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WO2025019326A2 - Amplification d'arn in situ - Google Patents

Amplification d'arn in situ Download PDF

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Publication number
WO2025019326A2
WO2025019326A2 PCT/US2024/037820 US2024037820W WO2025019326A2 WO 2025019326 A2 WO2025019326 A2 WO 2025019326A2 US 2024037820 W US2024037820 W US 2024037820W WO 2025019326 A2 WO2025019326 A2 WO 2025019326A2
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nucleic acid
rna
sample
determining
amplified
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WO2025019326A3 (fr
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Xiaowei Zhuang
Limor Cohen
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Harvard University
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Harvard University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

  • the present disclosure generally relates to the amplification of RNA, e.g., for MERFISH or other applications.
  • MERFISH is a tool that allows spatially resolved transcriptomic profiling of individual cells (Fig. 1). See, e.g., Int. Pat. Apl. Pub. No. WO 2016/018960, incorporated herein by reference in its entirety. MERFISH can detect thousands of RNA species with high spatial resolution in tissues through combinatorial labeling of single RNA molecules and sequential imaging with an error-robust encoding scheme capable of error detection and/or correction. In one embodiment of MERFISH, encoding (or primary) probes with readout sequences first bind to the target RNA in the tissue (Fig. 1A).
  • RNA species has a unique barcode
  • the encoding probes have a combination of readout sequences unique to each RNA species to create the barcode.
  • fluorescently labeled readout (or secondary) probes bind to the encoding probes in sequential rounds of hybridization (Figs. 1B-C).
  • the fluorescent signal is inactivated between rounds, for example by using chemical cleavage or photobleaching.
  • the presence or absence of a signal in each hybridization round represented by a bit of “1” or “0,” respectively, is used to assign a unique binary barcode to each RNA species.
  • RNA species can be identified, counted, and localized in a single cell using combinatorial labeling and sequential imaging.
  • MERFISH allows imaging of more than 10,000 RNA species (or genes) in individual cells.
  • MERIFSH can also be extended to enable spatially resolved 3D-genome imaging and epigenomic profiling of individual cells.
  • MERFISH has been used to map the spatial distribution of different cell types in various brain regions, for example, the motor cortex (Fig. ID) and the hypothalamus, the whole mouse brain, and human brain regions. See also Int. Pat. Apl. Pub. No. WO 2021/138078, incorporated herein by reference in its entirety.
  • the present disclosure generally relates to the amplification of RNA, e.g., for MERFISH or other applications.
  • the subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
  • a current limitation of MERFISH and other hybridization-based spatial transcriptomics methods is detecting short RNA sequences, thereby making it challenging to detect short RNAs and many RNA isoforms since their unique sequence is not sufficiently long. Certain aspects of the present disclosure are thus directed to overcoming these limitations.
  • the method comprises exposing a primer sequence to a target RNA, wherein the primer sequence is substantially complementary to at least a portion of the target RNA; using reverse transcriptase to synthesize a first nucleic acid portion comprising a portion substantially complementary to at least a portion of the target RNA, wherein the first nucleic acid portion is attached to the primer sequence; and using the reverse transcriptase to synthesize a second nucleic acid portion containing a promoter sequence, wherein the second nucleic acid portion is attached to the first nucleic acid portion, and wherein the primer sequence, the first nucleic acid portion, and the second nucleic acid portion defines a DNA strand.
  • the method in another set of embodiments, comprises exposing RNA contained within a sample to transcription reagents capable of transcribing the RNA into cDNA; and exposing the cDNA within the sample to amplification reagents capable of amplifying the cDNA within the sample.
  • the method comprises reverse transcribing a plurality of RNA molecules into cDNA within a sample; and amplifying the cDNA within the sample.
  • the method comprises exposing a primer sequence to a poly-A tail of an RNA comprising the poly-A tail, wherein the RNA is within a sample; using reverse transcriptase to synthesize a first nucleic acid portion substantially complementary to at least a portion of the RNA, wherein the first nucleic acid portion is attached to the primer sequence; using the reverse transcriptase to synthesize a second nucleic acid portion containing a promoter sequence, wherein the second nucleic acid portion is attached to the first nucleic acid portion, and wherein the primer sequence, the first nucleic acid portion, and the second nucleic acid portion defines a DNA strand; amplifying the DNA strand using the promoter sequence to produce amplified RNA; binding nucleic acid probes to at least some of the amplified RNA strands; and determining the nucleic acid probes within the sample.
  • the method comprises exposing RNA within a sample to a polyadenylation enzyme to produce RNA comprising a poly-A tail; exposing a primer sequence to the poly-A tail; using reverse transcriptase to synthesize a first nucleic acid portion substantially complementary to at least a portion of the RNA, wherein the first nucleic acid portion is attached to the primer sequence; using the reverse transcriptase to synthesize a second nucleic acid portion containing a promoter sequence, wherein the second nucleic acid portion is attached to the first nucleic acid portion, and wherein the primer sequence, the first nucleic acid portion, and the second nucleic acid portion defines a DNA strand; amplifying the DNA strand using the promoter sequence to produce amplified RNA; binding nucleic acid probes to at least some of the amplified RNA strands; and determining the nucleic acid probes within the sample.
  • the method comprises exposing a primer sequence to a poly-A tail of an RNA comprising the poly-A tail, wherein the RNA is within a sample; using reverse transcriptase to synthesize a first nucleic acid portion substantially complementary to at least a portion of the RNA, wherein the first nucleic acid portion is attached to the primer sequence; using the reverse transcriptase to synthesize a second nucleic acid portion containing a promoter sequence, wherein the second nucleic acid portion is attached to the first nucleic acid portion, and wherein the primer sequence, the first nucleic acid portion, and the second nucleic acid portion defines a DNA strand; and amplifying the DNA strand using the promoter sequence to produce amplified RNA.
