WO2025207561A1 - Isothermal amplification using novel synthetic oligonucleotides - Google Patents

Abstract

The present disclosure relates to isothermal amplification using novel synthetic oligonucleotides useful as primers and probes. These oligonucleotides are particularly useful in isothermal amplification methods wherein a 3'-blocked primer is activated by an endo-ribonuclease, such as RNase H1. The novel synthetic oligonucleotides include one or more 3'-modified ribonucleotides to control where in the oligonucleotides RNase H1 cleaves. The preferred isothermal amplification uses a strand-displacement polymerase, such as Bst 3.0, and cycling probe technology, which may also use RNase H1, for detection. The disclosure also provides combinations of primers, optionally within a cartridge, as well as a method, optionally carried out within a cartridge, and related kits.

Description

ISOTHERMAL AMPLIFICATION USING NOVEL

SYNTHETIC OLIGONUCLEOTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/570,477, filed March 27, 2024, which is incorporated by reference in its entirety for any purpose.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the area of nucleic acid amplification, especially isothermal amplification. In particular, the invention relates to the use of novel synthetic oligonucleotides for use in nucleic acid amplification.

BACKGROUND OF THE INVENTION

[0003] Nucleic acid amplification plays a central role in molecular diagnostics and in other applications, such as nucleic acid sequencing. Polymerase chain reaction (PCR) is the most common and widely known amplification method, but isothermal amplification is another option. PCR relies on thermal cycling, whereas isothermal amplification reaction occurs at a single temperature, which makes it more amenable to use in point-of-care devices. However, isothermal amplification is often less sensitive and/or specific than PCR, and the time-to-result can be longer for isothermal amplification than for PCR. One challenge facing isothermal methods is off- target priming, leading to amplification of non- target nucleic acids.

[0004] Whereas PCR uses heat to denature DNA duplexes, isothermal amplification takes place at a set temperature, so heat cannot be used to denature amplicons. To solve this problem, an endonuclease can be used to cleave the primer leaving a 3 ’-OH residue. That moiety can be used to initiate polymerization by a strand displacing polymerase which invades, extends, and displaces the amplified strand. Existing isothermal technologies employ a range of enzymes which nick or cleave. The present disclosure describes the first use of RNaseHl as the cleaving enzyme. Additionally, the present disclosure is the first to employ synthetic oligonucleotides containing N3’-P5’ ribophosphoramidates in an isothermal technology either by themselves with one or two 3’ terminal natural ribonucleotides or interspersed with natural ribonucleotides.

SUMMARY OF THE INVENTION

[0005] In various embodiments, the nucleic acid amplification assays described herein enhance the specificity of isothermal amplification in several ways. First, a 3 ’-blocked primer can be activated at high temperature by sequence- specific cleavage with an endonuclease (e.g., RNase Hl). High-temperature activation increases the fidelity of the cleaved primer since mismatched duplexes are less stable at high temperature. RNase Hl provides additional sequence-specificity since only perfect RNA/DNA hybrids are recognized and cleaved to release the 3 ’-OH for primer activation. Second, amplification assays can employ a new class of synthetic oligonucleotides containing a nuclease-resistant N3’-P5’ ribophosphoramidate core. This nuclease-resistant core facilitates control over where cleavage occurs in the primer. Third, “cycling probes” can provide additional sensitivity and sequence specificity to the amplification assays. Cycling probes can be designed as substrates for an endonuclease (e.g., RNase Hl). The cycling probes can also contain nuclease a nuclease-resistant N3’-P5’ribophosphoramidate core to afford greater control over probe cleavage. Assays can be designed that employ the same endonuclease (e.g., RNase Hl) for activating blocked primers, as well as cleaving cycling probe(s).

[0006] Various embodiments contemplated herein may include, but need not be limited to, one or more of the following.

[0007] Embodiment 1: An oligonucleotide comprising at least one ribonucleotide adjacent to at least one 3'-modified-ribonucleotide, wherein the 3 '-modified ribonucleotide comprises a hydrolytically stable modification at the 3 '-position that inhibits hydrolysis by RNase Hl.

[0008] Embodiment 2: The oligonucleotide of embodiment 1, wherein the at least one 3'-modified-ribonucleotide comprises a 3'-amino-ribonucleotide.

[0009] Embodiment 3: The oligonucleotide of embodiment 1 or embodiment 2, wherein the at least one 3'-amino-ribonucleotide comprises a non-natural nucleotide base.

[0010] Embodiment 4: The oligonucleotide of embodiment 3, wherein the nonnatural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5-nitro-3-(r-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, and 2-amino-8-(l’-beta-D- 2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one.

[0011] Embodiment 5: The oligonucleotide of any one of embodiments 1-4, wherein the 3'-amino-ribonucleotide comprises 3'-amino-ribothymidine nucleotide, 3'-amino- riboadenine nucleotide, or a combination thereof.

[0012] Embodiment 6: The oligonucleotide of any one of embodiments 1-5, wherein the at least one ribonucleotide comprises a natural ribonucleotide base.

[0013] Embodiment 7: The oligonucleotide of any one of embodiments 2-6, wherein the oligonucleotide comprises a cleavage cassette comprising the at least ribonucleotide and the at least one 3'-amino-ribonucleotide, wherein the cleavage cassette comprises: a 5’ end linked to a 5’ nucleotide sequence; and a 3 ’end linked to a 3’ nucleotide sequence.

[0014] Embodiment 8: The oligonucleotide of embodiment 7, wherein the cleavage cassette comprises a combination of at least five ribonucleotide(s) and 3'-amino- ribonucleotide(s), in total, optionally at least seven ribonucleotide(s) and 3'-amino- ribonucleotide(s), in total.

[0015] Embodiment 9: The oligonucleotide of any one of embodiments 1-8, wherein the ribonucleotide is located at the 3 ’ end of the cleavage cassette.

[0016] Embodiment 10: The oligonucleotide of embodiment 9, wherein the cleavage cassette comprises at least two ribonucleotides at the 3’ end of the cleavage cassette.

[0017] Embodiment 11: The oligonucleotide of any one of embodiments 1-10, wherein the 3'-amino-ribonucleotide is located 5’ of the ribonucleotide(s).

[0018] Embodiment 12: The oligonucleotide of any one of embodiments 1-11, wherein the cleavage cassette comprises a plurality of ribonucleotides and a plurality of 3'- amino-ribonucleotides .

[0019] Embodiment 13: The oligonucleotide of embodiment 12, wherein the cleavage cassette comprises two ribonucleotides at the 3’ end linked to a segment comprising a plurality of 3'-amino-ribonucleotides. [0020] Embodiment 14: The oligonucleotide of embodiment 13, wherein the plurality of 3'-amino-ribonucleotides comprises a least four 3'-amino-ribonucleotides, optionally at least five 3'-amino-ribonucleotides.

[0021] Embodiment 15: The oligonucleotide of embodiment 12 or embodiment 13, wherein the plurality of 3'-amino-ribonucleotides are adjacent to one another.

[0022] Embodiment 16: The oligonucleotide of any one of embodiments 7-15, wherein the cleavage cassette comprises at least rN-rN-arN-arN.

[0023] Embodiment 17: The oligonucleotide of embodiment 12, wherein the cleavage cassette comprises two ribonucleotides at the 3 ’end linked to a segment comprising a plurality of 3'-amino-ribonucleotides (arN) interspersed with a plurality of ribonucleotides (rN).

[0024] Embodiment 18: The oligonucleotide of embodiment 17, wherein the segment comprises at least rN-arN-rN-arN.

[0025] Embodiment 19: The oligonucleotide of any one of embodiments 7-18, wherein the cleavage cassette comprises a plurality of 3'-amino-ribonucleotides that comprise more than one type of base, optionally selected from a combination of thymine and adenine.

[0026] Embodiment 20: The oligonucleotide of any one of embodiments 7-18, wherein the cleavage cassette comprises a plurality of 3'-amino-ribonucleotides that comprise one type of base, optionally selected from thymine or adenine.

[0027] Embodiment 21: The oligonucleotide of any one of embodiments 7-20, wherein the cleavage cassette comprises a plurality of ribonucleotides that comprise more than one type of base.

[0028] Embodiment 22: The oligonucleotide of any one of embodiments 7-20, wherein the cleavage cassette comprises a plurality of ribonucleotides that comprise one type of base.

[0029] Embodiment 23: The oligonucleotide of any one of embodiments 7-22, wherein the oligonucleotide comprises a cleavable primer comprising a 3’ blocking group.

[0030] Embodiment 24: The oligonucleotide of any one of embodiments 7-20, wherein the 5’ nucleotide sequence and the 3’ nucleotide sequence both comprise deoxyribonucleotides . [0031] Embodiment 25: The oligonucleotide of any one of embodiments 7-23, wherein the cleavage cassette comprises one or more 2’-O-methyl ribonucleotides at the 5’ end.

[0032] Embodiment 26: A combination of at least two oligonucleotides according to any one of embodiments 7-25, wherein one oligonucleotide is a forward primer, and one oligonucleotide is a reverse primer for amplifying a target nucleic acid.

[0033] Embodiment 27: The combination of embodiment 26, wherein at least one oligonucleotide comprises the oligonucleotide of embodiment 13, and the other oligonucleotide comprises a cleavage cassette comprising two ribonucleotides at the 3 ’end linked to a segment comprising a plurality of 3'-amino-ribonucleotides (arN) interspersed with a plurality of ribonucleotides (rN), wherein the cleavage cassette also comprises: a 5’ end linked to a 5’ nucleotide sequence; and a 3’end linked to a 3’ nucleotide sequence.

[0034] Embodiment 28: The combination of embodiment 27, wherein at least one oligonucleotide comprises the oligonucleotide of embodiment 14, and the other oligonucleotide comprises a cleavage cassette comprising two ribonucleotides at the 3’end linked to a segment comprising at least rN-arN-rN-arN, wherein the cleavage cassette also comprises: a 5’ end linked to a 5’ nucleotide sequence; and a 3’end linked to a 3’ nucleotide sequence.

[0035] Embodiment 29: The combination of embodiment 27 or embodiment 28, wherein the at least one oligonucleotide comprises a cleavage cassette having at least two ribonucleotides at the 3’end linked to a segment comprising at least arN-arN-arN-arN.

[0036] Embodiment 30: The combination of any one of embodiments 27-29, wherein the 3'-amino-ribonucleotides comprise one type of base.

[0037] Embodiment 31 : The combination of any one of embodiments 27-29, wherein the 3'-amino-ribonucleotides comprise more than one type of base.

[0038] Embodiment 32: The combination of any one of embodiments 26-31 , wherein the combination comprises a plurality of said at least two oligonucleotides comprising the forward primer and the reverse primer, wherein each pair of forward and reverse primers detects a different target nucleic acid. [0039] Embodiment 33: The oligonucleotide of any one of embodiments 1-25 or the combination of any one of embodiments 26-32, wherein the oligonucleotide or combination is contained within a cartridge for detecting one or more target nucleic acids in a sample, the cartridge comprising: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality; an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reagent chamber comprising one or more of the oligonucleotide(s) and/or one or more of the combination(s); and a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; and a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel.

[0040] Embodiment 34: A method for amplifying a target nucleic acid, wherein the method comprises contacting sample nucleic acids with: a primer comprising the oligonucleotide of any one of embodiments 7-25 and another primer; or the combination of any one of embodiments 26-31 , wherein each oligonucleotide serves as a primer; in a reaction mixture under conditions suitable for primer extension.

