proteins, which are affinity reagents with a molecular weight of generally about 12-14 kDa. Affimer proteins are generally combined with targets (e.g., target proteins) with both high affinity and high specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive proteins. In some embodiments, affimer proteins are derived from cysteine protease inhibitors and include a peptide loop and a variable N-terminal sequence providing a binding site.

Antibodies may also refer to "epitope binding fragments" or "antibody fragments," as used herein, which generally refer to a portion of an intact antibody that is capable of binding to the same epitope as an intact antibody, although not necessarily to the same extent. Although multiple types of epitope binding fragments are possible, epitope binding fragments generally comprise at least one pair of heavy chain variable regions and light chain variable regions (VH and VL, respectively) bound together (e.g., by disulfide bonds) to retain an antigen binding site, and do not comprise all or a portion of an Fc region. Epitope binding fragments of antibodies can be obtained from a given antibody by any suitable technique (e.g., recombinant DNA techniques, or enzymatic or chemical cleavage of intact antibodies), and can generally be screened for specificity in the same manner as screening intact antibodies. In some embodiments, epitope binding fragments include F(ab') ₂ fragments, Fab' fragments, Fab fragments, Fd fragments, or Fv fragments. In some embodiments, the term "antibody" includes antibody-derived polypeptides, such as single-chain variable fragments (scFv), diabodies or other multimeric scFvs, heavy chain antibodies, single domain antibodies, or other polypeptides that contain a sufficient portion of an antibody (e.g., one or more complementarity determining regions (CDRs)) to confer specific antigen binding ability to the polypeptide.

(xi) Labels, detectable labels and optical labels

The terms "detectable label", "optical label" and "label" are used interchangeably herein and refer to a directly or indirectly detectable portion associated (e.g., conjugated) with a molecule to be detected (e.g., a probe for in situ determination). The detectable label can be directly detectable per se (e.g., a radioisotope label or a fluorescent label), or in the case of an enzymatic label, can be indirectly detectable, for example, by catalyzing a chemical change of a substrate compound or composition that is directly detectable. The detectable label can be suitable for small-scale detection and/or for high-throughput screening. Therefore, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds and dyes.

Detectable label can be detected qualitatively (for example, optically or spectrally), or can be quantified.Qualitative detection generally includes the detection method that wherein confirms the existence of detectable label or occurs, and quantifiable detection generally includes the detection method with quantifiable (for example, numerical value can report) value such as intensity, duration, polarization and/or other properties.In some embodiments, detectable label is combined with feature or the probe associated with feature.For example, the feature of detectably labeled can include the fluorescence, colorimetric or chemiluminescent label attached to bead (see, for example, Rajeswari et al., J.Microbiol Methods 139:22-28,2017 and Forcucci et al., J.Biomed Opt. 10:105010,2015, the entire contents of each of these documents are incorporated herein by reference).

In some embodiments, multiple detectable labels can be combined with the feature, probe, or composition to be detected. For example, detectable labels (e.g., Labeled nucleotides, such as -dCTP). Any suitable detectable label may be used. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore may be from the group consisting of: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), Alexa Fluor®, 350. Alexa 430. Alexa 488. Alexa 532. Alexa 546. Alexa 555. Alexa 568. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680、Alexa 700、Alexa 750, Allophycocyanin (APC), AMCA/AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG ^™ CBQCA, ATTO-TAG ^™ FQ, Auramine O-Folgen, BCECF (high pH), BFP (blue fluorescent protein), BFP/GFP FRET, BOBO ^™ -1/BO-PRO ^™ -1, BOBO ^™ -3/BO-PRO ^™ -3, FL, TMR, TR-X, 530/550, 558/568, 564/570, 581/591, 630/650-X, 650-665-X, BTC, Calcium, Calcium Blue, Calcium Crimson ^TM , CalciumGreen-1 ^TM , Calcium Orange ^TM , White, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthylfluorescein, 6-carboxyrhodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), carboxy-X-rhodamine (5-ROX), Cascade Cascade Yellow ^TM , CCF2 (GeneBLAzer ^TM ), CFP (cyan fluorescent protein), CFP/YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC alpha Cychrome (PE-Cy5), Dansylamide, Dansylcadaverine, Dansyl chloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18 (5)), DIDS, Dil (DilC18 (3)), DiO (DiOC18 (3)), DiR (DilC18 (7)), Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, -97 alcohol, eosin, erythrosine, ethidium bromide, ethidium homodimer-1 (EthD-1), europium (III) chloride, 5-FAM (5-carboxyfluorescein), fast blue, fluorescein-dT phosphoramidite, FITC, Fluo-3, Fluo-4, Fluoro-Gold ^TM (high pH), Fluoro-Gold ^TM (low pH), Fluoro-Jade, 1-43, Fura-2 (high calcium), Fura-2/BCECF, Fura Red ^TM (high calcium), Fura Red ^TM /Fluo-3, GeneBLAzer ^TM (CCF2), GFP Red Shifted (rsGFP), GFP wild type, GFP/BFP FRET, GFP/DsRed FRET, Hoechst 33342 & 33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine, JC-1, 6-JOE, JOJO ^TM -1/JO-PRO ^TM -1, LDS751 (+DNA), LDS 751 (+RNA), LOLO ^TM -1/LO-PRO ^TM -1, Fluorescent Yellow, LysoSensor ^TM Blue (pH5), LysoSensor ^TM Green (pH 5) LysoSensor ^TM yellow/blue (pH 4.2) green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green ^TM , Marina 4-Methylumbelliferone, mithramycin, green, orange, Red, NBD (amine), Nile Red, Oregon 488. Oregon 500. Oregon 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinocyanin Chlorophyll Protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (Propidium Iodide), PKH26, PKH67, POPO ^TM -1/PO-PRO ^TM -1, POPO ^TM -3/PO-PRO ^TM -3, Propidium Iodide (PI), PyMPO, Pyrene, Pyronine Y, Quantam Red (PE-Cy5), Quinacridone, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green ^TM 、RhodamineRed ^TM 、Rhodamine phalloidin、Rhodamine 110、Rhodamine 123、5-ROX(Carboxy-X-rhodamine)、S65A、S65C、S65L、S65T、SBFI、SITS、 -1 (high pH), -2, -1 (high pH), -1 (low pH), Sodium Green ^TM , #1, #2, 11. 13. 17. 45. blue, green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), -X, -X (NHS ester), thiadicarbocyanine, thiazole orange, -1/TO- -1, -3/TO- -3.TO- -5, Tri-color (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), -1/YO- -1, -3/YO- -3, 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTORho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5' 700, 5' 800, 5' 800CW (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye, 640 (NHS ester) and Dy 750 (NHS ester).

As mentioned above, in some embodiments, the detectable label is a luminescent or chemiluminescent portion or includes a luminescent or chemiluminescent portion. Common luminescent/chemiluminescent portions include, but are not limited to, peroxidases, such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. Given an appropriate substrate (e.g., an oxidizing agent plus a chemiluminescent compound), these protein moieties can catalyze chemiluminescent reactions. Many families of compounds are known to provide chemiluminescence under various conditions. Non-limiting examples of chemiluminescent compound families include 5-amino-6,7,8-trimethoxy-2,3-dihydro-1,4-phthalazinedione luminol and dimethylamino [ca] benzo analogs. These compounds can emit light in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of chemiluminescent compound families include, for example, 2,4,5-triphenylimidazole, p-dimethylamino and -methoxy substituents, oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinium esters, luciferin, lucigenin, or acridinium esters. In some embodiments, the detectable label is or includes a metal-based or mass-based label. For example, a small cluster of metal ions, metals, or semiconductors can serve as mass codes. In some instances, the metal can be selected from Groups 3-15 of the periodic table, such as Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, Bi, or a combination thereof.

Exemplary embodiments of the present disclosure include:

1A. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with a reaction mixture comprising one or more modified nucleotides or nucleotide analogs, wherein the one or more modified nucleotides or nucleotide analogs comprise modified nucleotides or nucleotide analogs having a hydrophobic modification,

(b) performing rolling circle amplification (RCA) on the circular nucleic acid template in the biological sample using a polymerase, thereby generating an RCA product incorporating the one or more modified nucleotides or nucleotide analogues, wherein the RCA product is not cross-linked by the one or more modified nucleotides or nucleotide analogues incorporated into the RCA product, and

(c) detecting said RCA product at a position in said biological sample which is not cross-linked by said one or more modified nucleotides or nucleotide analogues.

2A. The method of embodiment 1A, wherein the hydrophobic modification is a base modification.

3A. The method of embodiment 1A or 2A, wherein the hydrophobic modification comprises a carbon chain and/or a hydrocarbon ring.