  • the method comprises exposing RNA within a sample to a polyadenylation enzyme to produce RNA comprising a poly-A tail; exposing a primer sequence to the poly-A tail; using reverse transcriptase to synthesize a first nucleic acid portion substantially complementary to at least a portion of the RNA, wherein the first nucleic acid portion is attached to the primer sequence; using the reverse transcriptase to synthesize a second nucleic acid portion containing a promoter sequence, wherein the second nucleic acid portion is attached to the first nucleic acid portion, and wherein the primer sequence, the first nucleic acid portion, and the second nucleic acid portion defines a DNA strand; and amplifying the DNA strand using the promoter sequence to produce amplified RNA.
  • the method is a method of synthesizing a nucleic acid.
  • the method comprises binding a primer to a poly-A tail of an mRNA; using reverse transcriptase to synthesize a first nucleic acid portion substantially complementary to at least a portion of the mRNA, wherein the first nucleic acid portion is attached to the primer; using the reverse transcriptase to synthesize a second nucleic acid portion not complementary to the mRNA, wherein the second nucleic acid portion is attached to the first nucleic acid portion; annealing the second nucleic acid portion to a template oligonucleotide comprising a promoter; and using the reverse transcriptase to synthesize a third nucleic acid portion substantially complementary to the template oligonucleotide, wherein the third nucleic acid portion is attached to the second nucleic acid portion.
  • the method in another set of embodiments, is a method of synthesizing a nucleic acid in situ.
  • the method comprises binding a primer to a poly-A tail of an mRNA; using reverse transcriptase to synthesize a first nucleic acid portion substantially complementary to at least a portion of the mRNA, wherein the first nucleic acid portion is attached to the primer; using the reverse transcriptase to synthesize a second nucleic acid portion not complementary to the mRNA, wherein the second nucleic acid portion is attached to the first nucleic acid portion; annealing the second nucleic acid portion to a template oligonucleotide comprising a promoter; and using the reverse transcriptase to synthesize a third nucleic acid portion substantially complementary to the template oligonucleotide, wherein the third nucleic acid portion is attached to the second nucleic acid portion.
  • Figs. 1A-1D illustrate multiplexed error-robust fluorescence in situ hybridization or MERFISH, in one embodiment
  • Figs. 2A-2D illustrate RNA molecules amplified in situ, in another embodiment
  • Figs. 3A-3I illustrate MERFISH with in situ RNA amplification using 92 probes, in yet another embodiment
  • Figs. 4A-4C illustrate MERFISH measurements using eight probes per gene with amplification, in still another embodiment
  • Figs. 5A-5C illustrate an example of assay optimization, in yet another embodiment
  • Figs. 6A-6G schematically illustrate nucleic acid molecules amplified in situ, in accordance with another embodiment
  • Figs. 7A-7B illustrate MERFISH with in situ RNA amplification, in yet another embodiment
  • Figs. 8A-8D illustrate MERFISH with in situ RNA amplification with probes targeting the 3’ end and the 5’ end of transcript, in still another embodiment
  • Figs. 9A-9F illustrate MERFISH with in situ RNA amplification with six and four probes per gene, in yet other embodiments
  • Fig. 10 illustrates MERFISH with in situ RNA amplification for 4,425 genes using six probes per gene and in- situ RNA amplification, in still another embodiment
  • Fig. 11 illustrates MERFISH with in situ RNA amplification, with and without expansion microscopy, in another embodiment.
  • SEQ ID NO: 1 is /5Biosg/TAATACGACTCACTATAGGGAAATArGrG+G (TSO sequence 1, +G indicating a locked nucleic acid);
  • SEQ ID NO: 2 is /5Biosg/TAATACGACTCACTATAGGGAGArGrG+G (TSO sequence 2, +G indicating a locked nucleic acid);
  • SEQ ID NO: 4 is /5Biosg/TAATACGACTCACTATAGGGAAATA rNrG+G;
  • SEQ ID NO: 5 is /5Biosg/TAATACGACTCACTATAGGGAAATA rGrG+G;
  • SEQ ID NO: 6 is /5Biosg/TAATACGACTCACTATAGGGAGA rGrG+G;
  • SEQ ID NO: 7 is /5Biosg/TAATACGACTCACTATAGGGAGA rNrG+G;
  • SEQ ID NO: 8 is TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN;
  • SEQ ID NO: 9 is N-(SEQ ID NO: 9
  • the present disclosure generally relates to the amplification of RNA, e.g., for MERFISH or other applications.
  • One set of embodiments is generally directed to a method of synthesizing a nucleic acid.
  • Some embodiments are drawn to systems and methods for in situ amplification of RNA, which may allow genome- scale imaging of RNAs, including short RNAs and RNA isoforms that are differentiated by short sequences.
  • RNA such as mRNA may be transcribed into cDNA using a reverse transcriptase.
  • the reverse transcriptase can also be used to associate a promoter (for example, a T7 promotor) with the RNA, e.g., by using a template- switching oligonucleotide (TSO).
  • TSO template- switching oligonucleotide
  • the promotor sequence can then be used to amplify the RNA, e.g., using techniques such as in vitro transcription, which can be performed in situ. Having amplified or increased amounts of RNA in situ may be useful for certain applications, such as MERFISH, as the RNA is easier to detect.
  • Other aspects are generally related to methods for using such techniques, kits involving such techniques, or the like.
  • Some embodiments are generally directed to systems and methods to detect short RNA sequences.
  • the target RNAs may be first stained with encoding probes and then detected using fluorescently labeled readout probes (Fig. 1A). Since detection of short RNA sequence involves using fewer encoding probes and hence reducing the signal, an approach for signal amplification is needed, e.g., for use with MERFISH or other applications.
  • it can be difficult to detect nucleic acid in situ within a cell e.g., at sites within the cell where the nucleic acids are located, due to the small size of the cells, as well as the very small concentrations of nucleic acids that may be present. In some cases, for instance, only a single nucleic acid molecule may be present at a particular site within the cell, making detection of that molecule a significant challenge.