[0041] Embodiment 35: The method of embodiment 34, wherein the method comprises detecting the target nucleic acid, if detectable in the sample nucleic acids.

[0042] Embodiment 36: The method of embodiments 34 or 35, wherein the method comprises an isothermal method.

[0043] Embodiment 37: The method of any one of embodiments 34-36, wherein: the oligonucleotide comprises a cleavable primer comprising a 3’ blocking group; or the combination comprises an oligonucleotide comprising a cleavable primer comprising a 3’ blocking group; and the method comprises annealing the cleavable primer to its target nucleotide sequence and cleaving the cleavable primer with RNase Hl to release the blocking group.

[0044] Embodiment 38: The method of embodiment 37, wherein the RNase Hl cleaves at the 3’ end of the cleavage cassette. [0045] Embodiment 39: The method of embodiments 37 or 38, wherein the RNase Hl is a thermostable RNase Hl, optionally from Thermus thermophilus or Echerichia coli.

[0046] Embodiment 40: The method of any one of embodiments 34-39, wherein the reaction mixture comprises a probe.

[0047] Embodiment 41 : The method of embodiment 40, wherein the probe comprises a linear probe, and the primer that generates a nucleotide sequence to which the probe can bind is included in the reaction mixture in excess of the other primer.

[0048] Embodiment 42: The method of embodiments 40 or 41, wherein the probe comprises a fluorescent dye and a quencher molecule.

[0049] Embodiment 43 : The method of any one of embodiments 40-42, wherein the probe comprises a cycling probe.

[0050] Embodiment 44: The method of embodiment 43, wherein the cycling probe comprises not more than three ribonucleotides.

[0051] Embodiment 45: The method of embodiment 44, wherein the three ribonucleotides comprise a ribonucleotide core that is positioned near the 5 ’ end of the cycling probe.

[0052] Embodiment 46: The method of embodiment 44, wherein the not more than three ribonucleotides are flanked, on either end, with 2’-O-methyl-base-containing sequences.

[0053] Embodiment 47 : The method of any one of embodiments 34-46, wherein the reaction mixture comprises polymerase at a concentration of 100 mU/pL to 600 mU/pL, optionally wherein the polymerase comprises Bst 3.0.

[0054] Embodiment 48: The method of any one of embodiments 37-47, wherein the reaction mixture comprises the RNase Hl at a concentration of 700 mU/pL to 1300 mU/pL.

[0055] Embodiment 49: The method of any one of embodiments 34-48, wherein the method comprises multiplex amplification.

[0056] Embodiment 50: The method of any one of embodiments 37-49, wherein the method is carried out within a cartridge. [0057] Embodiment 51: The method of embodiment 50, wherein the cartridge comprises: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality; an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reagent chamber comprising one or more of the oligonucleotide(s) and/or one or more of the combination(s); and a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; and a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel.

[0058] Embodiment 52: A kit comprising the oligonucleotide of any one of embodiments 1-25 or the combination of any one of embodiments 26-32, wherein the kit comprises a strand-displacing polymerase.

[0059] Embodiment 53: The kit of embodiment 52, wherein the kit additionally comprises RNase Hl.

[0060] Embodiment 54: The kit of embodiments 52 or 53, wherein the oligonucleotide or combination wherein the oligonucleotide or combination is contained within a cartridge for detecting one or more target nucleic acids in a sample, the cartridge comprising: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality; an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reagent chamber comprising one or more of the oligonucleotide(s) and/or one or more of the combination(s); and a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; and a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel. [0061] Embodiment 55: The oligonucleotide or combination of embodiment 33, the method of embodiment 51, or the kit of embodiment 54, wherein a strand-displacing polymerase is disposed within one of the plurality of chambers.

[0062] Embodiment 56: The oligonucleotide or combination of embodiment 33, the method of embodiment 51 , or the kit of embodiment 54, wherein RNase Hl is disposed within one of the plurality of chambers.

[0063] Embodiment 57: The oligonucleotide or combination of embodiment 33, the method of embodiment 51, or the kit of embodiment 54, wherein a strand-displacing polymerase is disposed within one of the plurality of chambers, and RNase Hl is disposed within one of the plurality of chambers.

[0064] Embodiment 58: The oligonucleotide or combination of embodiment 33, or the method of embodiment 51, or the kit of embodiment 54, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

[0065] Embodiment 59: The oligonucleotide or combination of embodiment 33, or the method of embodiment 51, or the kit of embodiment 54, wherein the method is a point- of-care method.

[0066] Embodiment 60: The oligonucleotide or combination of embodiment 33, or the method of embodiment 51, or the kit of embodiment 54, wherein the cartridge comprises, a primer pair that selectively hybridizes to an exogenous control and/or an endogenous control, wherein the exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

[0067] Embodiment 61 : The oligonucleotide or combination of embodiment 33, or the method of embodiment 51, or the kit of embodiment 54, wherein the cartridge facilitates detection of a target nucleic acid in the sample within 30 minutes, within 20 minutes, or within 10 minutes from the time the sample is placed in a cartridge.

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG. 1: Comparison of 5’-strand cleavage by RNase H2 (5’-JRNase) and 3’- strand cleavage by RNase Hl (3’ JRNase). [0069] FIG. 2: Comparison of “gapmer” structures and mechanisms. Therapeutic antisense oligonucleotides (left) and the primers described herein (right) have a cleavage cassette including an enzyme binding “core” and tunable “wings.” In each case, RNase Hl binds to a DNA/RNA heteroduplex and cleaves the RNA-containing strand. RNase Hl cleavage leaves a 3’-terminal OH group that can be extended by a DNA polymerase (e.g., Bst) along the DNA template. N3’-P5’ -modified ribonucleotides can be employed in a cleavage cassette (“core”) to facilitate cleavage at specific ribonucleotide sites and allow isothermal amplification of template strands.

[0070] FIGS. 3A-3B: Schematic showing amplification of template DNA by RNase Hl-activated primers and Bst polymerase. FIG. 3A shows steps 1-4; FIG. 3B shows steps 5-15; and FIG. 3C shows steps 15 and 16.

[0071] FIG. 4 : Structure of N3’-P5’ oligoribonucleotide sugar-phosphate bond. The

3’-amino group of the N3’-P5’ modified ribonucleotide blocks hydrolysis by RNase Hl of the phosphoramidate bond. In contrast, the natural phosphate group of the 3 ’-nucleoside adjacent the N3’-P5’ modified ribonucleotide is hydrolyzed to release the 3’-OH group. The 2’-OH group in the cleavage cassette is available for binding of RNase Hl to facilitate cleavage.

[0072] FIG. 5: Exponential amplification with primer sequences containing a randomized RNA cassette. The number of copies of the target nucleotide sequence initially present in the amplification reaction is indicated for the various curves. The box depicts the limit of detection (LOD) of the assay. (See Example 1 .)

[0073] FIG. 6: Exponential amplification with primer sequences containing an allnatural RNA cassette with 5 rU and IrC. The number of copies of the target nucleotide sequence initially present in the amplification reaction is indicated for the various curves. The box = LOD. (See Example 2.)

[0074] FIG. 7: Linear amplification with different cassettes. (See Examples 2 and 3.)

[0075] FIG. 8: Exponential amplification with different cassettes. (See Example 3.)

[0076] FIG. 9 : Isothermal detection of le4 copies (ascending curve) of target using primers with T107 cassettes and cycling probe. Flat curve = no-template control (NTC). (See Example 4.) [0077] FIG. 10: Linear amplification detection using either a linear probe (moderate amplification) or a cycling probe with a cleavage cassette (faster amplification). Bottom curve = NTC for linear probe. Second- from-bottom curve = NTC for cycling probe. (See Example 5.)

[0078] FIG. 11: Linear amplification detection using either a cycling probe without 2’-O-methyl (moderate amplification) or with 2’-O-methyl (faster amplification). Bottom two curves - NTC. (See Example 6.)

[0079] FIG. 12: Linear amplification at 70°C (lel2 copies) comparing 4 primers with varying cleavage cassette sequences. (See Example 7.)

[0080] FIG. 13: Exponential amplification at 70°C of le6 copies (exponential amplification) and NTC (bottom curve). (See Example 7.)

[0081] FIG. 14: Synthesis of T107 phosphoramidite (Gryaznov, S., & Winter, H. [1998]). The method for synthesis of the T107 phosphoramidite was optimized to yield 2.8 g T107 from 3 g of Universal Sugar (56% overall yield for 7 steps).

[0082] FIG. 15: Synthesis of A107 phosphoramidite (Gryaznov, S., & Winter, H. [1998]).

[0083] FIG. 16: Use of a PAMAM dendrimer reduces multimerization seen with Bst 3.0 polymerase, as indicated by reduced laddering.

[0084] FIG. 17 -17C: Overview of a sample cartridge with a valve assembly configured for performing different sample processing steps, including chemical lysing of targets, which is configured for PCR and optional integrated nucleic acid analysis. FIG. 17A shows the sample cartridge body with reaction vessel, FIG. 17B shows an exploded view of the sample cartridge, and FIG. 17C shows components of the valve assembly, in accordance with some embodiments.

DETAILED DESCRIPTION

Definitions

[0085] Terms used in the claims and specification are defined as set forth below unless otherwise specified. [0086] The term “nucleic acid” refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides.

[0087] The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and noncoding RNA.

[0088] The term nucleic acid encompasses double- or triple- stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e, a double-stranded nucleic acid need not be double- stranded along the entire length of both strands).

[0089] The term nucleic acid also encompasses any modifications thereof, such as by methylation and/or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2’ -position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5-bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

[0090] More particularly, in some embodiments, nucleic acids, can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (see, e.g., Summerton and Weller ( 1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev. 7:1817-195; Okamoto et al. (2002) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement No. 2: 171-172), and other synthetic sequencespecific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entirety for their disclosure of LNAs.

[0091] The nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

[0092] As used herein, the term “gene” encompasses coding sequences, introns, and any associated control sequences that participate in the expression of the coding sequences.

[0093] As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single- stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0094] “Selective hybridization” or “selective annealing” refers to the binding of a nucleic acid to a target nucleic acid in the absence of substantial binding to other nucleic acids present in the hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated.

[0095] In some embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase “stringent hybridization conditions” generally refers to a temperature in a range from about 5 °C to about 20°C or 25 °C below the melting temperature (T_m) for a specific sequence at a defined ionic strength and pH. As used herein, the T_m is the temperature at which a population of double- stranded nucleic acid molecules becomes halfdissociated into single strands. Methods for calculating the T_m of nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) Methods in Enzymology, Vol.152: Guide to Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^nd ., Vols. 1-3, Cold Spring Harbor Laboratory), both incorporated herein by reference for their descriptions of stringent hybridization conditions). As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m =81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the primer or probe and nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, and the like), as well as the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol, betaine). The effects of these factors are well known and are discussed in standard references in the art. Illustrative stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60°C and a salt concentration of about 0.2 molar at pH7. T_m calculation for oligonucleotide sequences based on nearest-neighbors thermodynamics can carried out as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest- neighbor thermodynamics” John SantaLucia, Jr., PNAS February 17, 1998 vol. 95 no. 4, 1460-1465 (which is incorporated by reference herein for this description).

[0096] The term “oligonucleotide” is used to refer to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides. Typically, oligonucleotides are single- stranded DNA molecules.