4A. The method of any one of Embodiments 1A to 3A, wherein the hydrophobic modification comprises a triple bond.

5A. The method of any one of Embodiments 1A to 4A, wherein the hydrophobic modification comprises a vinyl or ethynyl group.

6A. The method of any one of embodiments 1A to 5A, wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is ethynyl-dUTP or vinyl-dUTP.

7A. The method of embodiment 6A, wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is 5-ethynyl-dUTP or 5-vinyl-dUTP.

8A. A method according to any one of embodiments 1A to 7A, wherein the diameter of the RCA product generated using the one or more modified nucleotides or nucleotide analogues is smaller than that of a reference RCA product produced using the same template when the one or more modified nucleotides or nucleotide analogues are not included in the reaction mixture.

9A. The method of any one of embodiments 1A to 8A, wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is added to the biological sample at a concentration of at least 1 μM, at least 1.25 μM, at least 2.5 μM, at least 5 μM, at least 10 μM, at least 40 μM, at least 80 μM or at least 100 μM.

10A. The method of any one of embodiments 1A to 9A, wherein the modified nucleotide or nucleotide analog is a modified dUTP, and the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 1:99, optionally wherein the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 40:60.

11A. The method of any one of embodiments 1A to 10A, wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is added to the biological sample at a concentration of about 50 μM to about 100 μM, optionally wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is added to the biological sample at a concentration of about 80 μM to about 100 μM.

12A. A method according to any one of embodiments 1A to 11A, wherein the median diameter of the RCA products generated using the one or more modified nucleotides or nucleotide analogues is smaller than the median diameter of a reference RCA product produced using the same template when the one or more modified nucleotides or nucleotide analogues are not included in the reaction mixture.

13A. The method of any one of embodiments 1A to 12A, wherein the median diameter of the RCA products generated using the one or more modified nucleotides or nucleotide analogs is less than 500 nm.

14A. A method according to any one of embodiments 1A to 13A, wherein the median diameter of the RCA products generated using the one or more modified nucleotides or nucleotide analogues is no more than 90% or no more than 80% of the median diameter of a reference RCA product produced using the same template when the one or more modified nucleotides or nucleotide analogues are not included in the reaction mixture.

15A. A method according to any one of embodiments 1A to 14A, wherein the average intensity, average signal-to-noise ratio and/or density of the RCA products incorporating the one or more modified nucleotides or nucleotide analogues are not significantly different from the average intensity, average signal-to-noise ratio and/or density of a reference RCA product produced using the same template when the one or more modified nucleotides or nucleotide analogues are not included in the reaction mixture.

16A. The method of any one of embodiments 1A to 15A, wherein said incorporation of said one or more modified nucleotides or nucleotide analogs into said RCA product increases the overall hydrophobicity of said RCA product.

17A. The method of any one of embodiments 1A to 16A, wherein the incorporation of the one or more modified nucleotides or nucleotide analogues into the RCA product promotes base stacking interactions between nucleotides in the RCA product.

18A. A method according to any one of embodiments 1A to 17A, wherein: the one or more modified nucleotides or nucleotide analogues incorporated into the RCA product do not contain an amine; and/or the one or more modified nucleotides or nucleotide analogues incorporated into the RCA product do not contain a detectable label, optionally wherein the detectable label is a fluorophore.

19A. A method according to any one of embodiments 1A to 18A, comprising, in (c), contacting the biological sample with a nucleic acid probe capable of hybridizing to the RCA product, optionally wherein the nucleic acid probe comprises a detectable label, and optionally wherein the detectable label is a fluorophore.

20A. The method of embodiment 19A, wherein the nucleic acid probe is an intermediate probe, and the method comprises contacting the biological sample with a detectably labeled probe that can hybridize to the intermediate probe.

21A. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with a reaction mixture comprising one or more nucleotides or nucleotide analogs,

(b) performing rolling circle amplification (RCA) on the circular nucleic acid template in the biological sample using a polymerase to generate an RCA product,

wherein the one or more nucleotides or nucleotide analogs comprise:

(i) a non-incorporable nucleotide or analog thereof, said non-incorporable nucleotide or analog thereof not being incorporated by said polymerase, and/or

(ii) an incorporable nucleotide or nucleotide analogue configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate, and

(c) detecting the RCA product at a certain position in the biological sample that is not cross-linked by nucleotides or nucleotide analogues.

22A. A method according to embodiment 21A, wherein in (c), the RCA product is not cross-linked to the RCA product itself, another molecule in the biological sample, or a matrix in which the biological sample is embedded via a nucleotide or nucleotide analogue incorporated into the RCA product.

23A. A method according to embodiment 21A or 22A, wherein: the one or more nucleotides or nucleotide analogues incorporated into the RCA product do not contain an amine; and/or the one or more nucleotides or nucleotide analogues incorporated into the RCA product do not contain a detectable label, optionally wherein the detectable label is a fluorophore.

24A. A method according to any one of embodiments 21A to 23A, comprising, in (c), contacting the biological sample with a nucleic acid probe capable of hybridizing to the RCA product, optionally wherein the nucleic acid probe comprises a detectable label, and optionally wherein the detectable label is a fluorophore, optionally wherein the nucleic acid probe is an intermediate probe, and the method comprises contacting the biological sample with a detectably labeled probe capable of hybridizing to the intermediate probe.

25A. A method according to any one of embodiments 21A to 24A, wherein the presence of the one or more modified nucleotides or nucleotide analogues in the reaction mixture reduces the polymerization rate of the polymerase and/or the size of the RCA product compared to a reference reaction mixture without the one or more modified nucleotides or nucleotide analogues, optionally wherein the reference reaction mixture contains only unmodified dATP, dTTP and/or dUTP, dCTP and dGTP.

26A. A method according to any one of embodiments 1A to 25A, comprising cross-linking the RCA product with itself or another molecule at one or more nucleotide residues other than: (i) the modified nucleotide or nucleotide analogue residue having the hydrophobic modification, and/or (ii) the incorporable nucleotide or nucleotide analogue incorporated into the RCA product, wherein the cross-linking is performed before and/or after detecting the RCA product.

27A. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with a circular probe or a circularizable probe or a probe set comprising one or more modified nucleotide or nucleotide analog residues, wherein the circular probe or the circularizable probe or the probe set comprises a hybridization zone that hybridizes to a target nucleic acid in the biological sample, and wherein the one or more modified nucleotide or nucleotide analog residues are outside the hybridization zone,

(b) performing rolling circle amplification (RCA) on the circular probe or the circularized probe generated from the circularizable probe or probe set using a polymerase to generate an RCA product,

wherein the presence of the one or more modified nucleotide or nucleotide analogue residues reduces the rate of polymerisation of the polymerase on the circle or circularisation probe and/or the size of the RCA product compared to a reference circular template without the one or more modified nucleotide or nucleotide analogue residues, and

(c) detecting the RCA product at a certain location in the biological sample.

28A. A method according to embodiment 27A, wherein the one or more modified nucleotides or nucleotide analog residues comprise modified deoxyribonucleotides (DNA) or DNA analog residues and/or modified ribonucleotides (RNA) or RNA analog residues.

29A. A method according to embodiment 27A or 28A, wherein the one or more modified nucleotide or nucleotide analog residues do not contain an amine and/or a detectable label, optionally wherein the detectable label is a fluorophore.

30A. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with: (i) a first circular probe or circularizable probe or probe set comprising one or more modified nucleotide or nucleotide analog residues, wherein the first circular probe or circularizable probe or probe set hybridizes to a first target nucleic acid in the biological sample, and (ii) a second circular probe or circularizable probe or probe set, wherein the second circular probe or circularizable probe or probe set hybridizes to a second target nucleic acid in the biological sample,

(b) performing rolling circle amplification (RCA) on the first circularized probe or the first circularized probe generated from the first circularizable probe or probe set using a polymerase to generate a first RCA product, and

(c) performing rolling circle amplification (RCA) on the second circularized probe or the second circularized probe generated from the second circularizable probe or probe set using a polymerase to generate a second RCA product,

wherein the presence of the one or more modified nucleotide or nucleotide analogue residues in the first circular or circularisation probe reduces the polymerisation rate of the polymerase using the first circular or circularisation probe as a template and/or the size of the RCA product, compared to a reference circular template without the one or more modified nucleotide or nucleotide analogue residues; and/or wherein the polymerisation rate of the polymerase using the first circular or circularisation probe as a template and/or the size of the RCA product is less than the polymerisation rate of the polymerase using the second circular or circularisation probe as a template and/or the size of the RCA product

1. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with a circular probe or a circularizable probe or a probe set that can hybridize to a target nucleic acid in the biological sample, wherein the circular probe or the circularized probe generated by the circularizable probe or the probe set comprises one or more modified nucleotides or nucleotide analog residues, and the one or more modified nucleotides or nucleotide analog residues are located outside the region that hybridizes to the target nucleic acid,

(b) performing rolling circle amplification (RCA) on the circular probe or the circularized probe using a polymerase to generate an RCA product,

wherein the presence of the one or more modified nucleotide or nucleotide analog residues in the circular probe or circularization probe reduces the polymerization rate of the polymerase using the circular probe or circularization probe as a template compared to a reference circular template without the one or more modified nucleotide or nucleotide analog residues.