  • detection of such molecules in situ within a cell may be improved by increasing the number of such molecules within the cell to be detected. If more molecules are present at a given location within a cell, then there would be more molecules, for example, for signaling entities to bind to, thereby increasing the observed signal produced by those signaling entities within the cell.
  • amplification of nucleic acids may occur in situ or within a cell.
  • those amplification reactions typically occur in controlled and normally homogenous environments, e.g., in test tubes, microwell plates, or other in vitro settings.
  • nucleic acid such as RNA within cells, i.e., where the amplification occurs in situ or in vivo.
  • target nucleic acid 20 within a cell environment 10 is to be amplified.
  • the nucleic acid may be located anywhere in the cell, e.g., in the nucleus or in the cytoplasm, etc.
  • a target nucleic acid within a cell may be RNA, such as mRNA, that is positioned at a specific location within a cell.
  • the target nucleic acid it is desired to determine where the target nucleic acid is located within cell (or even whether it is present or not within cell), and thus, by amplifying the amount of target nucleic acid present in situ within the cell, it may become easier to detect the target nucleic acid within the cell, for instance, using FISH, MERFISH, fluorescence labeling, or other established techniques for determining nucleic acids that are known to those of ordinary skill in the art.
  • target nucleic acid 20 is shown positioned within a cell.
  • the target nucleic acid may be, for example, mRNA having a poly-A tail.
  • the nucleic acid may be exposed to an enzyme, such as a polyadenylation enzyme (not shown in Fig. 6A), which is capable of adding a poly-A tail to the nucleic acid.
  • a polyadenylation enzyme not shown in Fig. 6A
  • the poly-A tail is shown as region 25 of target nucleic acid 20.
  • a primer sequence 30 is added to the cell and allowed to bind or hybridize (e.g., noncovalently) to poly-A tail 25.
  • the primer sequence may comprise a reverse transcriptase (RT) primer sequence (for example, a sequence rich in T’s) that is able to recognize or hybridize to the poly-A tail, e.g., as being substantially complementary.
  • RT reverse transcriptase
  • the primer may comprise a sequence of at least 7 consecutive T’s, at least 10 consecutive T’s, at least 50 consecutive T’s, etc.
  • target nucleic acid 20 is exposed to a reverse transcriptase (not shown), which is able to recognize the RT primer on the primer sequence. Additional mononucleotides may also be present (not shown) which allows the reverse transcriptase to synthesize cDNA 40 (complementary DNA) using target nucleic acid 20 as a template, attached to and extending from primer sequence 30.
  • the reverse transcriptase may also add a few non-templated nucleotides at the 5’ end of the cDNA, shown as sequence 45 in Fig. 6C. For instance, these may be added once the reverse transcriptase has gone past the end of target nucleic acid 20, as shown in this figure.
  • the non-templated nucleotides at the 5’ end of the cDNA may include one or more C’s.
  • sequence 50 comprising a promoter may be added, e.g., to the end of target nucleic acid 20, for example, by a reverse transcriptase.
  • the promoter may be useful, for example, for allowing the target nucleic acid sequence to be amplified (e.g., resulting in a lot of copies), as discussed below.
  • the promoter sequence may be a T7 promoter, or other promoter such as those disclosed herein.
  • the promoter may be part of a template switch oligonucleotide (TSO) sequence in some embodiments, which can hybridize to non-templated nucleotides 45.
  • TSO template switch oligonucleotide
  • the TSO sequence may comprise one or more G’s that can recognize non-templated nucleotides that include one or more C’s.
  • the reverse transcriptase may add sequence 50 to the end of target nucleic acid 20 based on non-templated nucleotides 45. As shown in Fig. 6D, sequence 50 may be attached to and extending from target nucleic acid 20.
  • the cDNA strand has been further extended to compliment sequence 50, e.g., by a reverse transcriptase.
  • the additional complimentary sequence 60 may be attached to and extending from sequence 50 comprising the promoter.
  • the target nucleic acid 20 has been extended to further include a promoter, such as a T7 promoter, and the reverse strand comprises a reverse transcriptase primer, cDNA substantially complementary to the target nucleic acid, and the other part of the T7 promoter (e.g., a double-stranded T7 promoter).
  • the promotor sequence is used to amplify or make multiple copies of target nucleic acid 20. As noted above, this may be performed in situ within the cell.
  • a variety of amplification techniques may be used to amplify the target nucleic acid.
  • the promoter is a T7 promoter
  • a T7 RNA polymerase may be used.
  • the amplification reagents may be added to the cell, e.g., after extending target nucleic acid 20 to further include a promoter.
  • the reagents may include a suitable polymerase (for example, a T7 RNA polymerase), suitable mononucleotides, etc.
  • such techniques may be used to produce a plurality of copies of the target nucleic acid 20, e.g., positioned at or near the original location of the target nucleic acid 20 within the cell, i.e., the amplification of the target nucleic acid may occur in situ.
  • the amplified target nucleic acid may be determined, qualitatively and/or quantitatively, in situ within the cell, in certain embodiments. It is believed that more copies of a target nucleic acid may allow more signaling entities to recognize and bind the target nucleic acid at a specific location, which may allow an increase in the signal that is observed due to the signaling entities. For example, as is shown schematically in Fig.
  • a signaling entity 70 has been added that is able to recognize a specific location on target nucleic acid 20. Because of the presence of multiple copies of target nucleic acid 20, multiple copies of signaling entity 70 are able to bind in situ, typically at or near the site of the original target nucleic acid thereby increasing the strength of the signal produced by the signaling entity.
  • a variety of techniques may be used for in situ determination, such as MERFISH or multiplexed error-robust fluorescence in situ hybridization. Other examples include FISH, fluorescence labeling, or the like. Non-limiting examples of such techniques, including but not limited to MERFISH, are disclosed in, for example, US Pat. No. 11,098,303 or Int. Pat. Apl. Pub. No. WO 2016/018960, each incorporated herein by reference in its entirety.