[0097] The term “primer” refers to an oligonucleotide that is capable of hybridizing (also termed “annealing”) with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, in some embodiments, range from 10 to 30 nucleotides, or, in some embodiments, from 10 to 60 nucleotides, in length. In some embodiments, primers can be, e.g., 15 to 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.

[0098] A primer is said to “anneal to” or “hybridize to” another nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes either completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to “anneal to” or be “specific for” a nucleotide sequence.” This description encompasses primers that anneal wholly to the nucleotide sequence, as well as primers that anneal partially to the nucleotide sequence.

[0099] The term “primer pair” refers to a set of primers including a 5’ “upstream primer” or “forward primer” that hybridizes with the complement of the 5’ end of the DNA sequence to be amplified and a 3’ “downstream primer” or “reverse primer” that hybridizes with the 3’ end of the sequence to be amplified. As will be recognized by those of skill in the art, the terms “upstream” and “downstream” or “forward” and “reverse” are not intended to be limiting, but rather provide illustrative orientations in some embodiments.

[0100] A “probe” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable moiety to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled, but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size, but are shorter than the amplicon.

[0101] As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target nucleic acid under suitable annealing conditions. [0102] The term “target” is used herein with reference to “target nucleic acids,” as well as “target organisms.” The former refers to nucleic acids to be detected, and the latter refers to organisms to be detected. The term, “target nucleic acid” is generally used herein to refer to a segment of nucleic acid that is defined by a primer pair and that gives rise to an amplicon produced in an amplification reaction; the term “amplification target” is also used herein to refer to this type of target nucleic acid. Primers and probes are also said to “target” nucleic acid sequences, and so these sequences can also be understood as “target nucleic acids.” Additionally, primers and probes are said to “target” or “be specific for” genes. In this usage, the primers and probes can be used to detect the presence of a particular gene by specifically hybridizing to a portion of the gene that indicates its presence. The meaning of “target” and “target nucleic acids” will be clear to one of skill in the art from the context in which the term is employed. In some embodiments, multiple target nucleic acids can be detected to detect a single target organism. In some embodiments, a single target nucleic acid can be detected to detect a single target organism. In some embodiments, an assay can employ multiple target nucleic acids for one or more target organisms and single target nucleic acids for one or more different target organisms.

[0103] Amplification according to the present teachings encompasses any means by which at least a part of at least one target nucleic acid is reproduced, typically in a templatedependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction — CCR), helicase-dependent amplification (I), and the like. Descriptions of such techniques can be found in, among other sources, Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.;4(l):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451 , Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web al7romegaega.com/geneticidproc/ussymp6proc/blegrad.html- ); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88: 188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20: 1691-96 (1992); Polstra et al., BMC Inf. Dis. 2: 18- (2002); Lage et al., Genome Res. 2003 Feb.;13(2):294-307, and Landegren et al., Science 241: 1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.;2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2): 165-74, Schweitzer et al., Curt Opin Biotechnol. 2001 Feb.;12(l):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat. No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

[0104] In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly- formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally.

[0105] As used herein, the term “amplification conditions” refers to conditions that promote amplification of a target nucleic acid in the presence of suitable primers.

[0106] As used herein, “in solution” means not immobilized on a substrate of any kind, for example, a bead or a surface in a cassette, such as a chamber wall.

[0107] A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.

[0108] A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators.

[0109] The term “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and/or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0110] The term “dye,” as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (e.g., a fluorescent signal).

[0111] The term “quencher,” as used herein generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0112] As used herein, the term “detecting” refers to “determining the presence of’ an item, such as a nucleic acid sequence.

[0113] As used herein, “Clinical Laboratory Improvement Amendments (CLIA)” refers to The Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations in effect as of the original filing date of the present application. The CLIA regulations include federal standards applicable to all U.S. facilities or sites that test human specimens for health assessment or to diagnose, prevent, or treat disease. A “CLIA-compliant” test is one that complies with these regulations. “CLIA- waived” tests include tests that does not comply with all of these regulations. For example, CLIA-waived tests include test systems cleared by the U.S. Food and Drug Administration for home use and those tests approved for waiver under the CLIA criteria.

[0114] An “endogenous control,” as used herein refers to a moiety that is naturally present in the sample to be used for detection. In some embodiments, an endogenous control is a “sample adequacy control” (SAC), which may be used to determine whether there was sufficient sample used in the assay, or whether the sample comprised sufficient biological material, such as cells. In some embodiments, an endogenous control is an RNA (such as an mRNA, tRNA, ribosomal RNA, etc.), such as a human RNA for a human sample. Nonlimiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA. In some embodiments, an endogenous control, such as an SAC, is selected that can be detected in the same manner as the target nucleic acid (e.g., RNA) is detected and, in some embodiments, simultaneously with the target nucleic acid (e.g., RNA).

[0115] An “exogenous control,” as used herein, refers to a moiety that is added to a sample or to an assay, such as a “sample processing control” (SPC). In some embodiments, an exogenous control is included with the assay reagents. An exogenous control is typically selected that is not expected to be present in the sample to be used for detection, or is present at very low levels in the sample such that the amount of the moiety naturally present in the sample is either undetectable or is detectable at a much lower level than the amount added to the sample as an exogenous control. In some embodiments, an exogenous control comprises a nucleotide sequence that is not expected to be present in the sample type used for detection of the target nucleic acid (e.g., RNA). In some embodiments, an exogenous control comprises a nucleotide sequence that is not known to be present in the species from whom the sample is taken. In some embodiments, an exogenous control comprises a nucleotide sequence from a different species than the subject from whom the sample was taken. In some embodiments, an exogenous control comprises a nucleotide sequence that is not known to be present in any species. In some embodiments, an exogenous control is selected that can be detected in the same manner as the target nucleic acid (e.g., RNA) is detected and, in some embodiments, simultaneously with the target nucleic acid (e.g., RNA). In some embodiments, the exogenous control is an RNA. In some such embodiments, the exogenous control is an Armored RNA®, which comprises RNA packaged in a bacteriophage protective coat. See, e.g., WalkerPeach et al, Clin. Chem. 45: 12: 2079-2085 (1999).

[0116] As used herein, the phase “one or more amino-ribonucleotides (arN) interspersed with one or more ribonucleotides (rN)” refers to a ribonucleotide sequence made up of at least one amino-ribonucleotide unit and at least one ribonucleotide unit, wherein the shortest of such sequence is three (3). The plurality of arN and plurality of rN can be distributed randomly, alternating, or as blocks in the ribonucleotide sequence. For example, the ribonucleotide sequence can be made up of repeating units of (arN-rN)_x, wherein “x” is an integer of at least 2 (i.e., the shortest such sequence is: arN-rN-arN-rN), i.e., an alternating ribonucleotide sequence. In other examples, the ribonucleotide sequence can be made up of a plurality of arN and a plurality of rN units in which the probability of finding a given repeating unit at any given site in the sequence is independent of the nature of the adjacent repeating units, i.e., a random ribonucleotide sequence. In further examples, the ribonucleotide sequence can be made up of a plurality of arN and a plurality of rN, wherein adjacent blocks are constitutionally different, i.e. adjacent blocks comprise repeating units derived from different species of monomer or from the same species of monomer but with a different composition or sequence distribution of repeating units.

[0117] The term “ribo/nucleotides” is used here to encompass ribonucleotides, nucleotides, or a combination of both.

[0118] As used herein, the phrase “at the 5’ end” or “at the 3’ end” refers to a location within three ribo/nucleotides of the 5’ or 3’ end (respectively) of a sequence of ribo/nucleotides.

[0119] As used herein, the phrase “near the 5’ end” or “near the 3’ end” refers to a location that is closer to the 5 ’ end than the 3 ’ end of a sequence of ribo/nucleotides or closer to the 3’ end than the 5’ end of a sequence of ribo/nucleotides.

[0120] As used herein with respect to “ribo/nucleotides,” the term “adjacent to” is used to describe a ribo/nucleotide that next to another ribo/nucleotide (i.e, the two ribonucleotides are not separated by any intervening ribo/nucleotides).

[0121] The terms “amino” and “amine” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: -NR1R2 or -NR1R2R3, wherein Ri, R2, and R3 each independently represent a hydrogen, an alkyl, an alkenyl, carbonyl, a heteroatom (including but not limited to O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quatemized), -(CH2)_m-R4, or Ri and R2 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R’3 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8.

[0122] With reference to an element of a primer, the term “blocking group” refers to a structure (typically, at the terminus of the primer) that cannot be extended by a polymerase. The primer bearing a blocking group, e.g., a 3’ terminal blocking group is said to be “capped.” A blocking group can be removed to allow the primer to prime the production of an extension product by a polymerase. In some embodiments, the blocked portion of the primer is removed by RNase Hl cleavage.

[0123] The term “cycling probe” refers to a probe as defined herein, that can be cleaved by an enzyme, particularly RNase Hl, after annealing to a target nucleic acid sequence, wherein such cleavage releases an intact target nucleic acid. A cycling probe enables a target nucleic acid to anneal to many molecules of the probe, thereby amplifying any signal associated with the probe. “Catalytic hybridization amplification” (CHA), alternatively known as “cycling probe technology,” is described in PCT publication no. WO 89/09284, and U.S. Pat. Nos. 5,011,769 and 4,876,187. Briefly, CHA is an improved hybridization assay method whereby the target sequence to be detected is able to capture many molecules of the probe in a repeating series of reactions (i.e., “cycling probe”). Essentially, enzyme-mediated cleavage of the probe within the probe target duplex results in release of the intact target sequence, which can repeatedly recycle through the reaction pathway. The target sequence serves as a catalytic cofactor for the cleavage of a complementary, labeled nucleic acid probe that is hybridized to the target. The detectable signal in this reaction results from cleavage of the probe, e.g., after repeated CHA cycles, one measures the labeled probe cleavage product. The CHA method is useful in detecting specific DNA or RNA sequences.

Isothermal Amplification Using Synthetic Oligonucleotides

[0124] The present disclosure describes nucleic acid amplification assays that employ novel synthetic oligonucleotides and, optionally, other elements to increase assay sensitivity and specificity, as well as time-to-result. Possible features of this assay are: (1) the use of a primer that is initially blocked to 3’ extension by a polymerase; (2) the use of an endonuclease, such as RNaseHl, to cleave and therefore activate the blocked primer for extension, and optionally to cleave a probe; (3) a strand-displacing polymerase, such as Bst DNA polymerase, to extend the cleaved primer; and (4) a 3’ blocked primer, and/or a probe with a cleavage cassette for primer/probe activation by and endonuclease, such as RNase Hl. Blocked Primer

[0125] A blocked primer is one wherein the 3’ end cannot serve as an initiation point for primer extension. Means for blocking primers are well known, and those of skill in the art can readily select a suitable approach for a particular application. However, choice of the blocker molecule or blocker modification (“blocker”) to the primer can have an impact on primer hybridization, the cleavage rate or removal of the blocker and subsequent primer activation and extension. In some embodiments, the blocker is chosen for its selectivity for RNase Hl as compared to other endonuclease enzymes. In some embodiments, the blocker is chosen based on the rate at which it can be cleaved, with particular advantages to blockers with fast cleavage rates. Aspects of the blocker that can impact cleavage include molecular structure, bonding, steric considerations, etc. In some embodiments, the rate-limiting step for processes described herein is the cleavage of the blocker. Commercial suppliers of custom oligonucleotide generally offer a number of modifications capable of preventing 3’ extension of a primer. Examples include a 3’-Spacer C3 CPG (l-dimethoxytrityloxy-propanediol-3- succinoyl)-long chain alkylamino-CPG), 3’ phosphate group, chemically reversed 3’ terminal nucleotide (3’ to 5’)/Inverted end, 2- aminopurine, hexandiol, 1’, 2 ’-dideoxyribose (dSpacer), PC (Photocleavable) spacer, Spacer 9 (triethylene glycol spacer), Spacer 18 (hexaethyleneglycol), 4-((4-(dimethylamino)phenyl)azo)benzoic Acid (dabcyl), and 4-(2',6'- dihydroxy-4'-dimethylaminophenylazo)-2-hydroxybenzoic acid (hydrodabcyl).