2. A method according to embodiment 1, wherein the one or more modified nucleotides or nucleotide analog residues comprise sugar-modified nucleotides.

3. A method according to embodiment 1 or 2, wherein the one or more modified nucleotides or nucleotide analog residues comprise a C2' modified nucleotide.

4. A method according to any one of embodiments 1 to 3, wherein the one or more modified nucleotides or nucleotide analog residues have a C3' internal folding conformation.

5. A method according to any one of embodiments 1 to 4, wherein the one or more modified nucleotide or nucleotide analog residues comprise one or more modified ribonucleotide residues.

6. The method according to any one of embodiments 1 to 5, wherein the circular probe or circularization probe does not contain unmodified ribonucleotide residues.

7. A method according to any one of embodiments 1 to 5, wherein the circular probe or circularization probe comprises one or more unmodified ribonucleotide residues.

8. A method according to any one of embodiments 1 to 7, wherein the one or more modified nucleotides or nucleotide analog residues are selected from the group consisting of 2′-O-methyl ribonucleic acid (2′-OMeRNA), locked nucleic acid (LNA), 2′-fluoro ribonucleic acid (2′-F RNA), and combinations thereof.

9. The method according to any one of embodiments 1 to 8, wherein the one or more modified nucleotide or nucleotide analog residues comprise 2′-OMeRNA.

10. The method according to any one of embodiments 1 to 9, wherein the one or more modified nucleotide or nucleotide analog residues comprise modified deoxyribonucleotide residues.

11. The method according to any one of embodiments 1 to 10, wherein the circular probe or circularization probe is mainly composed of unmodified deoxyribonucleotide residues.

12. A method according to any one of embodiments 1 to 11, wherein the circular probe or circularization probe contains no more than 20%, no more than 10%, no more than 5% or no more than 1% of modified nucleotide or nucleotide analog residues and/or unmodified ribonucleotide residues.

13. A method according to any one of embodiments 1 to 12, wherein the ring probe or circularization probe comprises no more than two, no more than three, no more than four or no more than five consecutive modified nucleotide or nucleotide analog residues and/or unmodified ribonucleotide residues.

14. A method according to any one of embodiments 1 to 13, wherein the one or more modified nucleotide or nucleotide analog residues comprise one or more triazole or thiol bonds instead of phosphodiester bonds.

15. A method according to embodiment 14, wherein the circular probe or circularization probe comprises one or more phosphorothioate bonds between residues.

16. A method according to embodiment 14 or 15, wherein the circular probe or circularization probe comprises one or more phosphorothioate bonds between residues.

17. A method according to any one of embodiments 1 to 16, wherein the method comprises contacting the biological sample with a circularizable probe or probe group comprising one or more thiol bonds, and the one or more thiol bonds are not located at the 5' or 3' end of the circularizable probe or probe group.

18. The method according to any one of embodiments 1 to 17, wherein the circular probe or circularization probe comprises two or more triazole bonds.

19. The method according to any one of embodiments 1 to 18, wherein the circular probe or circularization probe comprises two or more thiol bonds.

20. The method of embodiment 18 or 19, wherein at least two of the two or more triazole bonds or the two or more thiol bonds are separated by less than 100 nucleotides.

21. A method according to any one of embodiments 1 to 20, wherein the circular probe or circularizable probe or probe set is a first circular probe or circularizable probe or probe set, the circularized probe is a first circularized probe, and the target nucleic acid is a first target nucleic acid, and the method further comprises contacting the biological sample with a second circular or circularizable probe or probe set, wherein the second circular or circularizable probe or probe set hybridizes to a second target nucleic acid in the biological sample, and

The second circularized probe or the second circularized probe generated from the second circularizable probe or probe set is subjected to rolling circle amplification (RCA) using a polymerase to generate a second RCA product.

22. A method according to embodiment 21, wherein the second circular probe or circularization probe does not contain modified nucleotides or nucleotide analog residues, or wherein the second circular probe or circularization probe contains fewer modified nucleotides or nucleotide analog residues than the first circular probe or circularization probe.

23. A method according to embodiment 21 or 22, wherein the second circular probe or circularization probe comprises a modified nucleotide or nucleotide analog residue different from the first circular probe or circularization probe.

24. A method according to any one of embodiments 21 to 23, wherein the polymerization rate of the polymerase using the first circular or circularization probe as a template is slower than the polymerization rate of the polymerase using the second circular or circularization probe as a template.

25. The method of any one of embodiments 1 to 24, wherein the method comprises contacting the biological sample with a reaction mixture comprising one or more nucleotides or analogs thereof, wherein the one or more nucleotides or analogs thereof comprise:

(i) a non-incorporable nucleotide or analog thereof, which is configured to transiently bind to the polymerase but is not incorporated by the polymerase, and/or

(ii) an incorporable nucleotide or an analog thereof, the incorporable nucleotide or analog thereof being configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate.

26. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with a reaction mixture comprising one or more nucleotides or analogs thereof, and

(b) performing rolling circle amplification (RCA) on the circular nucleic acid template in the biological sample using a polymerase to generate an RCA product,

wherein the one or more nucleotides or analogs thereof comprise:

(i) non-incorporable nucleotides or analogs thereof, which are transiently bound to the polymerase but are not incorporated by the polymerase, and/or

(ii) an incorporable nucleotide or an analog thereof, the incorporable nucleotide or analog thereof being configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate.

27. A method according to embodiment 25 or 26, wherein the one or more nucleotides or their analogs include: non-incorporable nucleotides or their analogs, which are transiently bound to the polymerase but not incorporated by the polymerase; and incorporable nucleotides or their analogs, which are configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate.

28. A method according to any one of embodiments 25 to 27, wherein the average open time for incorporation of the incorporable nucleotide or its analog by the polymerase during polymerization is longer than the average open time for incorporation of the corresponding naturally occurring nucleoside triphosphate.

29. A method according to embodiment 28, wherein the average open time of the polymerase to incorporate the incorporable nucleotide during polymerization is at least 125%, 150%, 200%, 225% or 250% of the average open time to incorporate the corresponding nucleoside triphosphate.

30. The method of any one of embodiments 25 to 29, wherein the incorporable nucleotide or analog thereof comprises an α-thiol nucleotide, an alkyne-modified nucleotide, or an azide-modified nucleotide.

31. A method according to any one of embodiments 25 to 30, wherein the incorporable nucleotide or analog thereof comprises a diphosphate nucleotide.

32. A method according to embodiment 30, wherein the method does not include contacting the biological sample with deoxyribonucleoside triphosphates.

33. A method according to any one of embodiments 25 to 32, wherein no more than 50%, 40%, 30%, 20%, 10%, 5% or 1% of the nucleotides or their analogs in the reaction mixture are the non-incorporable nucleotides or their analogs and/or the incorporable nucleotides or their analogs.

34. A method according to any one of embodiments 25 to 33, wherein the non-incorporable nucleotide or analog thereof comprises a monophosphate nucleotide.

35. The method of any one of embodiments 25 to 34, wherein the non-incorporable nucleotide or analog thereof is dissociable from the polymerase.

36. A method according to any one of embodiments 25 to 35, wherein the average polymerization rate of the polymerase in the rolling circle amplification is inversely proportional to the concentration of the one or more incorporable or non-incorporable nucleotides or nucleotide analogs in the reaction mixture.

37. A method according to any one of embodiments 1 to 36, wherein the average polymerization rate of the polymerase in the rolling circle amplification is less than 2280nt/min, less than 2000nt/min, less than 1500nt/min, less than 1250nt/min, less than 1000nt/min, less than 750nt/min, less than 500nt/min or less than 250nt/min.

38. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with a reaction mixture comprising one or more nucleotide analogs having a hydrophobic modification, and

(b) performing rolling circle amplification (RCA) on the circular nucleic acid template in the biological sample using a polymerase, thereby generating an RCA product incorporating the one or more nucleotide analogs,

wherein the RCA product comprising the one or more nucleotide analogues is smaller than a reference RCA product not comprising the one or more nucleotide analogues.