  • techniques for determining target nucleic acids within a cell or in situ may be combined with techniques for amplifying signaling entities in some embodiments, for example, such as those disclosed in Int. Pat. Apl. Pub. No. WO 2020/123742 or U.S. Pat. Apl. Pub. No. 2022/0064697, each incorporated herein by reference in its entirety.
  • RNA e.g., in situ
  • various aspects are directed to various systems and methods for the amplification of RNA, e.g., for MERFISH or other applications.
  • one set of embodiments is generally directed to determining a sample, which may include a cell culture, a suspension of cells, a biological tissue, an organ, a biopsy, an organism, a biological specimen, or the like, in accordance with certain aspects.
  • the sample can also be cell-free but nevertheless contain nucleic acids in some cases.
  • the cell may be a human cell, or any other suitable cell, e.g., a mammalian cell, a fish cell, an insect cell, a plant cell, or the like. In some cases, only a single cell is student, although more than one cell may be present in other cases. If more than one cell is present, the cells may be of the same or different types.
  • a cell may be fixed, e.g., to preserve the positions of the nucleic acids or other targets within the cell.
  • Techniques for fixing cells are known to those of ordinary skill in the art.
  • a cell may be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, glyoxal, methacam, or the like.
  • a cell may be fixed using HEPES- glutamic acid buffer-mediated organic solvent (HOPE).
  • HOPE HEPES- glutamic acid buffer-mediated organic solvent
  • Enzymatic approaches typically rely on several enzymatic steps, such as ligation followed by amplification.
  • the advantage of enzymatic approaches is that the signal amplification can be very high, allowing many fluorophores to be detected.
  • there are several disadvantages including low efficiency of certain enzymes, which leads to false-negatives and lower detection efficiency) and high background signals, which leads to false-positives.
  • Enzymatic approaches can be used for spatially resolved single-cell transcriptomics measurements.
  • RNAs can be detected with 16 encoding probes, which in principle allows the detection of RNAs with ⁇ 200 nt length using an overlapping encoding probe design.
  • RNA molecules can be amplified in situ using a two-step enzymatic amplification approach (see, e.g., Fig. 2A).
  • reverse transcription (RT) with template switching may be used.
  • nucleic acids within a cell may be determined, e.g., after fixing the cell or otherwise preserving the positions of the nucleic acids or other targets within the cell, e.g., in situ. In some cases, at least some of the nucleic acids may be fixed at specific sites within the cell, e.g., where such nucleic acids are normally or endogenously found. The determination of the nucleic acids may be qualitative and/or quantitative. In addition, in some embodiments, expansion microscopy techniques may be used, e.g., to improve determination of the nucleic acids within a cell or other sample.
  • nucleic acids examples include DNA (for example, genomic DNA), RNA, or other nucleic acids that are present within a cell (or other sample).
  • the nucleic acids may be located anywhere within the cell, e.g., in the nucleus, in the cytoplasm, in the mitochondria, attached to specific organelles, or the like.
  • the nucleic acids may be endogenous to the cell, or added to the cell.
  • the nucleic acid may be viral, or artificially created.
  • the nucleic acid to be determined may be expressed by the cell.
  • the nucleic acid is RNA in some embodiments.
  • the RNA may be coding and/or non-coding RNA.
  • the RNA may encode a protein.
  • Non-limiting examples of RNA that may be studied within the cell include mRNA, siRNA, rRNA, miRNA, tRNA, IncRNA, snoRNAs, snRNAs, exRNAs, piRNAs, or the like.
  • a significant portion of the nucleic acid within the cell may be studied.
  • enough of the RNA present within a cell may be determined so as to produce a partial or complete transcriptome of the cell.
  • the transcriptome of a cell may be determined. It should be understood that the transcriptome generally encompasses all RNA molecules produced within a cell, not just mRNA. Thus, for instance, the transcriptome may also include rRNA, tRNA, siRNA, etc. in certain instances. In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, 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 transcriptome of a cell may be determined.
  • the nucleic acid within the cell may include a poly-A tail.
  • the poly-A tail may have at least 7, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, or at least 250 consecutive A’s in it.
  • the poly-A tail may have no more than 300, no more than 275, no more than 250, no more than 200, no more than 150, no more than 100, or no more than 50 consecutive A’s in it.
  • the poly-A tail may have consecutive A’s of between any of these ranges, e.g., the poly-A tail may have between 150 and 300 consecutive A’s, between 225 and 275 consecutive A’s, between 50 and 100 consecutive A’s, etc.
  • the nucleic acid may be an mRNA having a poly-A tail. In other embodiments, however, the nucleic acid may be other RNAs, e.g., including any of those disclosed herein. For instance, in some embodiments, polyadenylation of RNA may be used to improve template efficiency of reverse transcription, e.g., to determine RNAs that do not have a poly-A-tail, to determine samples that have fragmented or degraded RNA, or the like.
  • nucleic acids within a cell may be modified to include a poly-A tail.
  • a cell or other sample may be exposed to a polyadenylation enzyme, e.g., in situ, to a poly-A tail on the nucleic acid.
  • a polyadenylation enzyme is polynucleotide adenylyltransferase.
  • polynucleotide adenylyltransferases Various polyadenylation enzymes such as polynucleotide adenylyltransferases can be obtained commercially.
  • the polyadenylation enzyme may be added to a cell or other sample, and in some cases, along with ATP or adenosine triphosphate.
  • a primer sequence can be added using T4 RNA ligase.
  • a primer sequence may be ligated to the RNA at the 3’ end, and a complementary sequence may be added to primer the RT reaction.
  • the primer may be a gene-specific primer, in accordance with one embodiment.
  • the primer may be a random primer, e.g., a random pentamer primer, a random hexamer primer, a random heptamer primer, etc.
  • the random primer may be an 8-mer, a 9-mer, a 10-mer, or a higher-order primer.
  • the primer sequence is T(30)VN (SEQ ID NO: 3). In another embodiment, however, the primer sequence is a sequence that is not T(30)VN (SEQ ID NO: 3).