RNase Hl

[0126] RNase H enzymes are ribo-endonucleases that cleave adjacent to a ribonucleotide paired with a nucleotide in a nucleotide duplex. The activities of RNase H2 and Hl, while related, are distinct at the molecular level. FIG. 1 (Tannous, Kanaya, & Kanaya, 2015) depicts this difference in that the RNase H2 or 5’ JRNase cleaves on the 5’ end of a single (or a string of) ribobase(s) whereas RNase Hl (or 3’ JRNase) cleaves on the 3’ end of similar substrates. The 3’ cleavage activity of RNase Hl used in the methods disclosed herein allows for primer activation and subsequent extension from the resulting 3’- OH moiety left after the cleavage.

[0127] The effect of oligonucleotide structure on RNase Hl cleavage of RNA has been studied due to the use of this mechanism in “antisense” oligonucleotide therapeutics (FIG. 2). Antisense oligodeoxynucleotides target mRNA, and RNase H hydrolyzes RNA in the DNA/RNA duplex that forms. In the methods described here, the strand scission releases the 3 ’OH group to provide a substrate for DNA polymerases. DNA strands are susceptible to exonucleases in blood, and therefore antisense oligonucleotide “gapmers” with a DNA core and nuclease resistant “wings” have been developed as drug candidates. For example, locked nucleic acids, phosphorothioates, 2’-F, 2’-0Me, N3’-P5’ ODNs are phosphate-sugar backbone modifications that are used in the wings of therapeutic gapmers to provide nuclease stability. With the exception of phosphorothioates, the other backbone modifications listed above enhance stability of the duplexes and promote “RNA-like” A-form duplexes with a deep major groove and a shallow minor groove.

[0128] RNase Hl generally cleaves the 3’ end of a ribonucleotide at various positions within a longer string of RNA when it is hybridized to DNA. However, RNase Hl has been shown to also cleave a single ribonucleotide when MnCh is a component of the buffer (Tannous, Kanaya, & Kanaya, 2015). In the work described herein, buffer conditions were adjusted to allow RNase Hl to cleave one or more ribonucleotides, while also allowing for subsequent extension from that site by Bst DNA polymerase. In this system, RNase Hlcleaved a single ribonucleotide with a MnSO4 concentration of 10 mM, but the cleavage was slow, with less than 50% of substrate being measurable as cleaved in 15 minutes. To mitigate the slow cleavage, cleavage cassettes > 2-5 RNAs were tested as shown in Example 1.

Oligonucleotides Containing Cleavage Cassette

[0129] Unlike the antisense oligonucleotides (“oligos”) described above, novel RNase Hl -activatable primers (and cycling probes) described herein are generally designed to be cleaved at a single internal RNA nucleotide. (Antisense oligos have a DNA core that hybridizes and a long mRNA target, which RNase Hl can cleave at multiple positions.) More specifically, the novel cleavable primers described herein have an RNA “core” that can be designed to be cleaved at a single nucleotide position to provide a full-length primer. This RNA core is also referred to herein as a “cleavage cassette.”

[0130] Amino-deoxyribonucleotide oligos have been developed as wings for therapeutic antisense oligos, but no corresponding amino-ribonucleotide oligos have been developed. There is a single research report (Gryaznov and Winter, 1998) that describes the synthesis of properly protected A, T and U phosphoramidite reagents for automated synthesis of short phosphoramidate-containing RNA strands. Amino-ribonucleotide-containing oligos were found to form stable duplexes with DNA strands, but no applications were developed. The experimental work described herein discloses the preparation of the arT amidite (T107) and the discovery that DNA primer sequences with short (~5-nucleotide) amino- ribonucleotide/ribonucleotide cores hybridize to DNA targets and are cleaved by RNase Hl.

[0131] In some embodiments, oligos that can be used as primers or probes include a 5’ region that remains bound to its template strand at the reaction temperature. This 5’ region is linked to a ribonucleotide-containing cleavage cassette that can be designed to be cleaved rapidly and preferably at the 3’ end. The specific and rapid 3’ end cleavage is achieved through appropriate placement of ribonucleotides (e.g., natural ribonucleotides) and non- cleavable amino-ribonucleotides in the cassette. This design allows for multiple cleavage events and subsequent reconstitution of the cleavage substrate by a DNA polymerase. The cleavage cassette is linked to a 3’ region that also binds to the template strand at the reaction temperature and includes a 3’ blocking group to prevent non-specific primer extension.

[0132] Upon binding to a target nucleotide sequence, the oligo is cleaved by RNase Hl at the 3’ end of the cleavage cassette, providing the composition of ribonucleotides (e.g., natural ribonucleotides) and amino-ribonucleotides is appropriately constructed. FIG. 3 shows this and subsequent steps schematically (FIG. 3 steps 1-2). A DNA polymerase, such as Bst polymerase, can invade at the hydrolyzed RNA site created by RNase Hl and extend using the target nucleotide sequence as a template, displacing the 3 ’ -oligonucleotide fragment (FIG. 3, step 3). After extension, the RNase Hl cleavage site is reconstituted allowing RNaseHl to re-cleave the cassette, and subsequent extension by Bst polymerase allows strand displacement to occur (FIG. 3, steps 4-5). This process can occur indefinitely, provided that the cleavage is directed to the 3’ end of the cassette. Each cleavage and extension event will displace the previously extended product which can become a template for a detection probe and/or another primer.

[0133] If the cassette is comprised of only natural RNA nucleotides, RNaseHl will cleave at different locations within the cassette, eventually leading to the cassette becoming shortened to a length of less than two ribonucleotides. Cleavage of fewer than 2 ribonucleotides is inefficient, and the activation of those primers with such short cassettes will generally be too slow for the efficient exponential amplification desired for most embodiments. To prevent the shortening of the cleavage cassette, a ribonucleotide analog (N3’-P5’ phosphoramidate; Gryaznov & Winter, 1998), such as that shown in FIG. 4, can be employed. The combination of ribonucleotides (e.g., natural ribonucleotides), aminoribonucleotides, and the template DNA strand comprise a cleavage substrate for RNase Hl that can be rationally designed to facilitate the amplification schemes such as that shown in FIG. 3. Design of the cleavage cassette should include 2 natural ribonucleotides at the 3’ end. For primers that generate the probe template, the 5 ’ end of the cassette typically includes only amino ribonucleotides. For primers that do not generate the probe template, the 5’ end of the cassette typically includes a mixture of natural and amino ribonucleotides. When the 5’ end of the cassette includes a mixture of natural and amino ribonucleotides, there is preferably only 1 natural ribonucleotide in a row. It has been found that cassettes with greater diversity in base identity are cleaved more efficiently. Additionally, increasing the number of ribonucleotides in the cassette from 3 to 8 increases the rate of cleavage, and it can be extrapolated that increasing beyond 8 will further increase cleavage efficiency.

[0134] The experimental work below indicates that blocked primers with 2-3 ribonucleotides at the 3’ end can increase the specificity of amplification.

3’-Modified Ribonucleotides

[0135] RNase Hl requires specific features of the RNA/DNA hybrid for effective RNA cleavage of the 5'-phosphate and 3'-hydroxyl termini to be mediated by the enzyme. Lacy K.D. et al. in Molecular Therapy: Nucleic Acids 2022, Vol. 28 reports that the catalytic domain of RNase Hl contacts two 2'OH groups on either side of the scissile phosphate, requiring a total of four consecutive RNA nucleotides. On the DNA side, RNase Hl binds via a contorted phosphate-binding pocket and a DNA binding-channel, which can be achieved by adopting the unique B form geometry specific to RNA/DNA hybrids. Modifications to the RNA/DNA hybrid that distort this and/or other geometries, such as described herein, can prevent cleavage of the RNA strand. Particularly, 3 ’-RNA ribose modifications would likely affect duplex recognition by RNase Hl by potentially clashing with the RNase Hl binding interface or affecting the helical geometry of the duplex or hybridization with the RNA/DNA complex. As such, oligonucleotides comprising the 3 ’-RNA ribose modification renders the intemucleoside linkage between the 3 ’-modified ribonucleotide and the adjacent ribonucleotide hydrolytically stable, particularly, the modification inhibits hydrolysis by RNase Hl. In some embodiments, the 3 ’-modified ribonucleotide comprises a ribose ring in which the 3 ’-oxygen atom connecting the ribose sugar with the 5 ’phosphate is replaced with a hydrolytically stable functional group. For example, the 3’ -modified ribonucleotide can comprise a N3'^P5' phosphoramidate, a B3'^P5' boranophosphonate, a C3'^P5' alkylphosphonate, Si3'^P5', or Se3'— >P5'. Oligonucleotides comprising these 3’-modified ribonucleotides generally form stable duplexes with complementary single- stranded (ss) RNA and DNA, spatially and functionally mimic isosequential RNA structural element, and are resistant to cleavage by RNase Hl at the 3 ’-modified ribonucleotide position.

[0136] In illustrative embodiments, the 3 ’-modified ribonucleotides can be aminoribonucleotides, e.g., N3’-P5’ phosphoramidate oligoribonucleotides (amino-ribonucleotide oligos), which are thought to adopt an A-form helical conformation when paired to DNA strands, similar to properties exhibited by RNA (Lelyveld, O'Flaherty, Zhou, Izgu, & Szostak, 2019). These amino-ribonucleotides are recognized by the RNase Hl as ribonucleotides. Due to their 3 ’-amino modification, however, amino-ribonucleotides cannot be cleaved by RNase Hl. Illustrative N3’-P5’ phosphoramidate analogs of thymine (T107) and adenine (A107), and illustrative syntheses of the protected phosphoramidites required for automated synthesis, are shown in FIGS. 14 and 15, respectively.

[0137] The performance of other 3 ’-modifications in the oligonucleotides and methods described herein can be ascertained by testing such modifications in a cleavage assay, such as that described in Example 2, below.

Amplification

[0138] The novel cleavable primers described herein find particular application in isothermal amplification. Isothermal amplification does not employ a denaturation step to separate nucleic acid strands and thus must use different means to effect strand separation, such as a polymerase with strand displacement activity. For isothermal amplification, the novel primers are contacted with sample nucleic acids under conditions wherein the primers anneal to their template strands, if present.

[0139] Amplification reaction mixtures generally contain an appropriate buffer, a source of magnesium ions (Mg²⁺) in the range of about 1 to about 10 mM, e.g., in the range of about 2 to about 8 mM, nucleotides, and optionally, detergents, and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 30 mM preferred. In one embodiment, the TRIS buffer concentration is 20 mM in the reaction mix double-strength (2X) form. The reaction mix can have a pH range of from about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 as typical. Concentration of nucleotides can be in the range of about 25 mM to about 1000 mM, typically in the range of about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM. Detergents such as Tween 20, Triton X 100, and Nonidet P40 may also be included in the reaction mixture. Stabilizing agents such as dithiothreitol (DTT, Cleland’s reagent) or mercaptoethanol may also be included. In addition, master mixes may optionally contain dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). A master mix is commercially available from Applied Biosystems, Foster City, CA, (TaqMan® Universal Master Mix, cat. nos. 4304437, 4318157, and 4326708).