39. A method according to embodiment 38, wherein the hydrophobic modification is a base modification.

40. A method according to embodiment 38 or 39, wherein the hydrophobic modification comprises a carbon chain and/or a hydrocarbon ring.

41. The method of any one of embodiments 38 to 40, wherein the hydrophobic modification comprises a triple bond.

42. The method of any one of embodiments 38 to 41, wherein the hydrophobic modification comprises a vinyl or ethynyl group.

43. The method of any one of embodiments 38 to 42, wherein the nucleotide analog comprising the hydrophobic modification is ethynyl-dUTP or vinyl-dUTP.

44. The method of any one of embodiments 38 to 43, wherein the nucleotide analog comprising the hydrophobic modification is 5-ethynyl-dUTP or 5-vinyl-dUTP.

45. A method according to any one of embodiments 38 to 44, wherein the diameter of the RCA product generated using the one or more nucleotide analogues is smaller than that of a reference RCA product produced using the same circular nucleic acid template when the one or more nucleotide analogues are not included in the reaction mixture.

46. A method according to any one of embodiments 38 to 45, wherein the nucleotide analog comprising the hydrophobic modification is added to the sample at a concentration of at least 1 μM, at least 1.25 μM, at least 2.5 μM, at least 5 μM, at least 10 μM, at least 40 μM, at least 80 μM or at least 100 μM.

47. A method according to any one of embodiments 38 to 46, wherein the nucleotide analog is a modified dUTP and the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 1:99, optionally wherein the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 40:60.

48. A method according to any one of embodiments 38 to 47, wherein the nucleotide analog comprising the hydrophobic modification is added to the sample at a concentration of about 50 μM to about 100 μM, optionally wherein the nucleotide analog comprising the hydrophobic modification is added to the sample at a concentration of about 80 μM to about 100 μM.

49. A method according to any one of embodiments 38 to 48, wherein the median diameter of the RCA product into which the one or more nucleotide analogues have been incorporated is smaller than the median diameter of a reference RCA product generated using the same circular nucleic acid template when the one or more nucleotide analogues are not included in the reaction mixture.

50. A method according to any one of embodiments 38 to 49, wherein the median diameter of the RCA product incorporating the one or more nucleotide analogues is less than 500 nm.

51. A method according to any one of embodiments 38 to 50, wherein the median diameter of the RCA product incorporating the one or more nucleotide analogues is no more than 90% or no more than 80% of the median diameter of a reference RCA product generated using the same circular nucleic acid template when the one or more nucleotide analogues are not included in the reaction mixture.

52. A method according to any one of embodiments 38 to 51, wherein the average intensity, average signal-to-noise ratio and/or density of the RCA product incorporated with the one or more nucleotide analogues is not significantly different from the average intensity, average signal-to-noise ratio and/or density of a reference RCA product generated using the same circular nucleic acid template when the one or more nucleotide analogues are not included in the reaction mixture, optionally wherein the reference RCA product is generated using the same circular nucleic acid template when the one or more nucleotide analogues are not included in the reaction mixture, and optionally wherein the total number of nucleotides and nucleotide analogues in the RCA product is not less than the total number of nucleotides in the reference RCA product.

53. A method according to any one of embodiments 38 to 52, wherein said incorporation of said one or more nucleotide analogues into said RCA product increases the overall hydrophobicity of said RCA product.

54. A method according to any one of embodiments 38 to 52, wherein said incorporation of said one or more nucleotide analogues into said RCA product promotes base stacking interactions between nucleotides in said RCA product.

55. A method according to any one of embodiments 1A to 54, wherein the rolling circle amplification is performed at a temperature between 18°C and 30°C.

56. A method according to embodiment 55, wherein the rolling circle amplification is performed at 30°C.

57. The method of any one of embodiments 1A to 56, wherein the RCA product is generated using linear RCA, branched RCA, dendritic RCA, or any combination thereof.

58. A method according to any one of embodiments 1A to 57, wherein the RCA product is generated using a polymerase selected from the following: Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst DNA polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and any variants or derivatives of the foregoing.

59. The method of any one of embodiments 1A to 58, wherein the polymerase is Phi29 DNA polymerase, Vent DNA polymerase, or Bst DNA polymerase.

60. The method of any one of embodiments 1A to 59, wherein the polymerase is Phi29 DNA polymerase.

61. The method of any one of embodiments 1A to 60, wherein the RCA product is generated in situ in the biological sample or in a matrix in which the biological sample or molecules thereof are embedded.

62. A method according to any one of embodiments 1A to 61, wherein the RCA product is cross-linked to one or more other molecules in the biological sample and/or in a matrix in which the biological sample or molecules thereof are embedded.

63. The method of any one of embodiments 1A to 62, comprising detecting the RCA product in situ in the biological sample or in a matrix in which the biological sample or molecules thereof are embedded.

64. A method according to embodiment 63, wherein detecting the RCA product comprises contacting the biological sample with one or more detectably labeled probes, which directly or indirectly hybridize to one or more barcode sequences contained in the RCA product.

65. The method of any one of embodiments 1A to 64, wherein the signal associated with the RCA product is amplified in situ in the biological sample or in a matrix in which the biological sample or molecules thereof are embedded.

66. A method according to embodiment 65, wherein the signal amplification includes RCA of a probe directly or indirectly bound to the rolling circle amplification (RCA) product; a hybridization chain reaction (HCR) directly or indirectly on the RCA product; a linear oligonucleotide hybridization chain reaction (LO-HCR) directly or indirectly on the RCA product; a primer exchange reaction (PER) directly or indirectly on the RCA product; assembling a branched structure directly or indirectly on the RCA product; hybridizing multiple detectable probes directly or indirectly on the RCA product, or any combination thereof.

67. A method according to any one of embodiments 1A to 66, wherein the method further comprises terminating the rolling circle amplification by heat denaturation and/or by contacting the biological sample with a polymerase inhibitor.

68. A method according to embodiment 67, wherein terminating the rolling circle amplification includes contacting the biological sample with a polymerase inhibitor, wherein the polymerase inhibitor is selected from the group consisting of a pyrophosphate analogue, an allosteric inhibitor of the polymerase, a non-catalytic ion bound to the polymerase, and a chain-terminating nucleotide.

69. A method according to any one of embodiments 26 to 68, wherein the circular nucleic acid template is a circular probe or a circularized probe generated by a circularizable probe or a probe group, wherein the circular probe or the circularizable probe or the probe group hybridizes with the target nucleic acid in the biological sample.

70. The method of any one of embodiments 1A to 69, wherein the circularized probe is generated from the circularizable probe or probe set using the target nucleic acid as a ligation template.

71. The method of any one of embodiments 1A to 70, wherein the circularizable probe set comprises two, three or more probes.

72. The method of any one of embodiments 1A to 71, wherein the circularization probe is generated using enzymatic ligation and/or chemical ligation.

73. A method according to any one of embodiments 1A to 72, wherein the circularization probe is generated using template-dependent ligation and/or template-independent ligation.

74. The method of any one of embodiments 1A to 73, wherein the circularized probe is generated using a ligase having RNA-templated DNA ligase activity and/or RNA-templated RNA ligase activity.

75. The method of any one of embodiments 1A to 74, wherein the circularized probe is generated using a ligase selected from the group consisting of Chlorella viral DNA ligase (also known as PBCV-1 DNA ligase), T4 RNA ligase, T4 DNA ligase, and single-stranded DNA (ssDNA) ligase.

76. The method of any one of embodiments 1A to 75, wherein the circularization probe is generated using PBCV-1 DNA ligase or a variant or derivative thereof and/or T4 RNA ligase 2 (also known as T4Rnl2) or a variant or derivative thereof.

77. The method of any one of embodiments 1A to 76, wherein the RCA product comprises one or more barcode sequences or their complements.

78. A method according to embodiment 77, wherein the one or more barcode sequences or their complementary sequences correspond to the target nucleic acid or a portion thereof.

79. A method according to any one of embodiments 1A to 78, wherein the RCA product comprises between about 10 and about 100, between about 100 and about 1,000, between about 1,000 and about 5,000, between about 5,000 and about 10,000 or more than 10,000 copies of the circular nucleic acid template or the circular or circularized probe.

80. A method according to any one of embodiments 1A to 79, wherein the RCA product is in the form of nanospheres having a diameter between about 0.1 μm and about 3 μm, optionally wherein the diameter is between about 0.1 μm and about 0.5 μm, between about 0.5 μm and about 1 μm, between about 0.8 μm and about 1.3 μm, or between about 1 μm and about 1.5 μm.