  • the primer may include an LNA, or a locked nucleic acid.
  • the LNA may be a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2’ oxygen and 4, carbon, thereby “locking” the nucleic acid conformation.
  • the poly-A tail of the nucleic acid may be exposed to a sequence comprising a primer, such as a reverse transcriptase (RT) primer.
  • a primer such as a reverse transcriptase (RT) primer.
  • RT reverse transcriptase
  • a primer binds to the poly-A tail of the mRNA.
  • the sequence may comprise a primer that is able to recognize the poly-A tail.
  • primers are available commercially from a variety of sources.
  • the primer may be substantially rich in T’s, which may be able to hybridize the A’s in the poly-A tail.
  • the primer may be at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% T’s.
  • the primer may have at least 7, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, or at least 250 consecutive T’s in it.
  • the primer may have no more than 300, no more than 275, no more than 250, no more than 200, no more than 150, no more than 100, or no more than 50 consecutive T’s in it. In some cases, the primer may have consecutive T’s of between any of these ranges, e.g., the primer may have between 150 and 300 consecutive T’s, between 225 and 275 consecutive T’s, between 50 and 100 consecutive T’s, etc.
  • a primer is a nucleic acid (e.g., DNA or RNA) that serves as a starting point for nucleic acid synthesis, allowing polymerase enzymes such as nucleic acid polymerase to extend the primer and replicate the complementary strand.
  • a primer may be complementary to and to hybridize to a target nucleic acid.
  • the primer is a synthetic primer.
  • a primer is a non-naturally-occurring primer.
  • a primer typically has a length of 10 to 50 nucleotides.
  • a primer may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides.
  • a primer has a length of 18 to 24 nucleotides.
  • the primers may be used as a primer for an enzyme such as reverse transcriptase.
  • the reverse transcription may occur with template switching, where a first template (e.g., the nucleic acid target) is replaced with a second template (e.g., a sequence containing a promoter), such as is discussed below, e.g., during transcription. This may be useful, for example for attaching a sequence comprising a promoter to the nucleic acid target, for use in amplification of the nucleic acid target or the like.
  • reverse transcription may be performed using an RT enzyme such as reverse transcriptase.
  • the reverse transcriptase may be a viral reverse transcriptase, e.g., M-MLV reverse transcriptase, AMV reverse transcriptase, or the like.
  • M-MLV reverse transcriptase M-MLV reverse transcriptase
  • AMV reverse transcriptase A variety of reverse transcriptase enzymes are commercially available. Those of ordinary skill in the art will be aware of suitable conditions for causing reverse transcription to occur.
  • the reverse transcriptase may be able to recognize or hybridize to the RT primer on the primer sequence, thereby allowing reverse transcription to occur.
  • a reverse transcriptase may be added along with mononucleotides (e.g., dNTPs or deoxyribose nucleoside triphosphates), which allows the reverse transcriptase to synthesize DNA (e.g., cDNA or complementary DNA) using the target nucleic acid as a template.
  • mononucleotides e.g., dNTPs or deoxyribose nucleoside triphosphates
  • RNA molecules within a sample may be reverse transcribed into cDNA.
  • the reverse transcriptase enzyme may synthesize complementary DNA (cDNA) and optionally, add a few non-templated nucleotides at the 5’ end of the mRNA template, in accordance with one set of embodiments. These may be added once the reverse transcriptase has gone past the end of the target nucleic acid.
  • the reverse transcriptase enzyme may add 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides that are not complementary. In some cases, these may be attached to an end of the cDNA sequence.
  • the non-templated nucleotides at the 5’ end of the cDNA may include one or more C’s.
  • C the non-templated nucleotides at the 5’ end of the cDNA
  • some types of reverse transcriptase enzyme once the target strand has ended, will begin adding one or more C’s to the cDNA strand, e.g., in the absence of any target strand.
  • the cDNA strand may, in certain embodiments, contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more C’s on the end of the cDNA strand.
  • the non-templated nucleotides then may be annealed or hybridized in one set of embodiments to a template oligonucleotide, e.g., comprising a promoter.
  • a template oligonucleotide comprising a promoter may be a template switching oligonucleotide (TSO), for example, comprising a T7 promoter sequence.
  • TSO template switching oligonucleotide
  • T7 promoter other suitable promoters that may be used include, but are not limited to, T3 promoters or SP6 promoters.
  • more than one promoter may be added.
  • the template oligonucleotide or TSO may have any length.
  • the template oligonucleotide or TSO may have a length of 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, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides.
  • the length may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides.
  • the length may be between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
  • the non-templated nucleotides at the end of the cDNA are hybridized to a template switch oligonucleotide (TSO) sequence, for example, if the non- templated nucleotides comprise one or more C’s and the TSO sequence comprises one, two, three, or more consecutive G’s.
  • the TSO may include a promoter, such as a T7 promoter.
  • promoters include T3 promoters or SP6 promoters.
  • the TSO may be an oligonucleotide sequence that is able to hybridize to the untemplated C nucleotides added by the reverse transcriptase during reverse transcription.
  • the TSO may be useful for cDNA amplification, e.g., due to the presence of the promoter sequence.
  • the TSO sequence may comprise one, two, three, four or more consecutive A’s, T’s, C’s or G’s, in various embodiments.
  • the template switching oligonucleotide sequence may comprise a sequence rNrG+G, wherein N is A, C, G, or T; +G is a locked nucleic acid; and r is any suitable integer, e.g., 1, 2, 3, 4, 5, 6, or more.
  • Other locked nucleic acids may also be used in other embodiments within the TSO.
  • the template switching oligonucleotide sequence may comprise a sequence rNrX+X, wherein N and X are each independently any suitable nucleotide (with the + indicating a locked nucleotide), and r is any suitable integer, e.g., 1, 2, 3, 4, 5, 6, or more.
  • this may lead the reverse transcriptase enzyme to switch templates from the target nucleic acid to the TSO or other template oligonucleotide, e.g., during synthesis.