[0140] The reaction mixture also includes a ribo-endonuclease, such as RNase Hl for activating the 3’ blocked primer that includes a cleavage cassette. The ribo-endonuclease unblocks the primers, preferably by cleaving 3’ of a ribonucleotide paired with a nucleotide in a nucleotide duplex, leaving a 3 ’-OH that can be extended by a DNA polymerase. RNase Hl is typically used within a concentration range of 0.5 to 1 U/pL. In various embodiments the RNase Hl can be about: 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 U/pL or can fall within any range bounded by any of these values. A higher concentration of RNase Hl can be used when the cleavage cassette is not optimal for the enzyme.

Polymerase

[0141] The polymerase is generally a DNA polymerase that lacks single-strand specific 5’ to 3’ exonuclease activity. Conveniently, the polymerase is capable of displacing the strand complementary to the template strand, a property termed “strand displacement.” Strand displacement results in synthesis of multiple copies of the target sequence per template molecule. In some embodiments, the DNA polymerase for use in the disclosed methods is highly processive. Illustrative DNA polymerases include variants of Taq DNA polymerase that lack 5’ to 3’ exonuclease activity, e.g., the Stoffel fragment of Taq DNA polymerase (ABI), SD polymerase (Bioron), mutant Taq lacking 5’ to 3’ exonuclease activity described in USPN 5474920, Bea polymerase (Takara), Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene). If thermocycling is to be carried out (as in PCR), the DNA polymerase is preferably a thermostable DNA polymerase. Table 1 below lists polymerases available from New England Biolabs that have no 5’ to 3’ exonuclease activity, but that have strand displacement activity accompanied by thermal stability.

Table 1- Thermostable Stand-Displacing Polymerases Lacking 5’ to 3’ Exonuclease

Activity

In some embodiments, the DNA polymerase comprises a fusion between Taq polymerase and a portion of a topoisomerase, e.g., TOPOTAQ™ (Fidelity Systems, Inc.).

[0142] Strand displacement can also be facilitated through the use of a strand displacement factor, such as a helicase. DNA polymerases that can perform strand displacement in the presence of a strand displacement factor are suitable for use in the disclosed methods, even if the DNA polymerase does not perform strand displacement in the absence of such a factor. Strand displacement factors useful in the methods described herein include BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68(2): 1158- 1164 (1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA binding proteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)). Helicase and SSB are available in thermostable forms and therefore suitable for use in PCR.

[0143] The work described herein employed a Bst polymerase, which is a DNA polymerase from Bacillus stearo thermophilus. Bst polymerase has 5' 3 ' polymerase and double-strand specific 5 ' — > 3' exonuclease activity but lacks 3' — > 5' exonuclease activity. Different types of readily available Bst polymerase include Bst DNA Polymerase, Full Length, BST Max Isothermal DNA Polymerase, Bst 3.0 DNA Polymerase, and Bst 2.0 DNA Polymerase.

Primer and Probe Concentrations

[0144] The concentration of primers in the amplification reaction typically ranges from 0.2 pM to 2 pM, e.g., about: 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 pM, or falls within any range bounded by these values. Primer concentrations are generally determined by their cassette sequence. The concentration of primers that generate the probe template typically ranges from 1 pM to 2 pM, e.g., about 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 pM, or falls within any range bounded by these values. The concentration of primers that do not generate the probe template ranges from 0.2 pM to 1 pM, e.g., about 0.2, 0.4, 0.6, 0.8, or 1.0 pM, or falls within any range bounded by these values. The asymmetry is beneficial due to the difference in cleavage efficiencies and can be determined empirically. The probe concentration typically ranges from 0.1 to 0.5 pM, e.g., about: 0.1, 0.2, 0.3, 0.4, or 0.6 pM, or falls within any range bounded by these values; the probe is generally only added at amounts needed for instrument detection of the fluorescence. Probe concentration is typically kept low to reduce the risk detecting off-target amplification.

Samples

[0145] Nucleic acid-containing samples can be obtained from biological sources and prepared using conventional methods known in the art. In particular, nucleic acid samples useful in the methods described herein can be obtained from any source, including unicellular organisms and higher organisms such as plants or non-human animals, e.g., canines, felines, equines, primates, and other non-human mammals, as well as humans. In some embodiments, samples may be obtained from an individual suspected of being, or known to be, infected with a pathogen (e.g., viral, bacterial, fungal or parasitic), an individual suspected of having, or known to have, a disease, such as cancer, or a pregnant individual.

[0146] Nucleic acids can be obtained from cells, bodily fluids (e.g., blood, a blood fraction, urine, etc.), or tissue samples by any of a variety of standard techniques. In some embodiments, the method employs samples of plasma, serum, spinal fluid, lymph fluid, peritoneal fluid, pleural fluid, oral fluid, and external sections of the skin; samples from the respiratory, intestinal genital, or urinary tracts; samples of tears, saliva, blood cells, stem cells, or tumors. Samples can be obtained from live or dead organisms or from in vitro cultures. Illustrative samples can include single cells, paraffin-embedded tissue samples, and needle biopsies. In some embodiments, the nucleic acids analyzed are obtained from a single cell.

[0147] Nucleic acids of interest can be isolated using methods well known in the art. The sample nucleic acids need not be in pure form but are typically sufficiently pure to allow the steps of the methods described herein to be performed.

Target Nucleic Acids

[0148] Any target nucleic acid that can detected by nucleic acid amplification can be detected using the methods described herein. In typical embodiments, at least some nucleotide sequence information will be known for the target nucleic acids. For example, if the amplification reaction employed is PCR, sufficient sequence information is generally available for each end of a given target nucleic acid to permit design of suitable amplification primers.

[0149] The targets can include, for example, nucleic acids associated with pathogens, such as viruses, bacteria, protozoa, or fungi; RNAs, e.g., those for which over- or underexpression is indicative of disease, those that are expressed in a tissue- or developmental- specific manner; or those that are induced by particular stimuli; genomic DNA, which can be analyzed for specific polymorphisms (such as SNPs), alleles, or haplotypes, e.g., in genotyping. Of particular interest are genomic DNAs that are altered (e.g., amplified, deleted, and/or mutated) in genetic diseases or other pathologies; sequences that are associated with desirable or undesirable traits; and/or sequences that uniquely identify an individual (e.g., in forensic or paternity determinations).

Labeling Strategies

[0150] Any suitable labeling strategy can be employed in the methods described herein. Where the reaction is analyzed for presence of a single amplification product, a universal detection probe can be employed in the amplification mixture. Suitable universal detection probes include double- stranded DNA-binding dyes, such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, OR), Eva Green (Biotium), ethidium bromide, and the like (see Zhu et al., 1994, Anal. Chem. 66: 1941-48).

[0151] In some embodiments, one or more target-specific probes (i.e., specific for a target nucleotide sequence to be detected) is employed in the amplification mixtures to detect amplification products. By judicious choice of labels, analyses can be conducted in which the different labels are excited and/or detected at different wavelengths in a single reaction (“multiplex detection”). See, e.g., Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Colour and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992); and Linck et al. (2017) “A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species,” PLOS One 12(5).

[0152] In some embodiments, it may be convenient to include labels on one or more of the primers employed in an amplification mixture.

Cycling Probes

[0153] In some embodiments, a cycling probe can be used for detection and, optionally, quantification of target nucleic acids in the methods described herein. Cycling probes have been used for years as a way of amplifying signal in amplification assays. Cycling probes are described in, e.g., PCT Publication No. WO 89/09284, and U.S. Patent Nos. 5,011,769 and 4,876,187, which are incorporated herein by reference for this description.

[0154] U.S. Patent No. 5,763,181 describes the use of fluorescently labeled cycling probes to detect target nucleic acids. Generally, the disclosed method employs a fluorescently labeled oligonucleotide cleavage substrate containing a nucleotide sequence that is recognized by the enzyme that catalyzes the cleavage reaction. The oligonucleotide substrate can be DNA, RNA, or both (e.g., a hybrid basepair including one deoxynucleotide and one deoxyribonucleotide) and can be single- or double-stranded. The oligonucleotide can be labeled with a single fluorescent label or with a fluorescent pair (donor and acceptor) on a single strand of DNA or RNA. The choice of single- or double-label can depend on the efficiency of the enzyme employed in the method of the invention. There is no limitation on the length of the oligonucleotide substrate, so long as the fluorescent probe is labeled sufficiently far (e.g., 6-7 nucleotides) away from the enzyme cleavage site. Examples of fluorophores commonly used in this method include fluorescein isothiocyanate, fluorescein amine, eosin, rhodamine, dansyl, and umbelliferone. Other fluorescent labels will be known to the skilled artisan. Some general guidance for designing sensitive fluorescently labeled polynucleotide probes can be found in Heller and Jablonski's U.S. Patent No. 4,996,143. This patent discusses parameters that can be considered when designing fluorescent probes. The cycling probe cleavage reaction can be catalyzed by such enzymes as DNases, RNases, helicases, exonucleases, restriction endonucleases, or retroviral integrases. Other enzymes that effect nucleic acid cleavage are known to the skilled artisan and can be employed to cleave cycling probes having their cognate cleavage sites.

[0155] In some embodiments, the cycling probe can be a probe comprising a cleavage cassette, as described above, which is cleavable using ribo-endonuclease such as RNase Hl. The intact cycling probe has low fluorescence, but when hybridized to the amplified DNA strand it forms a substrate for RNase Hl. Hydrolysis separates the 5’ Fluor from the 3’- quencher molecules and releases fluorescence. A cleavage cassette including three ribonucleotides (RRR) provides a good balance between increased background (R>3) and reduced cleavage (R<3). Other modifications in the regions flanking the cleavage cassette are well tolerated, and the position of the RRR core toward the 5 ’-end of the probe affords good results.

[0156] Modifications in one or both of the regions flanking the cleavage cassette can he included in a cycling probe, e.g., to increase cleavage speed. 2’-0Me/DNA duplexes adopt A-form structures that mimic the natural RNA/DNA substrate for RNase Hl. In fact, the first published gapmer was a 2’-OMe-DNA-2’-OMe structure (Inoue, et al., 1987) that showed sequence-dependent hydrolysis using RNase H. The 2’-0Me modified 5’ flanking primer sequence was found to be a poor substrate for Bst polymerase and could not be “backfilled” efficiently (FIG. 3, step 12). However, other enzymes capable of extending against 2’-0Me have been identified, such as Superscript III reverse transcriptase. If 2’-0Me nucleotides are used in the 5 ’ flanking region of the primers to speed up cleavage of the cassette, a reverse transcriptase can be added in addition to Bst polymerase. [0157] Other “RNA-like” oligonucleotide modifications can be used in the 5 ’-and/or 3’ flanking sequence(s). For example, 2’-fluoro modification is less bulky than the 2’-O- methyl modification and may be better tolerated by Bst. However, “reverse phosphoramidites” are required for incorporation of N3’-P5’ phosphoramidates, and “reverse” 2’-F analogs are not commercially available. It is also possible to use the N3’-P5’ phosphoramidates in one or both of these flanking sequence(s). Since the experimental work below shows that T107 was tolerated in the primers, N3’-P5’ phosphoramidates, such asT107 and A107 “sprinkled” into the 5’- and/or 3’-flanking sequence(s) are expected to speed the assay.