81. A method according to any one of embodiments 1A to 80, wherein the length of the RCA product is between about 1 kilobase and about 15 kilobases, between about 15 kilobases and about 25 kilobases, between about 25 kilobases and about 35 kilobases, between about 35 kilobases and about 45 kilobases, between about 45 kilobases and about 55 kilobases, between about 55 kilobases and about 65 kilobases, between about 65 kilobases and about 75 kilobases, or more than 75 kilobases.

82. A method according to any one of embodiments 1A to 81, wherein the target nucleic acid comprises DNA and/or RNA, optionally wherein the target nucleic acid is a reporter oligonucleotide of genomic DNA, RNA, mRNA, cDNA, or a marker that binds directly or indirectly to the analyte in the biological sample.

83. The method according to any one of embodiments 1A to 82 further includes cross-linking the RCA product with itself, with one or more other molecules in the biological sample and/or with a matrix in which the biological sample or its molecules are embedded, optionally wherein the cross-linking reduces the mobility of the RCA product in the biological sample and/or in the matrix.

84. The method of any one of embodiments 1A to 83, wherein the biological sample is a fixed and/or permeabilized biological sample.

85. The method of any one of embodiments 1A to 84, wherein the biological sample is a non-homogenized tissue sample or tissue section.

86. The method of any one of embodiments 1A to 85, wherein the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

87. A method according to any one of embodiments 1A to 86, wherein the biological sample is a tissue section having a thickness between about 1 μm and about 50 μm, optionally wherein the thickness of the tissue section is between about 5 μm and about 35 μm.

88. A method according to any one of embodiments 1A to 87, wherein the biological sample is cross-linked.

89. The method of any one of embodiments 1A to 88, wherein the biological sample is embedded in a matrix, optionally wherein the matrix is a hydrogel.

90. The method of any one of embodiments 1A to 89, wherein the biological sample is cleared.

91. A method for analyzing a biological sample, comprising:

(a) contacting the biological sample with: (i) a first circular probe or circularizable probe or probe set comprising one or more modified nucleotide or nucleotide analog residues, wherein the first circular probe or circularizable probe or probe set hybridizes to a first target nucleic acid in the biological sample, and (ii) a second circular probe or circularizable probe or probe set, wherein the second circular probe or circularizable probe or probe set hybridizes to a second target nucleic acid in the biological sample, and

(b) performing rolling circle amplification (RCA) on the first circularized probe or the first circularized probe generated from the first circularizable probe or probe set using a polymerase to generate a first RCA product, and

performing rolling circle amplification (RCA) on the second circularized probe or the second circularized probe generated by the second circularizable probe or probe set using a polymerase to generate a second RCA product,

wherein the presence of the one or more modified nucleotide or nucleotide analog residues in the first circular or circularizing probe reduces the polymerization rate of the polymerase using the first circular or circularizing probe as a template, compared to a reference circular template without the one or more modified nucleotide or nucleotide analog residues, and/or

wherein the polymerization rate of the polymerase using the first circle or circularization probe as a template is slower than the polymerization rate of the polymerase using the second circle or circularization probe as a template.

92. A method according to embodiment 91, wherein the second circular or circularized probe does not contain modified nucleotides or nucleotide analog residues.

93. A method according to embodiment 91, wherein the second circular or circularized probe contains fewer modified nucleotides or nucleotide analog residues than the first circular or circularized probe.

94. A method according to any one of embodiments 91 to 93, wherein the first target nucleic acid is more abundant in the biological sample than the second target nucleic acid.

95. A kit for performing rolling circle amplification, the kit comprising:

(a) a circular probe or a circularizable probe or a probe set comprising one or more modified nucleotide or nucleotide analog residues, wherein the circular probe or the circularizable probe or the probe set comprises a target hybridizing region complementary to a target sequence of a target nucleic acid; and

(b) polymerase;

wherein the one or more modified nucleotides or nucleotide analog residues reduce the polymerization rate of the polymerase in a rolling circle amplification reaction using the circular probe or the circularized probe generated by the circularizable probe or probe set as a template, compared to a reference circular template without the one or more modified nucleotides or nucleotide analog residues.

96. The kit according to embodiment 95 further comprises a ligase for generating the circularized probe from the circularizable probe or probe set.

97. A kit according to embodiment 95 or 96, wherein the one or more modified nucleotides or nucleotide analog residues comprise sugar-modified nucleotide residues and/or backbone-modified nucleotides or nucleotide analog residues.

98. A kit according to any one of embodiments 95 to 97, wherein the one or more modified nucleotides or nucleotide analog residues are selected from the group consisting of: 2′-O-methyl ribonucleic acid (2′-OMeRNA), locked nucleic acid (LNA) nucleotides, 2′-fluoro ribonucleic acid (2′-FRNA), thiophosphate backbone nucleotides, phosphorothioate backbone nucleotides, triazole-modified nucleotides, and combinations thereof.

99. The kit according to any one of embodiments 95 to 98, further comprising:

one or more non-incorporable nucleotides or analogs thereof, wherein the one or more non-incorporable nucleotides or analogs thereof are configured to transiently bind to the polymerase but not be incorporated by the polymerase, and/or

One or more incorporable nucleotides or analogs thereof, the one or more incorporable nucleotides or analogs thereof configured to be incorporated by the polymerase at a slower rate than a corresponding nucleoside triphosphate.

100. A kit for performing rolling circle amplification (RCA), the kit comprising:

(a) polymerase;

(b) one or more non-incorporable nucleotides or analogs thereof, wherein the one or more non-incorporable nucleotides or analogs thereof are configured to transiently bind to the polymerase but are not incorporated by the polymerase, and/or

one or more incorporable nucleotides or analogs thereof configured to be incorporated by the polymerase at a slower rate than a corresponding nucleoside triphosphate; and

(c) one or more circular probes for use as RCA templates, one or more circularisable probes or probe sets for generating circularised probes for use as RCA templates, and/or one or more reagents for generating circularised templates for RCA.

101. A kit according to embodiment 99 or 100, wherein the one or more non-incorporable nucleotides or analogs thereof and/or one or more incorporable nucleotides or analogs thereof comprise α-thiol nucleotides, alkyne-modified nucleotides, azide-modified nucleotides, diphosphate nucleotides and/or monophosphate nucleotides.

102. The kit of any one of embodiments 95 to 101, further comprising unmodified deoxyribonucleotide triphosphates.

103. A kit according to any one of embodiments 100 to 102, wherein any combination of the one or more non-incorporable nucleotides or analogs thereof, the one or more incorporable nucleotides or analogs thereof and/or the unmodified deoxyribonucleotide triphosphates are contained in the reaction mixture.

104. A kit for performing rolling circle amplification, the kit comprising:

(a) polymerase;

(b) one or more modified nucleotides or nucleotide analogs comprising one or more hydrophobic modifications and configured to be incorporated by the polymerase; and

(c) one or more circular probes for use as templates for rolling circle amplification (RCA), one or more circularizable probes or probe sets for generating circularized probes for use as templates for RCA, and/or one or more reagents for generating circularized templates for rolling circle amplification.

105. A kit according to embodiment 104, wherein the one or more modified nucleotides or nucleotide analogs comprise modified dUTPs, wherein each modified dUTP is independently selected from ethynyl-dUTP and vinyl dUTP.

106. A kit according to embodiment 104 or 105, wherein the one or more modified nucleotides or nucleotide analogs are provided in a reaction mixture comprising the one or more nucleotide analogs and unmodified deoxyribonucleotide triphosphates.

107. The kit of embodiment 105 or 106, wherein the ratio of the modified dUTP to unmodified dTTP in the reaction mixture is at least about 80:20.

It should be understood that all combinations of the described methods and systems are considered part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing in this disclosure are considered part of the inventive subject matter disclosed herein.

Example

The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1: In situ analysis using probes containing modified nucleotide or nucleotide analog residues to reduce the rate of rolling circle amplification

This embodiment discloses an exemplary method for in situ target nucleic acid detection using rolling circle amplification (RCA). In some aspects, improved target detection and/or image analysis can be achieved by reducing the polymerization rate of in situ RCA. In some instances, the methods disclosed herein facilitate providing RCA products with less intensity and/or smaller size.

In some examples, reducing the polymerisation rate of RCA (eg RCA performed in situ in a cell or tissue sample) may be achieved by using a circular probe or circularisable probe or probe set comprising one or more modified nucleotide or nucleotide analogue residues.