  • the resulting cDNA that is produced may have a promoter sequence such as a T7 promoter sequence.
  • the reverse transcriptase may then be used the TSO as a second template, producing a sequence on the cDNA that is substantially complementary to the sequence of the TSO or other template oligonucleotide.
  • the promoter sequence may be one that, when transcribed (e.g., by the reverse transcriptase), produces a double- stranded promoter sequence.
  • the promotor sequence may be a double- stranded T7 promoter (for example, as shown in Fig. 2A).
  • amplified nucleic acids may be produced, e.g., using the promoter, to initiate amplification.
  • the sequence that is to be amplified may include, for example, the sequence comprising the TSO (e.g., comprising a promoter) and the target nucleic acid sequence, e.g., as discussed above.
  • the target nucleic acid may be amplified, for example, by exposing the target nucleic acid to one or more polymerases, for example, in the presence of mononucleotides (e.g., dNTPs or deoxyribose nucleoside triphosphates), which allows the polymerase to synthesize nucleic acids (e.g., DNA or RNA) using the target nucleic acid as a template.
  • mononucleotides e.g., dNTPs or deoxyribose nucleoside triphosphates
  • nucleic acids may be produced or amplified using an RNA polymerase such as T7 RNA polymerase, or using a DNA polymerase such as T7 DNA polymerase.
  • the amplification reaction may be facilitated, in certain embodiments, by a promoter (e.g., a T7 promoter). This may result in generation of many copies of the target nucleic acid, e.g., downstream of the promoter.
  • a promoter e.g., a T7 promoter
  • a variety of RNA polymerases are available commercially, including T7, T3, or SP6 RNA polymerases.
  • RNA polymerases include RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, or RNA polymerase V.
  • the RNA polymerase may arise from any suitable source, e.g., bacteria, viruses, or eukaryotes.
  • DNA polymerases are available commercially, including T7 DNA polymerase.
  • Other examples of DNA polymerases include DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV, DNA polymerase V, or DNA polymerases alpha, beta, lambda, gamma, sigma, mu, delta, epsilon, eta, iota, kappa, xi, theta, or Revl.
  • TdT is another non-limiting example.
  • oligonucleotides may include promoter sequences, such as one or more of T7, T3, or SP6 promoter sequences, that can be used to facilitate the transcription process.
  • promoter sequences such as one or more of T7, T3, or SP6 promoter sequences
  • the amount of nucleic acid production can be controlled in some embodiments by controlling the amount and/or concentration of nucleotides and/or cofactors that are present as well as the duration of the transcription reaction.
  • RNA may then be removed or selectively degraded, relative to the DNA, for example, through alkaline hydrolysis, enzymatic digestion, or other techniques.
  • relatively large quantities or masses of oligonucleotides can be produced as is discussed herein, e.g., at least about 10' 3 pmol, at least about 10' 2 pmol, at least about 10' 1 pmol, at least about 10° pmol, at least about 10 1 pmol, at least about 10 2 pmol, at least about 10 3 pmol, etc.
  • At least about 100, at least about 300, at least about 500, at least about 1,000, at least about 3,000, at least about 5,000, at least about 10,000, at least about 30,000, at least about 50,000 at least about 100,000, at least about 300,000, at least about 500,000, at least about 1,000,000 copies, at least about 3,000,000 copies, at least about 5,000,000 copies, at least about 10,000,000 copies, at least about 30,000,000 copies, at least about 50,000,000 copies, or at least about 100,000,000 copies of the oligonucleotide may be produced.
  • FISH fluorescence in situ hybridization
  • fluorescence labeling e.g., of nucleic acid probes
  • Yn-situ Y-branched probe in situ hybridization
  • SFO switchable fluorescent oligonucleotide
  • RNAScope or the like.
  • Still other examples include hybridization chain reactions, branched DNA.
  • sequencing can also be used as a readout.
  • sequencing-based spatial transcriptomics that in situ amplification of RNA techniques such as those described herein can be used to enhance include, but are not limited to, Visium Spatial Gene Expression or droplet-based single-cell RNA sequencing.
  • rRNA ribosomal RNA
  • rRNA radioactive ISH of ribosomal RNA
  • FISH of rRNA immunological FISH with biotin-labeled probe
  • FISH of actin mRNA Drosophila enhancer trap
  • WM ISH in Drosophila ES cell enhancer and gene trap in mice
  • in situ reporter in C. elegans or the like.
  • RNA capping may be used to improve template switching efficiency.
  • single-stranded binding proteins may be used to improve T7 amplification efficiency.
  • capped TSO may be used to prevent further template switching, which can reduce background spots.
  • locked nucleic acids LNA may be incorporated into the T7 amplification step to improve probe binding affinity.
  • protein digestion may be used prior to reverse transcription to improve efficiency of reverse transcription.
  • polyadenylation of RNA may be used to improve template efficiency of reverse transcription, to image RNAs that do not have a poly-A-tail, and/or to image samples that have fragmented or degraded RNA.
  • probes may be anchored into a gel matrix using alkylating agents, such as Melpha-X and Label-X, to improve the performance of MERFISH over multiple rounds of hybridization and imaging. See also Int. Pat. Apl. Pub. No. WO 2018/089445, incorporated herein by reference in its entirety.
  • multiple rounds of amplification via repeating the RT and T7 amplification steps may be used to further enhance the signal (Fig. 5C).
  • in situ amplification techniques such as those described herein may allow for the detection of short RNA sequences, such as RNA isoforms that differ by short sequences, as well as short genes and non-coding RNAs including microRNAs (miRNAs). This may be useful, for example, to increase the genomic coverage of spatially resolved single-cell transcriptomics measurements. These amplification techniques could also help to reduce the cost of spatial transcriptomics measurements in some cases, because a smaller number of oligonucleotide probes would be needed with this amplification method. This method may also allow the detection of RNA editing, such as A- to-I editing events, in a multiplexed manner in certain embodiments. Additional applications include, but are not limited to detection of single-nucleotide variations for various applications, such as cancer research and lineage tracing.