Illustrative Automation and Systems

[0158] Tn some embodiments, a target nucleic acid is detected using an automated sample handling and/or analysis platform. In some embodiments, commercially available automated analysis platforms are utilized. For example, in some embodiments, the GeneXpert® system (Cepheid, Sunnyvale, CA) is utilized.

[0159] The methods described herein are illustrated for use with the GeneXpert® system. Exemplary sample preparation and analysis methods are described below. However, the present invention is not limited to a particular detection method or analysis platform. One of skill in the art recognizes that any number of platforms and methods may be utilized.

[0160] The GeneXpert® utilizes a self-contained, single use cartridge. Sample extraction, amplification, and detection may all be carried out within this self-contained “laboratory in a cartridge” (available from Cepheid - see www.cepheid.com). One of skill in the art will recognize that the methods disclosed herein are suitable for use with other cartridge-based systems comprising a cartridge having a plurality of fluidly connected chambers housed within a single disposable cartridge body that provides for automated sample preparation, nucleic acid extraction, amplification, and detection. In some embodiments, the cartridge allows for storage of dried reagents and provides for automated fluidic movements, reagent rehydration, and mixing at the time of sample processing. In some embodiments, the cartridge comprises multiple fluidic pathways to prevent or limit bubble trapping and contamination, while allowing for thermal cycling and optical monitoring of reaction progress in a reaction chamber that extends from the body of the cartridge. [0161] Components of the cartridge include, but are not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. In some embodiments, a rotary valve enables fluid transfer from chamber to chamber and contains nucleic acids lysis and filtration components. An optical window enables real-time optical detection. A reaction tube enables very rapid thermal cycling.

[0162] In some embodiments, the GeneXpert® system includes a plurality of modules for scalability. Each module is configured with fluid sample handling and analysis components.

[0163] After the sample is added to the cartridge, the sample is contacted with lysis buffer and released nucleic acid is bound to a nucleic acid-binding substrate such as a silica or glass substrate. The sample supernatant is then removed and the nucleic acid eluted in an elution buffer such as a Tris/EDTA buffer. The eluate may then be processed in the cartridge to detect target genes as described herein. In some embodiments, the eluate is used to reconstitute at least some of the reagents, which are present in the cartridge as lyophilized particles.

[0164] In some embodiments, an off-line centrifugation is used to improve assay results with samples with low cellular content. The sample, with or without the buffer added, is centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of supernatant, buffer, or other liquid. The resuspended pellet is then added to a GeneXpert® cartridge as previously described.

Illustrative Assay Cartridge

[0165] As shown in FIG. 17A, the assay cartridge 100 comprises a cartridge body 102 containing a plurality of chambers 108 for reagents or buffers and sample processing. The chambers are disposed around a central syringe barrel 106 that is in fluid communication with a valve body 110 (see FIGS. 17B and 17C) and that is sealed with a gasket 104. The valve body 110 can include a cap 112 and the entire cartridge body can be supported on a cartridge base 101. The valve body typically contains one or channels or cavities (chamber(s) 114) that can contain a filter as described herein that can function to bind and elute a nucleic acid. In some embodiments the cartridge further comprises one or more temperature- controlled channels or chambers that can, in certain embodiments, function as thermocycling chambers. A “plunger” not shown can be operated to draw fluid into the syringe barrel 106 and rotation of the valve body 110 provides selective fluid communication between the various reagent chambers 108 and channels, reaction chamber(s), mixing chambers, and optionally, any temperature-controlled regions. Thus, the various reagent chambers 108, reaction chambers, filter material(s), and temperature-controlled chambers or channels are selectively in fluid communication by rotation of the plunger and reagent movement (e.g., chamber loading or unloading) is operated by the “syringe” action of the plunger within the valve assembly. In other embodiments, the various reagent chambers, reaction chambers, filter material, and temperature-controlled chambers or channels are selectively in fluid communication by linear progression (e.g., by forced movement) of the reagents and sample from one chamber to the next.

[0166] While the methods described herein are described primarily with reference to the GeneXpert® cartridge by Cepheid Inc. (Sunnyvale, Calif.) (see, e.g., FIG. 17A), it will be recognized, that in view of the teachings provided herein the methods can be implemented on other cartridge/microfluidic systems, including alternative cartridge designs having valve assemblies that involve multiple interfacing components, as well as cartridge body defined by multiple interfacing components to form the multiple chambers of the cartridges, for example, those described in Korean Application No. 102293717B1 and KR 102362853B1 , cartridges that utilizes ultrasonic waves to lyse cells in a biological sample, for example, those described in International Application No. WO2021/245390A1, cartridges and systems that utilizes an electrowetting grid for microdroplet manipulation and electrosensor arrays configured to detect analytes of interest, for example, those described in International Application No. WO2016/077341 A2, cartridges that facilitate movement of nucleic acid from one chamber to the next chamber by opening a vent pocket, for example, those described in International Application No. WO2012/145730A2, multiplexed assay systems comprising a plurality of thermocycling units such that individual chambers can be heated, cooled, and/or compressed to mix fluid within the chamber or to propel fluid in the chamber into another chamber, for example, those described in International Application No. WO2015/138343A1, and as well as systems for rapid amplification of nucleic acids facilitated by flexible portions of the sample cartridge aligned to accomplish temperature cycling for nucleic acid amplification, for example, those described in International Application No.WO2017/147085Al. Such cartridge/microfluidic systems can include, for example microfluidic systems implemented using soft lithography, micro/nano-fabricated microfluidic systems implemented using hard lithography, and the like.

Kits

[0167] Also contemplated is a kit for carrying out the methods described herein. Such kits include one or more reagents useful for practicing any of these methods. A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow. The kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay.

[0168] Kits preferably include instructions for carrying out one or more of the screening methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user can be employed. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.

EXAMPLES

[0169] Thermostable versions of RNase Hl sourced from NEB or Lucigen were used in the studies described below. Even though the enzymes may be from different species (T. thermophilus and E. coli, respectively) they both have performed equivalently in our system.

Example 1; Amplification using primers containing a natural ribonucleotide cleavage cassette

[0170] Primers with a randomized natural ribonucleotide cassette were tested in polymerase chain reaction (PCR) method, as shown in FIG. 4. Excipients were added to enhance efficiency. The excipients added included 1 M Betaine, 0.01% Tween-20, 1 mM Manganese Chloride, 0.01% PAMAM Dendrimer, generation 1. The results are shown in FIG. 5. [0171] Linear amplification speed and endpoints were found to be positively correlated with the number of ribonucleotides in the cassette. With a mixed ribonucleotide cassette, we were able to amplify and detect le8 copies of template in 30 mins. However, with a linear probe, a sharp drop-off in amplification detection was observed between le8 and le7 copies of template (FIG. 5).

[0172] The primer sequences used to detect le8 copies in 30 mins contained a 6- ribonucleotide cassette with a random sequence (see below, bold = ribonucleotide cassette).

FWD: 5’ TGTGCTCAGCCCrArGrCrCrUrCAGTGCCTCGCATCATTGTGCT 3’ S03 (SEQ ID N0:1)

REV 5’ GCAGATAGCCACTrCrCrCrCrGrCCTGCCAGTACCTCACACACCC 3’ S03 (SEQ ID N0:2)

(S03 refers to a 3’ blocking group to prevent non-specific extension Modified bases can be employed in primers to increase the stability of base pairs and therefore the duplex as a whole and/or to suppress non-specific primer extension. Suitable modified bases are described in US Publication No. 2023/0096557, which is incorporated herein by reference in its entirety.)

[0173] Since linear amplification with an all-natural ribonucleotide cassette reached a plateau, we hypothesized that the drop-off observed with exponential amplification was due to the primers being whittled to < 3 ribonucleotides. These results are consistent with other studies that show the length of RNA/DNA for efficient binding/cleavage by RNase Hl is 5- 6 bp (Yazbeck, et al. NAR 2002, 3015).

Example 2: Amplification using primers containing amino-ribonucleotides in a cleavage cassette

[0174] To ensure the cleavage cassette remained long enough for fast and continuous cleavage, the amplification method of claim 1 was repeated using a non-cleavable RNA analog in the cleavage cassette.

[0175] The RNA analog has a substitution of the 3’ hydroxyl for a 3’ amino group (an N3’-P5’ phosphoramidate). This modification prevents cleavage of the base and, when placed in the 5’ region of the cassette, forces the RNaseHl to cleave at the 3’ end of the cassette. For convenience, only the T base of the N3 ’ -P5 ’ phosphoramidate was synthesized and tested, named T107 (T). However, before testing the T107 cassettes, we ordered primers with cassette sequences to act as controls for the T107 sequences. The control primer had rU bases instead of T107 bases. The control primers for other T107 primers’ sequences are shown below. The cleavage cassette was changed, and 4 nucleotides were added to the 5’ end of the primer (italics) to ensure the Tm remained the same.

FWD: 5’ CGGATGTGCTCAGCCCrUrUrUrUrUrCAGTGCCTCGCATCATTGTGCT 3’ S03 (SEQ ID N0:3)

REV :5'CGGAGCAGATAGCCACTrUrUrUrUrUrCCTGCCAGTACCTCACACACCC 3’ S03 (SEQ ID N0:4)

[0176] FIG. 6 shows the results of amplification using primers containing an allnatural ribonucleotide cassette composed of a sequence of 5 rUs linked at the 3’ end to 1 rC.

[0177] Surprisingly, we saw that changing the cleavage cassettes from the ones shown above with mixed bases to rUrUrUrUrUrC (to act as a control for our future T107 primers) reduced sensitivity by 2 logs (FIG. 7). This suggests that RNase Hl may exhibit sequence specificity, and this has been studied recently in the context of antisense oligonucleotide efficiency (Kielpinshi, Hagedorn, Lindow, & Vinther, 2017).

[0178] Discussion: The original T107 cleavage cassette design was TTTTTrC and no cleavage was observed. This was surprising because we assumed the T107s would sufficiently resemble RNAribobases to mimic a canonical cleavage site for RNaseHl. Our next step was to replace one of the T107s with a rC (TTTTrCrC), and this did allow for cleavage and linear amplification which did not plateau. This was surprising because we thought RNaseHl would cleave 50% of the time at either of the 2 rCs, leading to an eventual plateau. Similarly, we saw that interspersing rUs within the T107s (rUTrUTrCrC) also led to robust linear amplification which plateaued much later than the all-natural ribobase cassette. These data led us to hypothesize that the T107s have some sort of protective effect on the natural ribonucleotidebases adjacent to them, perhaps extending across both ribonucleotidbases. In any case, the results indicate that using N3’-P5’ phosphoramidates can mitigate the tendency of linear amplification with all natural primers to reach a plateau.

Example 3: Amplification of cleavage cassettes with different combinations of natural and amino-ribonucleotides

[0179] We tested the impact of the length of the ribonucleotide cassette on the rate of cleavage and linear amplification in amplification reactions according to Example 1 and found that this rate decreased as the number of ribonucleotides was reduced from 5 ribonucleotides. [0180] Variations of a cleavage cassette with natural ribonucleotide and aminoribonucleotides (N3’-P5’ phosphoramidates) were also tested. The results are shown in Table 2 (U,C = natural ribonucleotides; T = T107).