A biological sample (e.g., a processed or cleared biological sample, a tissue sample, a sample embedded in a hydrogel, etc.) is contacted with a circular probe or a circularizable probe or probe set (e.g., a padlock probe, a SNAIL probe, a RollFISH probe, a PLAYR probe, etc.). Under separate experimental conditions, the circularizable probe or probe set (a) does not contain a modified nucleotide or nucleotide analog residue (e.g., as shown in FIG. 1B ), (b) contains one or more 2'-OMeRNA nucleotide residues (e.g., as shown in FIG. 1A and FIG. 1C , comparing the structure of 2'-OMeRNA with an unmodified nucleotide residue), or (c) contains one or more triazole bonds (e.g., as shown in FIG. 1D ). In some examples, the circularizable probe or probe set contains any combination of modified nucleotide or nucleotide analog residues. The modified nucleotide or nucleotide analog residue is capable of serving as a template for RCA, but results in a reduced polymerization rate. In examples where different polymerisation rates of RCA probes are exhibited individually, the nucleotide sequences of the circularisable probes or probe sets in (a), (b) and (c) may be identical except for the modified nucleotide or nucleotide analogue residues.

Each probe design is subjected to RCA reaction under the same amplification conditions. The circularizable probe is hybridized with the target nucleic acid sequence (such as mRNA or cDNA) in the biological sample and connected to generate a circularized probe. The RCA primer is hybridized with the connected (circularized) probe, and the RCA reaction mixture (containing Phi29 reaction buffer, dNTP and Phi29 polymerase) is added to the sample. The sample is incubated at an incubation temperature (e.g., 30°C or 37°C) for a defined time (e.g., 3 hours) so that the closed loop is used as a template for RCA and amplified by DNA polymerase to generate an RCA product. The RCA reaction is terminated by washing the sample with TE buffer.

The RCA products under each condition were hybridized with a fluorophore-labeled (e.g., Cy5-labeled) detectable probe at room temperature and imaged to assess the size of the RCA products. Prior to imaging, tissue samples were treated with Gold Antifade Mounting medium to optimize the light path and prolong sample integrity. Imaging was performed using an Orca Fusion camera at 40x magnification.

RCA products are expected to be reduced in size (e.g., have a smaller average diameter) under conditions where the probe comprises 2'-OMeRNA nucleotide residues or triazole bonds compared to conditions where the probe does not comprise modified nucleotide or nucleotide analogue residues.

In another example, a biological sample can be contacted with a plurality of circularizable probes, wherein each circularizable probe hybridizes with a target nucleic acid in the sample. In one example, the first target nucleic acid (mRNA 1) is highly abundant in the sample (e.g., present at high density), while the second target nucleic acid (mRNA 2) is less abundant in the sample (e.g., present at low density). The sample can be contacted with the following probes: a first circularizable probe comprising one or more modified nucleotides or nucleotide analog residues (e.g., comprising one or more 2'-OMeRNA and/or triazole bonds) and hybridized with a target sequence contained in mRNA 1, and a second circularizable probe that does not comprise a modified nucleotide or nucleotide analog residue and hybridizes with a target sequence contained in mRNA 2. The circularizable probes can be connected, and RCA can be performed as described above to generate a first RCA product and a second RCA product in the sample. The polymerization rate on the first circularization probe is less than the polymerization rate on the second circularization probe, thereby producing a first RCA product smaller than the second RCA product (e.g., as schematically depicted in Figures 1A and 1B).

Example 2: Use of nucleotides or their analogs in the reaction mixture to reduce the rate of rolling circle amplification

This embodiment discloses an exemplary method for improving in situ target detection and image analysis using rolling circle amplification (RCA). Improved target detection and image analysis can be achieved by reducing the polymerization rate of in situ RCA, thereby providing RCA products with smaller intensity and size. Reducing the polymerization rate of in situ RCA can be achieved by including one or more nucleotides or their analogs in the reaction, the one or more nucleotides or their analogs comprising (i) non-incorporable nucleotides or their analogs, which are transiently bound to the polymerase but not incorporated by the polymerase, and/or (ii) incorporable nucleotides or their analogs, which are configured to be incorporated by the polymerase at a slower rate than the corresponding nucleoside triphosphate.

This example provides an exemplary method for analyzing a biological sample using RCA. Specifically, this example provides an exemplary method for including nucleotides or analogs thereof in the RCA reaction mixture to reduce the rate of polymerization and thereby reduce the size of the resulting RCA product.

In some instances, a biological sample (e.g., a biological sample treated or transparent, a tissue sample, a sample embedded in a hydrogel, etc.) is contacted with a circular probe or a circularizable probe or a probe set (e.g., a padlock probe). For example, a circularizable probe is hybridized with a target nucleic acid sequence (such as mRNA or cDNA) in a biological sample, and connected to generate a circularized probe by a circularizable probe. RCA primers are hybridized with the circularized probe. In some instances, a circular probe or a circularizable probe or a probe set does not include any modified nucleotides or nucleotide analog residues. In other instances, a circular or circularizable probe or a probe set may be used, which includes a modified nucleotide or nucleotide analog residue (e.g., as described in Example 1) that reduces the polymerization rate of a polymerase on a template.

In other examples, the circular template for RCA can be a circularized cDNA molecule (e.g., generated using CircLigase ^™ , a thermostable ATP-dependent ligase ^that catalyzes the intramolecular ligation (e.g., circularization) of ssDNA templates having 5′-phosphate and 3′-hydroxyl groups). RCA primers can similarly be hybridized to the circularized cDNA molecule.

An RCA reaction mixture containing Phi29 reaction buffer, Phi29 polymerase, and unmodified dNTPs (dATP, dTTP, dCTP, dGTP) is added to the sample. Under separate experimental conditions, the reaction mixture also contains (a) no additional nucleotides or their analogs, (b) α-thiol nucleotides, (c) diphosphate nucleotides, or (d) monophosphate nucleotides. In other examples, the reaction mixture can contain any combination of α-thiol nucleotides, diphosphate nucleotides, and/or monophosphate nucleotides.

The sample is incubated at an incubation temperature (eg, 30°C or 37°C) for a defined time (eg, 3 hours) to allow the circularized probe to serve as a template for RCA and to be amplified by a DNA polymerase to generate an RCA product. The RCA reaction is terminated by washing the sample with TE buffer.

The RCA product of each condition is hybridized to a fluorophore-labeled (e.g., Cy5-labeled) detectable probe at room temperature and imaged to assess the size of the RCA product. Prior to imaging, tissue samples can be treated with Gold Antifade Mounting Medium to optimize the light path and prolong sample integrity. Imaging can be performed using an Orca Fusion camera at 40x magnification with a 4×4 field of view (FOV) per section.

The size of the RCA product is expected to be reduced when alpha-thiol nucleotides, diphosphate nucleotides or monophosphate nucleotides are included in the reaction mixture compared to conditions containing only unmodified dNTPs.

Example 3: Use of modified nucleotides in a reaction mixture to reduce the rate of in situ rolling circle amplification

Example 2 above describes an exemplary method for reducing the rate of rolling circle amplification (RCA) using various incorporable nucleotides or their analogs, which are incorporated by a polymerase at a slower rate than the corresponding unmodified nucleoside triphosphates. This example demonstrates a method for analyzing a biological sample using rolling circle amplification (RCA), wherein exemplary incorporable nucleotides 5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP are included in the RCA reaction mixture to reduce the polymerization rate. The reduction in the polymerization rate results in a smaller size and intensity of the RCA product.

Sections of fresh/frozen mouse brain were prepared for in situ analysis by formalin fixation and permeabilization.

Padlock probes targeting genes Prox1 and Satb2 were added to the samples and allowed to hybridize overnight at 37°C. After probe hybridization, the samples were washed in formamide and SSC to remove unbound probes. For padlock probe ligation, the samples were incubated with 1.25U/μL SplintR ligase in SplintR ligase buffer at 37°C for 2 hours. Primers for rolling circle amplification (RCA) were added to the samples and allowed to hybridize with the circularized padlock probes at 30°C for 30 minutes.

To each sample, add the RCA reaction mixture containing Phi29 polymerase, Phi29 reaction buffer and dNTP. Under separate conditions, allow RCA to proceed for 2 hours or 4 hours. Control conditions include only unmodified dNTPs (dATP, dGTP, dCTP, dTTP), and experimental conditions include modified dNTP 5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP. For experimental conditions, each modified nucleotide replaces 75% of the unmodified corresponding nucleotides (100% of the dATP nucleotides are unmodified, 100% of the dGTP nucleotides are unmodified, 25% of the dCTP nucleotides are unmodified dCTP and 75% are 5-azido-PEG ₄ -dCTP, and 25% of the dTTP nucleotides are unmodified and 75% are 5-ethynyl-dUTP).

The experimental conditions were set as shown in Table 1 below.