  • the techniques described herein could be applied to multiplexed protein imaging.
  • antibodies can be tagged with oligonucleotide barcodes that can then be amplified in situ. Endogenous RNAs can also be amplified and detected simultaneously for integrated transcriptomics and proteomics.
  • the techniques described herein could be used for detection of artificial nucleic acid barcodes that are introduced into cells, for example for imaging-based genetic screening, lineage tracing, neuronal projection mapping, or connectivity mapping.
  • two nucleic acids that are substantially complementary may differ by no more than 5, 4, 3, 2, or 1 nucleotides. In some cases, the two nucleic acids may have identical nucleic acid sequences.
  • RNA This example illustrates one embodiment demonstrating amplification of RNA.
  • a cell-type specific gene Sst was selected and the signal detected using singlemolecule FISH (smFISH) without amplification and with an embodiment of the amplification methods described herein.
  • smFISH singlemolecule FISH
  • Fig. 2B In situ RNA amplification provides brighter signal than unamplified smFISH
  • Fig. 2C A second gene (Slcl7a7) was also selected and a 100 bp segment was detected using four probes with an embodiment of the amplification methods described herein (Fig. 2C).
  • five exons of the Vip gene were detected using sequential rounds of hybridization and signal cleavage (Fig. 2D).
  • a 92-probe per gene library for -250 genes was used, which was previously used to classify cell types in the mouse primary motor cortex (MOp). These -250 genes were measured in the mouse MOp and compared to the previously published results without amplification (Fig. 3). The probe number was then reduced to eight probes per gene for these -250 genes and measured in the mouse MOp using MERFISH with the amplification method described here (Fig. 4).
  • Fig. 2 shows in situ RNA amplification using reverse transcription and transcription.
  • the first step is reverse transcription (RT) with template switching.
  • a primer binds to the poly-A tail of the mRNA.
  • the RT enzyme synthesizes complementary cDNA and adds a few non-templated nucleotides at the 5’ end of the mRNA template.
  • the non-templated nucleotides then anneal to a template switching oligo (TSO) with a T7 promoter sequence, leading the RT enzyme to switch templates from the mRNA to the TSO.
  • TSO template switching oligo
  • the resulting cDNA has a T7 promoter sequence, which is complementary to the sequence on the TSO, attached to the 3' end.
  • the second step is generation of RNA amplicons using T7 RNA polymerase. This step is facilitated by the presence of the double stranded T7 promoter region.
  • Fig. 2B shows the detection of Sst in mouse brain tissues using 30, 5, and 1 probes with and without amplification. Asterisk indicates enhanced contrast.
  • Fig. 2C shows the detection of a 100 bp segment of Slcl7a7 in mouse brain tissue with amplification using two different channels to confirm colocalization.
  • Fig. 2D shows the detection oiVip exons using two probes per exon with amplification. Sequential rounds of hybridization were used to detect different exons. Control images confirm that the signal has been cleaved between hybridization rounds.
  • Fig. 3 shows MERFISH with in situ RNA amplification using 92 probes using methacam fixation (Figs. 3A, 3D, and 3G), glyoxal fixation (Figs. 3B, 3E, and 3H), and formaldehyde fixation (Figs. 3C, 3F, and 31).
  • Figs. 3A-3C show the correlation between MERFISH data with amplification and MERFISH data without amplification. Each dot is the RNA counts per cell measured for each imaged gene.
  • Figs. 3D-3F show the cortical layer structure in mouse brains determined using MERFISH data with amplification.
  • Figs. 3G-3I shows correspondence between cell types determined using MERFISH data with amplification and those determined with the published MERFISH data without amplification.
  • Fig. 4 shows MERFISH measurements of the MOp using eight probes per gene with amplification.
  • Fig. 4A shows correlation between amplified, eight-probe MERFISH data and unamplified, 92-probe MERFISH. Each dot is the RNA counts per cell measured for each imaged gene.
  • Fig. 4B shows the cortical layer structure in mouse brains determined from the amplified, eight-probe MERFISH data.
  • Fig. 4C shows the correspondence between cell types determined using amplified, eight-probe MERFISH data and those determined with the published 92-probe MERFISH data without amplification.
  • Fig 5 shows assay optimization.
  • Fig. 5A shows smFISH of Sst using RNA amplification with (1) 5’ mRNA capping, which can enhance template- switching efficiency, (2) single-stranded binding proteins (SSB), which can improve T7 amplification, and (3) capped TSO, which prevents additional template switching from occurring.
  • Fig. 5A shows smFISH of Sst using RNA amplification with (1) 5’ mRNA capping, which can enhance template- switching efficiency, (2) single-stranded binding proteins (SSB), which can improve T7 amplification, and (3) capped TSO, which prevents additional template switching from occurring.
  • SSB single-stranded binding proteins
  • FIG. 5B shows smFISH of Sst using combination of TSO sequences (TSO sequence 1: /5Biosg/TAATACGACTCACTATAGGGAAATArGrG+G (SEQ ID NO.: 1), TSO sequence 2: /5Biosg/TAATACGACTCACTATAGGGAGArGrG+G (SEQ ID NO.: 2)) and enzymes from different vendors (T7 1: ThermoFisher, T7 2: New England Biolabs).
  • Fig. 5C shows a signal of one round of RT-T7 amplification (top) and two rounds of RT-T7 amplification. Representative images from the same round of an eight-probe MERFISH experiment. For the experiments in Figs.
  • sequences that can be used include, but are not limited to, /5Blosg/FAATACGACTCACTATAGGG AATA rNrG+G (SEQ ID NO: 4), /5Biosg/TAATACGACTCACTATAGGGAAAT rGrG+G (SEQ ID NO: 5), /5Biosg/TAATACGACTCACTATAGGGAGA rGrG+G (SEQ ID NO: 6), or /5Biosg/TAATACGACTCACTATAGGGAGA rNrG+G (SEQ ID NO: 7) (with and without the biotin or /5Bisog/ cap).