Table 2 - Amplification Results for Different Cleavage Cassettes

*If the 5’ end of the cassette is cleaved, the cleavage cassette will be cleaved less efficiently and the linear amplification plateaus.

[0181] Within the cassette, the location and number of T107s affected the location and speed of cleavage. More than 1 natural ribonucleotide on the 5’ end led to cleavage on the 5’ end and to earlier plateaus. Including only 1 rC on the 3’ end didn’t allow for cleavage. However, unexpectedly, 2 rC nucleotides allowed for more efficient cleavage when the T107 residues were positioned 5’ to the rC. Higher numbers of T107s 5’ of the 2 natural rCs positively correlated with the speed of linear amplification (see FIG. 7). If the part of the primer 5’ to the cleavage cassette consisted of 2’-0-Me RNA analogs, the rate of linear extension significantly increased. (This modification is not feasible when using Bst 3.0 as the polymerase because Bst 3.0 cannot transcribe across 2’-O-Methyl bases, which is preferable for the amplification scheme to work well. Enzymes such as Superscript III reverse transcriptase were screened and found to be able to extend across 2’-O-Methyl bases.) It is hypothesized that faster cleavage by RNase Hl is due to the 2’-0-Me bases forcing the DNA:RNA duplex into the A form. Finally, interspersing rU bases between T107 bases speeds up linear amplification to the rate of a fully natural RNA cassette (See FIG. 7). However, when the 5’ ribonucleotide is a natural RNA this does appear to cause a plateau due to the primer being cleaved and leading to a dead end due to that cleavage event. This plateau in a mixed (T107) and natural ribonucleotide cassette is reached much later than the full natural ribonucleotide cassette and its impact on exponential amplification will be explored in the future.

[0182] Thus, the composition of the cleavage cassette within the primer affects the speed of cleavage and whether or not the cassette will be cleaved between the two bases on the 5’ end. When such cleavage at that position occurs, it leads to the primer no longer being activatable by subsequent RNase H cleavage events and results in a plateau during a linear amplification reaction (as shown in FIG. 7). A cleavage cassette comprised of only natural ribonucleotides is cleaved very quickly; however, a plateau is seen with linear amplification due to the ability of RNase Hl to cleave between the two RNAs at the 5’ end of the cassette. A cleavage cassette with 4 (TTTTrCrC) or 5 (TTTTTrCrC) T107 bases 5’ of 2 natural ribonucleotides, leads to a continuing linear amplification, as shown by the lack of a plateau. The presence of consecutive T107 bases in the cleavage cassette does however slow down the rate of cleavage. Interspersing natural ribonucleotides with T107 preceding the 2 natural ribonucleotides speeds up cleavage and delays the plateau in the reaction (rUTrUTrCrC).

[0183] It was determined that pairing a primer containing the TTTTTrCrC cassette with a primer containing the TrUTrUTrCrC cassette produced a fast and sensitive amplification assay (as shown in FIG. 8). The primer with the TTTTTCC cassette was added at 1500 nM and acted as the primer which will generate the probe binding (cognate) sequence. The other primer with the TUTUTCC cassette was added at 500 nM. This primer combination resulted in the detection of Ie2 copies of template in a little over an hour.

[0184] Discussion: The results demonstrate that slow cleavage of the N3’-P5’ phosphoramidate RNA cassette can be sped up by interspersing with non-modified ribobases.

Example 4: Amplification using a cycling probe containing a cleavage cassette

[0185] The use of a cycling probe containing a cleavage cassette further enhances assay speed and sensitivity (see FIG. 9). The assay results shown in FIG. 9 were generated using the following oligonucleotides:

FWD (probe generating) primer:

3' Dab CTGGCCTCTAATGACGTrCrC(T107)(T107)rU(T107)(T107)CAGCGCAATAACGCAAC 5' (SEQ ID N0:5) REV primer:

3' Dab AAAACGCTTCTGGACAGrCrC(T107)rU(T107)rU(T107)GCGACACTGCATCTAGG 5' (SEQ ID N0:6) Probe:

(Fluor)(2OM-T)rGrCrG(2OM-C) (2OM-T) CTGTGGTTCGTCC-Quencher (SEQ ID NO:7)

(“Dab” refers to Dabcyl: Fluor refers to fluorophore; 20M refers to 2’-0-methyl . )

[0186] The concentrations of the forward (probe generating) and reverse primers were 1500 nM and 750 nM, respectively. The probe concentration was 250 nM. The remainder of the components in the amplification reaction mixture were: 50 mM Tris-HCl (pH 8.5), 12.5 mM NaCl, 0.5 mM MnCl₂, 4.5 mM MgCl₂, 0.5 M betaine, 2.5% PEG-8000, 5 ng/pL ET SSB (single stranded binding protein), 2 mM dNTPs, 0.1% Triton-X, 1000 mU/pL RNase Hl, 125 mU/pL Bst 3.0. The isothermal amplification reaction was carried out at 70 °C. This reaction mixture was arrived at after assay optimization that showed that decreasing the concentration of Bst 3.0 from 750 mU/pL to 125 mU/pL and increasing Rnase Hl concentration from 600 mU/pL to 1000 mU/pL was beneficial to the overall speed of the exponential amplification reaction, enabling detection of le4 copies can be in 20 minutes.

Example 5: Amplification comparing a cycling probe containing a cleavage cassette versus a linear probe

[0187] The primers of Example 4 were used (at the same concentrations) in amplifications comparing the results obtained with a cycling probe containing a cleavage cassette with a conventional linear probe. The two probe sequences were:

Linear probe sequence: (Fluor)TGCGCTCTGTGGTTCGTCC-Quencher (SEQ ID N0:8)

Cycling probe sequence: (Fluor)TrGrCrGCTCTGTGGTTCGTCC-Quencher (SEQ ID N0:9)

[0188] The probe concentrations were 100 nM. The other components of the amplification reaction mixture were: 50 mM Tris-HCl (pH 8), 25 KC1, 1 mM MnCh, 7.5 mM MgCl₂, 1 M betaine, 2 mM dNTPs, 0.1% Triton-X, 200 mU/uL RNase Hl, 500 mU/uL Bst 3.0.

[0189] The linear probe which was quenched when unbound and generated maximal fluorescence upon hybridization to its template strand. However, linear probes can subsequently be displaced by the polymerase. This feature required the assay to be optimized to produce an excess of the strand serving as the template for the probe, which resulted in a high degree of primer asymmetry and a concomitant decrease in the overall efficiency of the assay. To mitigate this, the method of detection was switched from a linear probe to a cycling probe. The cycling probe resembled the linear probe, but 3 of the nucleotides on the 5’ end were switched to ribonucleotides. This allowed the cycling probe to be cleaved upon binding to its template, resulting in a more robust increase in fluorescence, as seen in FIG. 10. Additionally, with the cycling probe, multiple probes of a single template can yield multiple cleavage events, resulting in a greater degree of fluorescence compared to the linear probe.

Example 6: Impact of 2’O-methyl sequence in cycling probe

[0190] Probes should generally be designed to avoid oligonucleotide interactions, e.g., probe-primer or probe-probe interactions. In addition, probes should generally be designed to avoid non-specific interaction of the cleavage cassette with the sample nucleic acid. Specificity can be increased by increasing the length of the cleavage cassette; however, in many applications, it is preferable to keep the length of the cleavage cassette to three ribonucleotides. However, the fastest cleavage by RNase Hl has been observed under the reaction conditions tested so far when the cleavage cassette is greater than four ribonucleotides. This tension can be addressed by including 2’0-methyl ribonucleotides in the cleavage cassette, preferably flanking (on both sides) of the three-nucleotide cleavage site.

[0191] The primers of Example 4 were used (at the same concentrations) in amplifications comparing the results obtained with cycling probe containing a cleavage cassettes, wherein one probe contains 2’-O-methyl-modified ribonucleotides and one probe does not. Linear isothermal amplification was carried out using the following probes:

Cycling probe with 2’-O-Me sequence:

(Fluor)(2OM-T)rGrCrG(2OM-C) (2OM-T) CTGTGGTTCGTCC-Quencher (SEQ ID NO:10)

Cycling probe with no 2’-0-Me sequence:

(Fluor)TrGrCrGCTCTGTGGTTCGTCC-Quencher (SEQ ID N0:11)

[0192] FIG. 11 demonstrates that cleavage rates were faster for the 2’-O-methyl- containing cleavage cassette. Example 7 : Impact of cleavage cassette sequence diversity

[0193] To test the effect of cleavage cassette sequence diversity on amplification, the A analog of the N3’-P5’ phosphoramidate was synthesized and incorporated into primers. Three new primers with varying cleavage cassette sequences (mixture of A107 and T107) were compared to a primer with all T107; these primers are shown below:

Primer D= 3’ Dabcyl CTGGCCTCTAATGACGTrCrC(T107)(T107)(T107)(T107)(T107)CAGCGCAATAACGCAAC 5’

(SEQ ID N0:12)

Primer A= 3’ Dabcyl CTGGCCTCTAATGACGTrCrC(T107)(A107)(T107)(A107)(A107)CAGCGCAATAACGCAAC 5’

(SEQ ID N0:13)

Primer B = 3’ Dabcyl CTGGCCTCTAATGACGTrGrC(A107)(A107)(T107)(T107)(A107)CAGCGCAATAACGCAAC 5’

(SEQ ID N0:14)

Primer C = 3’ Dabcyl CTGGCCTCTAATGACGTrCrC(A107)(A107)(T107)(T107)(A107)CAGCGCAATAACGCAAC 5’ (SEQ ID N0:15)

[0194] Amplification was detected using a linear probe (125 nM) with the sequence:

(Fluor)TGCGCTCTGTGGTTCGTCC - Quencher (SEQ ID N0:16)

[0195] The other components of the amplification reaction mixture were: 75 mM Tris, 12.5 mM NaCl, 2.5% PEG 8000, 0.5 mM MnCl₂, 4.5 mM MgCl₂, 0.1% Triton-X, 2 mM dNTPs, 1 U/pL RNaseHl, and 0.125 U/pL Bst 3.0.

[0196] All three new primers performed equally or better to the primer with all T107s (see FIG. 12).

[0197] The best mixed cassette forward primer:

3’ Dabcyl CTGGCCTCTAATGACGTrCrC(A107)(A107)(T107)(T107)(A107)CAGCGCAATAACGCAAC 5’) (SEQ ID N0:15) was paired with the best reverse primer:

3’ Dabcyl AAAACGCTTCTGGACAGrCrC(T107)rU(T107)rU(T107)GCGACACTGCATCTAGG 5’ (SEQ ID NO; 6)

(at 1750 nM and 500 nM, respectively), along with cycling probe (125 nM):

(Fluor)(2OM-T)rGrCrG(2OM-C) (2OM-T) CTGTGGTTCGTCC - Quencher (SEQ ID N0:17) in a reaction mixture including the components given above (in this example). In this exponential amplification, le6 copies was detected in 11 mins (see FIG. 13).

[0198] Discussion: The combination of forward and reverse primers resulting in efficient and fast exponential amplification was unexpected. Our current fastest time to result for an amplification reaction is 20 minutes but we expect further shortening of this time to result is feasible and will be achieved through modifications of the primer or enzyme systems used.