Table 1

condition RCA Time Presence of modified dNTPs 1 2 hours no 2 2 hours Yes (75%) 3 4 hours no 4 4 hours Yes (75%)

The RCA reaction is terminated, and an intermediate probe (e.g., L-shaped probe) corresponding to Prox1 or Satb2 is hybridized to the corresponding RCA product (RCP). Each intermediate probe comprises a region that hybridizes to the target sequence of the corresponding RCA product and an overhang for binding a fluorescently labeled probe (e.g., a detection oligonucleotide, DO). The fluorescently labeled probe is then hybridized to the intermediate probe. The sample is imaged by fluorescence microscopy to assess the density, size, and intensity of detectable signals associated with RCP in the dentate gyrus and cortex.

As shown in Figure 3, the density of RCP was not significantly affected by the presence of modified dNTPs. However, as shown in Figure 4, the size of RCP decreased under the condition of the presence of modified nucleotides in the RCA reaction, whether it was 2 hours or 4 hours. As shown in Figure 5, the intensity of RCP decreased under the condition of the presence of modified nucleotides in the RCA reaction, whether it was 2 hours or 4 hours.

The results indicate that inclusion of modified dNTPs can reduce the rate of RCA, resulting in a decrease in the size of the RCP and/or the size and intensity of the detectable signal associated with the RCP.

Example 4: Quantitative RCA shows that the use of modified nucleotides reduces the polymerization rate

Example 3 above demonstrates that the use of modified incorporable nucleotides for in situ RCA can reduce the size and/or intensity of the resulting RCA product. This example provides quantitative RCA results showing that the polymerisation rate is reduced in a reaction mixture comprising the modified dNTPs 5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP in the RCA reaction mixture, thereby reducing the polymerisation rate.

Quantitative RCA (qRCA) was performed to determine the effect of specific modified dNTPs on the RCA rate. The reaction included Phi29 polymerase, Phi29 reaction buffer, circular RCA template oligonucleotide, RCA primer, and dNTPs containing 0%, 50%, or 100% modified nucleotides, as shown in Table 2. As an example, 50% 5-azido-PEG ₄ -dCTP indicates that 50% of the dCTP nucleotides are unmodified dCTP and 50% are 5-azido-PEG ₄ -dCTP. Negative controls included conditions without circular oligonucleotides, dNTPs, or Phi29 polymerase. The reaction also included SYBR Gold to facilitate quantification of the polymerization rate. The reaction was incubated at 37°C and imaged every 30 seconds for 1 hour.

Table 2.

As shown in Figure 6B, 5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP, either alone or in combination, were included to reduce the RCA rate in a concentration-dependent manner. The effect on the RCA rate was cumulative, wherein for the reaction incorporating both 5-azido-PEG ₄ -dCTP and 5-ethynyl-dUTP, the maximum reduction in the RCA rate was observed (Figure 6B, left figure). Compared with 5-ethynyl-dUTP (Figure 6B, right figure), 5-azido-PEG ₄ -dCTP (Figure 6B, middle figure) has a stronger effect on the modified nucleotide alone. Since 5-azido-PEG ₄ -dCTP is the larger of the two modifications (Figure 6A), these results indicate that the reduction in polymerization rate may be related to the size of the base modification.

The above results indicate that modified dNTPs or their analogs can reduce the rate of RCA reactions in a concentration- and modification-dependent manner. Therefore, the results support the use of incorporable nucleotides or their analogs to control the rate of RCA reactions, which are configured to be incorporated by the polymerase at a slower rate than dNTPs (e.g., dATP, dTTP/dUTP, dCTP or dGTP).

Example 5: Use of modified nucleotides in the reaction mixture for in situ reduction of RCA product size

This embodiment discloses an exemplary method for improving in situ target detection and image analysis using rolling circle amplification (RCA). Improved target detection and image analysis can be achieved by reducing the size of the RCA product. Reducing the size of the RCA product can be achieved by including one or more nucleotides or their analogs in the reaction (e.g., including hydrophobic modified nucleotides or their analogs, such as 5-ethynyl-dUTP (5-EdUTP) and/or 5-vinyl-dUTP).

The above examples describe exemplary methods for reducing the rate of RCA. This example provides an exemplary method for analyzing a biological sample using RCA, wherein an exemplary nucleotide (5-ethynyl-dUTP (5-EdUTP) or 5-vinyl-dUTP) with an added hydrophobic group is included in the RCA reaction mixture to promote compression of the RCA product. The inclusion of modified nucleotides results in a smaller size of the RCA product.

Sections of fresh/frozen mouse brain were prepared for in situ analysis by formalin fixation and permeabilization. Padlock probes targeting the genes Prox1 and Satb2 were added to the samples and allowed to hybridize overnight at 37°C. After probe hybridization, the samples were washed in formamide and SSC to remove unbound probes. For padlock probe ligation, the samples were incubated with SplintR ligase and RNase inhibitor in ligase buffer at 37°C for 2 hours. Primers for rolling circle amplification (RCA) were added to the samples and allowed to hybridize with the circularized padlock probes at 37°C for 30 minutes.

An RCA reaction mixture containing Phi29 polymerase and 100 μM dNTPs was added to each sample (100 μM dATP, 100 μM dCTP, 100 μM dGTP, and 100 μM combined dTTP and modified dUTP). The modified dUTP 5-ethynyl-dUTP (5-EdUTP) or 5-vinyl-dUTP was added to the reaction mixture at the following concentrations: 0 μM (control), 1 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, or 100 μM, wherein the modified dUTP replaced the unmodified dTTP in the reaction mixture to a combined total concentration of 100 μM for the modified dUTP and dTTP. The structural formulas of 5-EdUTP and 5-vinyl-dUTP are shown in FIG7 (where the hydrophobic modification is shown with a dashed circle).

RCA was allowed to proceed at 37°C for 2 hours. The RCA reaction was terminated and an intermediate probe corresponding to Prox1 or Satb2 was hybridized to the corresponding RCA product (RCP). Each intermediate probe contained a region hybridized to the target sequence of the corresponding RCA product and an overhang for binding a fluorescently labeled probe. A fluorescently labeled (Cy3) probe was then hybridized to the intermediate probe. The sample was imaged by fluorescence microscopy to assess the density, size and intensity of the detectable signal associated with the RCP.

As shown in Figure 8A, the density of RCPs was not significantly affected by the presence of modified dNTPs. The signal intensity above the local background (Figure 8B), the local signal-to-noise ratio (Figure 8C), and the signal-to-background ratio (Figure 8D) were also not affected by the presence of modified dNTPs.

As shown in Figures 9A to 9B, when 5-EdUTP or 5-vinyl-dUTP is included, the distribution of RCP size (e.g., measured by diameter) is reduced, wherein when 80 μM 5-EdUTP is included, the RCP size is significantly reduced. As shown in Figures 10A to 10B, for different groups of concentrations, when 5-EdUTP or 5-vinyl-dUTP is included, the distribution of RCP size is reduced, wherein when 100 μM 5-EdUTP is included, the RCP size distribution is significantly reduced.

The results indicate that inclusion of nucleotide analogs with hydrophobic modifications can reduce the size of the RCP and/or the size of the detectable signal associated with the RCP.

The scope of the present disclosure is not intended to be limited to the specific embodiments disclosed, which are provided, for example, to illustrate the various aspects of the present disclosure. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such changes may be practiced without departing from the true scope and essence of the present disclosure, and such changes are intended to fall within the scope of the present disclosure.

Claims (30)

1. A method for analyzing a biological sample, comprising:

(a) Contacting the biological sample with a reaction mixture comprising one or more modified nucleotides or nucleotide analogs comprising modified nucleotides or nucleotide analogs having a hydrophobic modification,

(B) Performing Rolling Circle Amplification (RCA) of a circular nucleic acid template in the biological sample using a polymerase, thereby generating an RCA product incorporating the one or more modified nucleotides or nucleotide analogs, wherein the RCA product is not crosslinked to another molecule via the one or more modified nucleotides or nucleotide analogs incorporated into the RCA product, and

(C) Detecting the RCA product at a location in the biological sample that is not crosslinked by the one or more modified nucleotides or nucleotide analogs.

2. The method of claim 1, wherein the hydrophobic modification is a base modification.

3. The method of claim 1 or 2, wherein the hydrophobic modification comprises a carbon chain and/or a hydrocarbon ring.

4. A method according to any one of claims 1 to 3, wherein the hydrophobic modification comprises a triple bond.

5. The method of any one of claims 1 to 4, wherein the hydrophobic modification comprises a vinyl or acetylene group.

6. The method of any one of claims 1 to 5, wherein the modified nucleotide or nucleotide analogue comprising the hydrophobic modification is ethynyl-dUTP or vinyl-dUTP.