  • RT primer sequences that could be used include, but are not limited to, TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN (SE Q ID N0: 8) TTACACTCCATCCACTCAATT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TTTTTTTTTTTTTTTTTTTTTTTVN (SEQ ID NO: 9), or TTACACTCCATCCACTCAATT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TTTTT (SEQ ID NO: 10).
  • Fig. 7 shows MERFISH with in situ RNA amplification using 92 probes per gene.
  • Fig. 7A shows correlation between MERFISH data with amplification and MERFISH data without amplification. Each dot is the RNA counts per cell measured for each imaged gene.
  • Fig. 7B shows correspondence between cell types determined using MERFISH data with amplification and those determined with the published MERFISH data without amplification.
  • Fig. 8 shows MERFISH with in situ RNA amplification with probes targeting the 3’ end and the 5’ end of transcript.
  • Fig. 8 A shows correlation between MERFISH data with amplification using 12 probes per gene targeting the 5’ end of the transcript and published MERFISH data without amplification using 92 probes per gene. Each dot is the RNA counts per cell measured for each imaged gene.
  • Fig. 8B shows correspondence between cell types determined using MERFISH data with amplification using 12 probes per gene targeting the 5’ end of the transcript and those determined with the published MERFISH data without amplification.
  • Fig. 8 A shows correlation between MERFISH data with amplification using 12 probes per gene targeting the 5’ end of the transcript and published MERFISH data without amplification using 92 probes per gene. Each dot is the RNA counts per cell measured for each imaged gene.
  • Fig. 8B shows correspondence between cell types determined using MERFISH data with amplification using 12 probes per gene targeting
  • FIG. 8C shows correlation between MERFISH data with amplification using 12 probes per gene targeting the 3’ end of the transcript and published MERFISH data without amplification using 92 probes per gene. Each dot is the RNA counts per cell measured for each imaged gene.
  • Fig. 8D shows correspondence between cell types determined using MERFISH data with amplification using 12 probes per gene targeting the 3’ end of the transcript and those determined with the published MERFISH data without amplification.
  • Fig. 9 shows MERFISH with in situ RNA amplification with six and four probes per gene.
  • Fig. 9A shows correlation between MERFISH data with amplification using six probes per gene and the published MERFISH data without amplification using 92 probes per gene. Each dot is the RNA counts per cell measured for each imaged gene.
  • Fig. 9B shows the cortical layer structure in mouse brains determined using MERFISH data with amplification. The images show transcriptionally distinct cell clusters in different colors, with each cell color-coded or shaded by its cluster identity.
  • Fig. 9C shows correspondence between cell types determined using MERFISH data with amplification using six probes per gene and those determined with the published MERFISH data without amplification using 92 probes per gene.
  • Fig. 9D shows correlation between MERFISH data with amplification using four probes per gene and the published MERFISH data without amplification using 92 probes per gene. Each dot is the RNA counts per cell measured for each imaged gene.
  • Fig. 9E shows the cortical layer structure in mouse brains determined using MERFISH data with amplification. The images show transcriptionally distinct cell clusters in different colors, with each cell color-coded or shaded by its cluster identity.
  • Fig. 9F shows correspondence between cell types determined using MERFISH data with amplification using four probes per gene and those determined with the published MERFISH data without amplification using 92 probes per gene.
  • Fig. 10 shows MERFISH with in situ RNA amplification for 4,425 genes using six probes per gene and in-situ RNA amplification. Correlation between 4,425 gene MERFISH data with amplification using six probes per gene and RNA sequencing data.
  • Fig. 11 shows MERFISH with in situ RNA amplification, with and without expansion microscopy. Correlation is between MERFISH data with amplification, with and without expansion using six probes per gene. Each dot is the RNA counts per cell measured for each imaged gene.
  • a challenge with imaging a large number of genes is the high density of RNA molecules, which prevents neighboring RNA molecules from being resolved from each other using MERFISH.
  • expansion microscopy can be used following in-situ RNA amplification to physically separate RNA molecules in an expandable gel for imaging a large number of genes.
  • MERFISH probes were hybridized, and the samples were imaged using MERFISH.
  • MERFISH probes targeted 242 genes described in Zhang, Meng, el al., '‘Spatially resolved cell atlas of the mouse primary motor cortex by MERFISH,” Nature, 598(787$?): 137- 143, 2021.
  • samples were expanded prior to imaging. See generally Xia, Chenglong, et al. “Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression,” Proc. Natl. Acad. Set. USA, 116(39):19490-19499, 2019.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as “and/or” as defined above.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

La présente divulgation concerne de manière générale l'amplification d'ARN, par exemple, pour MERFISH ou d'autres applications. Un ensemble de modes de réalisation concerne de manière générale un procédé de synthèse d'un acide nucléique. Certains modes de réalisation concernent des systèmes et des procédés d'amplification d'ARN in situ, qui peuvent permettre une imagerie à l'échelle du génome d'ARN, y compris des ARN courts et des isoformes d'ARN qui sont différenciées par des séquences courtes. Dans certains modes de réalisation, l'ARN tel que l'ARNm peut être transcrit en ADNc à l'aide d'une transcriptase inverse. La transcriptase inverse peut également être utilisée pour associer un promoteur (par exemple, un promoteur T7) à l'ARN, par exemple, à l'aide d'un oligonucléotide de commutation de modèle (TSO). La séquence promotrice peut ensuite être utilisée pour amplifier l'ARN, par exemple, à l'aide de techniques telles que la transcription in vitro, qui peuvent être effectuées in situ. Des quantités amplifiées ou accrues d'ARN in situ peuvent être utiles pour certaines applications telles que MERFISH, étant donné que l'ARN est plus facile à détecter. D'autres aspects concernent en général des méthodes d'utilisation de telles techniques, des kits impliquant de telles techniques, ou analogues.
PCT/US2024/037820 2023-07-14 2024-07-12 Amplification d'arn in situ Pending WO2025019326A2 (fr)

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