Example 8: Reduction in Bst 3.0-related multimerization

[0199] Bst 3.0 is known to produce multimers when polymerizing short fragments (Hafner, Wolter, Stafford, & Giffard, 2001). It has been shown that this nonspecific polymerase activity by Bst could be reduced by the addition of poly(aspartic) acid (Sakhabutdinova, et al., 2021). We determined that a PAMAM dendrimer, ethylenediamine core ([NH2(CH2)2NH2]:(G=0.5); dendri PAMAM(NHCH2CH2COONa)₈). When added to our reaction, we saw a reduction in multimerization as shown by less laddering seen in le8 with 0.01% PAMAM than le8 with 0% PAMAM (see FIG. 16).

Claims

CLAIMS What is claimed is:

1. An oligonucleotide comprising at least one ribonucleotide adjacent to at least one 3'-modified-ribonucleotide, wherein the 3 '-modified ribonucleotide comprises a hydrolytically stable modification at the 3 '-position that inhibits hydrolysis by RNase Hl.

2. The oligonucleotide of claim 1, wherein the at least one 3'-modified- ribonucleotide comprises a 3'-amino-ribonucleotide.

3. The oligonucleotide of claim 1 or claim 2, wherein the at least one 3'- amino-ribonucleotide comprises a non-natural nucleotide base.

4. The oligonucleotide of claim 3, wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5- nitro-3-(r-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, and 2-amino-8-(r-beta-D-2’- ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one.

5. The oligonucleotide of any one of claims 1-4, wherein the 3 '-aminoribonucleotide comprises 3'-amino-ribothymidine nucleotide, 3'-amino-riboadenine nucleotide, or a combination thereof.

6. The oligonucleotide of any one of claims 1-5, wherein the at least one ribonucleotide comprises a natural ribonucleotide base.

7. The oligonucleotide of any one of claims 2-6, wherein the oligonucleotide comprises a cleavage cassette comprising the at least ribonucleotide and the at least one 3'-amino-ribonucleotide, wherein the cleavage cassette comprises: a 5’ end linked to a 5’ nucleotide sequence; and a 3 ’end linked to a 3’ nucleotide sequence.

8. The oligonucleotide of claim 7, wherein the cleavage cassette comprises a combination of at least five ribonucleotide(s) and 3'-amino-ribonucleotide(s), in total, optionally at least seven ribonucleotide(s) and 3'-amino-ribonucleotide(s), in total.

9. The oligonucleotide of any one of claims 1-8, wherein the ribonucleotide is located at the 3’ end of the cleavage cassette.

10. The oligonucleotide of claim 9, wherein the cleavage cassette comprises at least two ribonucleotides at the 3’ end of the cleavage cassette.

11. The oligonucleotide of any one of claims 1-10, wherein the 3'-amino- ribonucleotide is located 5’ of the ribonucleotide(s).

12. The oligonucleotide of any one of claims 1-11, wherein the cleavage cassette comprises a plurality of ribonucleotides and a plurality of 3'-amino-ribonucleotides.

13. The oligonucleotide of claim 12, wherein the cleavage cassette comprises two ribonucleotides at the 3’ end linked to a segment comprising a plurality of 3'- amino-ribonucleotides .

14. The oligonucleotide of claim 13, wherein the plurality of 3'-amino- ribonucleotides comprises a least four 3'-amino-ribonucleotides, optionally at least five 3'- amino-ribonucleotides .

15. The oligonucleotide of claim 12 or claim 13, wherein the plurality of 3'-amino-ribonucleotides are adjacent to one another.

16. The oligonucleotide of any one of claims 7-15, wherein the cleavage cassette comprises at least rN-rN-arN-arN.

17. The oligonucleotide of claim 12, wherein the cleavage cassette comprises two ribonucleotides at the 3 ’end linked to a segment comprising a plurality of 3'- amino-ribonucleotides (arN) interspersed with a plurality of ribonucleotides (rN).

18. The oligonucleotide of claim 17, wherein the segment comprises at least rN-arN-rN-arN.

19. The oligonucleotide of any one of claims 7-18, wherein the cleavage cassette comprises a plurality of 3'-amino-ribonucleotides that comprise more than one type of base, optionally selected from a combination of thymine and adenine.

20. The oligonucleotide of any one of claims 7-18, wherein the cleavage cassette comprises a plurality of 3'-amino-ribonucleotides that comprise one type of base, optionally selected from thymine or adenine.

21. The oligonucleotide of any one of claims 7-20, wherein the cleavage cassette comprises a plurality of ribonucleotides that comprise more than one type of base.

22. The oligonucleotide of any one of claims2-20, wherein the cleavage cassette comprises a plurality of ribonucleotides that comprise one type of base.

23. The oligonucleotide of any one of claims 7-22, wherein the oligonucleotide comprises a cleavable primer comprising a 3’ blocking group.

24. The oligonucleotide of any one of claims2-20, wherein the 5’ nucleotide sequence and the 3’ nucleotide sequence both comprise deoxyribonucleotides.

25. The oligonucleotide of any one of claims 7-23, wherein the cleavage cassette comprises one or more 2’-O-methyl ribonucleotides at the 5’ end.

26. A combination of at least two oligonucleotides according to any one of claims 7-25, wherein one oligonucleotide is a forward primer and one oligonucleotide is a reverse primer for amplifying a target nucleic acid.

27. The combination of claim 26, wherein at least one oligonucleotide comprises the oligonucleotide of claim 13, and the other oligonucleotide comprises a cleavage cassette comprising two ribonucleotides at the 3 ’end linked to a segment comprising a plurality of 3'-amino-ribonucleotides (arN) interspersed with a plurality of ribonucleotides (rN), wherein the cleavage cassette also comprises: a 5’ end linked to a 5’ nucleotide sequence; and a 3 ’end linked to a 3’ nucleotide sequence.

28. The combination of claim 27, wherein at least one oligonucleotide comprises the oligonucleotide of claim 14, and the other oligonucleotide comprises a cleavage cassette comprising two ribonucleotides at the 3 ’end linked to a segment comprising at least rN-arN-rN-arN, wherein the cleavage cassette also comprises: a 5’ end linked to a 5’ nucleotide sequence; and a 3 ’end linked to a 3’ nucleotide sequence.

29. The combination of claim 27 or claim 28, wherein the at least one oligonucleotide comprises a cleavage cassette having at least two ribonucleotides at the 3’end linked to a segment comprising at least arN-arN-arN-arN.

30. The combination of any one of claims 27-29, wherein the 3'-amino- ribonucleotides comprise one type of base.

31. The combination of any one of claims 16-29, wherein the 3'-amino- ribonucleotides comprise more than one type of base.

32. The combination of any one of claims 26-31, wherein the combination comprises a plurality of said at least two oligonucleotides comprising the forward primer and the reverse primer, wherein each pair of forward and reverse primers detects a different target nucleic acid.

33. The oligonucleotide of any one of claims 1-25 or the combination of any one of claims 26-32, wherein the oligonucleotide or combination is contained within a cartridge for detecting one or more target nucleic acids in a sample, the cartridge comprising: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality; an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reagent chamber comprising one or more of the oligonucleotide(s) and/or one or more of the combination(s); and a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; and a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel.

34. A method for amplifying a target nucleic acid, wherein the method comprises contacting sample nucleic acids with: a primer comprising the oligonucleotide of any one of claims 7-25 and another primer; or the combination of any one of claims 26-31, wherein each oligonucleotide serves as a primer; in a reaction mixture under conditions suitable for primer extension.

35. The method of claim 34, wherein the method comprises detecting the target nucleic acid, if detectable in the sample nucleic acids.

36. The method of claims 34 or 35, wherein the method comprises an isothermal method.

37. The method of any one of claims 34-36, wherein: the oligonucleotide comprises a cleavable primer comprising a 3 ’ blocking group; or the combination comprises an oligonucleotide comprising a cleavable primer comprising a 3’ blocking group; and the method comprises annealing the cleavable primer to its target nucleotide sequence and cleaving the cleavable primer with RNase Hl to release the blocking group.

38. The method of claim 37, wherein the RNase Hl cleaves at the 3’ end of the cleavage cassette.

39. The method of claims 37 or 38, wherein the RNase Hl is a thermostable RNase Hl, optionally from Thermits thermophilus or Echerichia coli.

40. The method of any one of claims 34-39, wherein the reaction mixture comprises a probe.

41. The method of claim 40, wherein the probe comprises a linear probe, and the primer that generates a nucleotide sequence to which the probe can bind is included in the reaction mixture in excess of the other primer.

42. The method of claims 40 or 41, wherein the probe comprises a fluorescent dye and a quencher molecule.

43. The method of any one of claims 40-42, wherein the probe comprises a cycling probe.

44. The method of claim 43, wherein the cycling probe comprises not more than three ribonucleotides.

45. The method of claim 44, wherein the three ribonucleotides comprise a ribonucleotide core that is positioned near the 5 ’ end of the cycling probe.

46. The method of claim 44, wherein the not more than three ribonucleotides are flanked, on either end, with 2’-O-methyl-base-containing sequences.

47. The method of any one of claims 34-46, wherein the reaction mixture comprises polymerase at a concentration of 100 mU/pL to 600 mU/u L, optionally wherein the polymerase comprises Bst 3.0.

48. The method of any one of claims 37-47, wherein the reaction mixture comprises the RNase Hl at a concentration of 700 mU/pL to 1300 mU/pL.

49. The method of any one of claims 34-48, wherein the method comprises multiplex amplification.

50. The method of any one of claims 37-49, wherein the method is carried out within a cartridge.

51. The method of claim 50, wherein the cartridge comprises: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality; an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reagent chamber comprising one or more of the oligonucleotide(s) and/or one or more of the combination(s); and a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; and a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel.

52. A kit comprising the oligonucleotide of any one of claims 1-25 or the combination of any one of claims 26-32, wherein the kit comprises a strand-displacing polymerase.

53. The kit of claim 52, wherein the kit additionally comprises RNase Hl.

54. The kit of claims 52 or 53, wherein the oligonucleotide or combination wherein the oligonucleotide or combination is contained within a cartridge for detecting one or more target nucleic acids in a sample, the cartridge comprising: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality; an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reagent chamber comprising one or more of the oligonucleotide(s) and/or one or more of the combination(s); and a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; and a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel.

55. The oligonucleotide or combination of claim 33, the method of claim 51, or the kit of claim 54, wherein a strand-displacing polymerase is disposed within one of the plurality of chambers.

56. The oligonucleotide or combination of claim 33, the method of claim 51, or the kit of claim 54, wherein RNase Hl is disposed within one of the plurality of chambers.

57. The oligonucleotide or combination of claim 33, the method of claim 51, or the kit of claim 54, wherein a strand-displacing polymerase is disposed within one of the plurality of chambers, and RNase Hl is disposed within one of the plurality of chambers.

58. The oligonucleotide or combination of claim 33, or the method of claim 51, or the kit of claim 54, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

59. The oligonucleotide or combination of claim 33, or the method of claim 51, or the kit of claim 54, wherein the method is a point-of-care method.

60. The oligonucleotide or combination of claim 33, or the method of claim 51, or the kit of claim 54, wherein the cartridge comprises, a primer pair that selectively hybridizes to an exogenous control and/or an endogenous control, wherein the exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

61. The oligonucleotide or combination of claim 33, or the method of claim 51 , or the kit of claim 54, wherein the cartridge facilitates detection of a target nucleic acid in the sample within 30 minutes, within 20 minutes, or within 10 minutes from the time the sample is placed in a cartridge.