7. The method of claim 6, wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is 5-ethynyl-dUTP or 5-vinyl-dUTP.

8. The method of any one of claims 1 to 7, wherein the diameter of the RCA product generated using the one or more modified nucleotides or nucleotide analogs is smaller than a reference RCA product generated using the same template without the one or more modified nucleotides or nucleotide analogs included in the reaction mixture.

9. The method of any one of claims 1-8, wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is added to the biological sample at a concentration of at least 1 μΜ, at least 1.25 μΜ, at least 2.5 μΜ, at least 5 μΜ, at least 10 μΜ, at least 40 μΜ, at least 80 μΜ or at least 100 μΜ.

10. The method of any one of claims 1 to 9, wherein the modified nucleotide or nucleotide analog is a modified dUTP and the ratio of the modified dUTP to unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 1:99, optionally wherein the ratio of the modified dUTP to the unmodified dUTP or dTTP in the reaction mixture is between about 80:20 and about 40:60.

11. The method of any one of claims 1-10, wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is added to the biological sample at a concentration of about 50 μΜ to about 100 μΜ, optionally wherein the modified nucleotide or nucleotide analog comprising the hydrophobic modification is added to the biological sample at a concentration of about 80 μΜ to about 100 μΜ.

12. The method of any one of claims 1 to 11, wherein the median diameter of an RCA product generated using the one or more modified nucleotides or nucleotide analogs is less than the median diameter of a reference RCA product generated using the same template without the one or more modified nucleotides or nucleotide analogs included in the reaction mixture.

13. The method of any one of claims 1 to 12, wherein the RCA product generated using the one or more modified nucleotides or nucleotide analogues has a median diameter of less than 500nm.

14. The method of any one of claims 1 to 13, wherein the median diameter of the RCA product generated using the one or more modified nucleotides or nucleotide analogs is no more than 90% or no more than 80% of the median diameter of a reference RCA product generated using the same template without the one or more modified nucleotides or nucleotide analogs included in the reaction mixture.

15. The method of any one of claims 1 to 14, wherein the average intensity, average signal-to-noise ratio, and/or density of the RCA product incorporating the one or more modified nucleotides or nucleotide analogs is not significantly different from the average intensity, average signal-to-noise ratio, and/or density of a reference RCA product produced using the same template without the one or more modified nucleotides or nucleotide analogs in the reaction mixture.

16. The method of any one of claims 1 to 15, wherein the incorporation of the one or more modified nucleotides or nucleotide analogs into the RCA product increases the overall hydrophobicity of the RCA product.

17. The method of any one of claims 1 to 16, wherein the incorporation of the one or more modified nucleotides or nucleotide analogs into the RCA product promotes base stacking interactions between nucleotides in the RCA product.

18. The method of any one of claims 1 to 17, wherein:

the one or more modified nucleotides or nucleotide analogs incorporated into the RCA product do not comprise an amine; and/or

The one or more modified nucleotides or nucleotide analogs incorporated into the RCA product do not comprise a detectable label, optionally wherein the detectable label is a fluorophore.

19. The method of any one of claims 1 to 18, comprising in (c) contacting the biological sample with a nucleic acid probe capable of hybridizing to the RCA product, optionally wherein the nucleic acid probe comprises a detectable label, and optionally wherein the detectable label is a fluorophore.

20. The method of claim 19, wherein the nucleic acid probe is an intermediate probe, and the method comprises contacting the biological sample with a detectably labeled probe capable of hybridizing to the intermediate probe.

21. A method for analyzing a biological sample, comprising:

(a) Contacting the biological sample with a reaction mixture comprising one or more nucleotides or nucleotide analogs,

(B) Rolling Circle Amplification (RCA) of the circular nucleic acid template in the biological sample using a polymerase, thereby producing an RCA product,

Wherein the one or more nucleotides or nucleotide analogs comprise:

(i) A non-incorporable nucleotide or analogue thereof which is not incorporated by the polymerase, and/or

(Ii) An incorporable nucleotide or nucleotide analogue configured to be incorporated by the polymerase at a slower rate than a corresponding nucleoside triphosphate, and

(C) Detecting the RCA product at a position in the biological sample that is not crosslinked by a nucleotide or nucleotide analogue.

22. The method of claim 21, wherein in (c) the RCA product is not crosslinked to the RCA product itself, another molecule in the biological sample, or a matrix embedding the biological sample via a nucleotide or nucleotide analogue incorporated into the RCA product.

23. The method according to claim 21 or 22, wherein:

The one or more nucleotides or nucleotide analogs incorporated into the RCA product do not comprise an amine; and/or

The one or more nucleotides or nucleotide analogs incorporated into the RCA product do not comprise a detectable label, optionally wherein the detectable label is a fluorophore.

24. The method according to any one of claims 21 to 23, comprising in (c) contacting the biological sample with a nucleic acid probe capable of hybridizing to the RCA product, optionally wherein the nucleic acid probe comprises a detectable label, and optionally wherein the detectable label is a fluorophore,

Optionally wherein the nucleic acid probe is an intermediate probe and the method comprises contacting the biological sample with a detectably labeled probe capable of hybridizing to the intermediate probe.

25. The method of any one of claims 21 to 24, wherein the presence of the one or more modified nucleotides or nucleotide analogs in the reaction mixture reduces the polymerization rate of the polymerase and/or the size of the RCA product compared to a reference reaction mixture without the one or more modified nucleotides or nucleotide analogs, optionally wherein the reference reaction mixture comprises only unmodified dATP, dTTP and/or dUTP, dCTP and dGTP.

26. The method of any one of claims 1 to 25, comprising cross-linking the RCA product to itself or another molecule at one or more nucleotide residues other than: (i) The modified nucleotide or nucleotide analogue residue having the hydrophobic modification, and/or (ii) the incorporable nucleotide or nucleotide analogue incorporated into the RCA product, wherein the cross-linking is performed before and/or after detection of the RCA product.

27. A method for analyzing a biological sample, comprising:

(a) Contacting the biological sample with a circular probe or circularisation probe or set of probes comprising one or more modified nucleotide or nucleotide analogue residues, wherein the circular probe or circularisation probe or set of probes comprises a hybridization region to a target nucleic acid in the biological sample, and wherein the one or more modified nucleotide or nucleotide analogue residues are outside the hybridization region,

(B) Performing Rolling Circle Amplification (RCA) on the circular probe or circularized probes generated from the circularizable probe or probe set using a polymerase, thereby generating RCA products,

Wherein the presence of the one or more modified nucleotide or nucleotide analogue residues reduces the rate of polymerization of the polymerase on the circular or circularized probe and/or the size of the RCA product, compared to a reference circular template without the one or more modified nucleotide or nucleotide analogue residues, and

(C) Detecting the RCA product at a location in the biological sample.

28. The method of claim 27, wherein the one or more modified nucleotide or nucleotide analogue residues comprise a modified Deoxyribonucleotide (DNA) or DNA analogue residue and/or a modified Ribonucleotide (RNA) or RNA analogue residue.

29. The method of claim 27 or 28, wherein the one or more modified nucleotide or nucleotide analogue residues do not comprise an amine and/or a detectable label, optionally wherein the detectable label is a fluorophore.

30. A method for analyzing a biological sample, comprising:

(a) Contacting the biological sample with: (i) A first cyclic probe or circularisation probe or probe set comprising one or more modified nucleotide or nucleotide analogue residues, wherein the first cyclic probe or circularisation probe or probe set hybridizes to a first target nucleic acid in the biological sample, and (ii) a second cyclic probe or circularisation probe or probe set, wherein the second cyclic probe or circularisation probe or probe set hybridizes to a second target nucleic acid in the biological sample,

(B) Performing Rolling Circle Amplification (RCA) on the first circular probe or the first circularized probe generated from the first circularizable probe or probe set using a polymerase, thereby generating a first RCA product, and

(C) Performing Rolling Circle Amplification (RCA) on the second circular probe or a second circularized probe generated from the second circularizable probe or probe set using a polymerase, thereby generating a second RCA product,

Wherein the presence of the one or more modified nucleotide or nucleotide analogue residues in the first circular or circularized probe reduces the rate of polymerization and/or the size of the RCA product of the polymerase using the first circular or circularized probe as a template compared to a reference circular template without the one or more modified nucleotide or nucleotide analogue residues; and/or wherein the rate of polymerization of the polymerase and/or the size of the RCA product using the first circular or circularized probe as a template is less than the rate of polymerization of the polymerase and/or the size of the RCA product using the second circular or circularized probe as a template.