WO2013119827A1

WO2013119827A1 - Direct rna capture with molecular inversion probes

Info

Publication number: WO2013119827A1
Application number: PCT/US2013/025167
Authority: WO
Inventors: Thomas Clarke; Alexander SHEH; Sarah GRUSZKA
Original assignee: Pathogenica, Inc.
Priority date: 2012-02-07
Filing date: 2013-02-07
Publication date: 2013-08-15

Abstract

The invention provides methods of direct capture of RNA sequences using circularizing capture oligonucleotide probes (also known as molecular inversion probes— MIPs) without requiring generating an intervening full-length cDNA molecule. The invention also provides kits and compositions for performing these methods.

Description

DIRECT RNA CAPTURE WITH MOLECULAR INVERSION PROBES

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/596,170, filed on February 7, 2012.

The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Direct capture of RNA molecules, in the absence of generating an intermediate cDNA, for, e.g., sequencing or quantifying particular species

(molecules/ transcripts) is technically challenging, but is scientifically important for providing, for example, important diagnostic tools in a wide variety of applications from environmental monitoring to human diagnostics. Existing platforms using molecular inversion probes, particularly probes that capture a region of interest two nucleotides or more in length, are limited to only DNA templates, as enzymes have not been demonstrated capable of correctly substituting the gap-filling and ligation steps for an RNA template when the gap size is >1 nucleotide. Demonstrating the application of molecular inversion probes with a >1 nucleotide gap size on RNA would be an important advance in enabling sequencing of segments of RNA without introducing a separate cDNA generation step. The problems associated with the cDNA step are: 1) Practical complexity in requiring additional enzymatic step and clean up to generate a template for DNA specific reagents and 2) Introduction of inherent bias due to primer binding or reverse transcriptase sequence preferences, or differential stability of some RNA sequences over others which may lead to over or under representation in the cDNA pool.

Accordingly, a need exists for easy methods of capturing sequences of target RNA molecules using molecular inversion probes (MIPs), preferably in the absence 671491.6 of generating a full intervening cDNA molecule. This is challenging for at least the reason that the polymerase activity of reverse transcriptase is unusual in its ability to displace a non-template strand (e.g., the ligation arm of a MIP, which is necessary for carrying out the circularizing capture reaction) while simultaneously carrying out DNA synthesis with no requirement for accessory proteins like DNA helicases or single-strand DNA binding proteins. See, e.g. the discussion in Paulson et al., Virology 366:361-76 (2007).

SUMMARY OF THE INVENTION

The invention provides, inter alia, easy methods of capturing sequences of target RNA molecules using molecular inversion probes (MIPs) in the absence of generating a full-length intervening cDNA molecule. The invention also provides kits and compositions for performing these methods. As the skilled artisan will appreciate, the methods provided by the invention are not traditional reverse transcription reactions, which typically employ random priming to generate full- length cDNAs of unknown templates or, in some cases, use a conventional oligonucleotide primer (i.e. which consists of a single, contiguous complementary sequence to a template) specific for a target sequence to, again, generate a full- length cDNA that is subsequently analyzed.

In some embodiments, the methods use of a molecular inversion probe to selectively target RNA template from any source (viral, prokaryotic, and eukaryotic) to obtain information including amount of transcript and sequence data.

This process includes

Hybridization of two homer arms of MIP to RNA template Copying of sequence flanked by two MIP homer arms by enzymatic extension from the 3' end of the MIP

• Ligation of captured sequence to the 5' end of the MIP

In some embodiments this method utilizes specific concentrations of MnS04, MgC12 and dNTPs, as well as multiple enzymes to accomplish this and the direct capture from RNA does not require the separate formation of complementary DNA product. This is done through a DNA:R A hybrid state, that does not require the separate formation of a full-length complementary DNA product.

Some methods provided by the invention use direct RNA capture to detect viruses, bacteria or fungi from a clinical sample where the viral, bacterial or fungal molecules are present a level below that which could be detected by using DNA capture.

The invention also provides methods of generating of a circular DNA molecule from a probe as defined herein, using an RNA template such that the resulting molecule contains the probe and reverse complement of the targeted RNA sequence. Also contemplated are methods based on use of a polymerase capable of synthesizing a complementary strand of cDNA from an RNA template, and a ligase capable of ligating DNA to DNA to circularize a template and in particular embodiments, use of Tth polymerase and T4 ligase to generate the circular DNA molecules or in other embodiments, use of Tth polymerase and Taq ligase to generate the circular DNA molecules; and in still other embodiments, use of truncated or specifically mutagenized polymerase or ligase enzymes, such as mutants derived or related to, Tth polymerase or Taq Ligase to generate the circular probes.

In some particular embodiments, the methods provided by the invention use the circular DNA molecules from an RNA template to detect variants or mutations present in an RNA transcript in a clinical sample, or a sample containing both human and non-human nucleic acid. In other embodiments, the methods use the circular DNA molecules from an RNA template to detect spliced RNA transcripts in a clinical sample, or a sample containing both human and non-human nucleic acid. In some embodiments, the methods encompass deployment of a library of >1 and up to 10,000 probes (as defined below) that form complementary DNA product from RNA without and intermediate step for the formation of full-length cDNA. Also encompassed herein is the combination of two or more methods of Direct RNA capture, as disclosed herein, together with i) cDNA capture, ii) ds DNA capture or iii) protein conjugated DNA capture, to quantify nucleic acids and or proteins in a biological sample.

In any of the disclosed methods, the backbone of the circular molecule contains binding sites for primers that can be used to amplify, label, and/or sequence the population of molecules produced from a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

FIGs. 1A-1C illustrate the assay to determine 5' binding arm displacement activity by RT polymerase candidates. This figure shows a schematic outline of qPCR assay to 5' MIP arm displacement by candidate RT polymerase (1A, assay set up). qPCR amplicons were designed to detect two cDNA products; ampliconl detects a product within the capture region of a molecular inversion probe (IB, perfect junction example), and amplicon 2 detects the cDNA product produced if the 5 'arm of the probe is displaced by read through of the RT polymerase (1C, overextension example).

FIG. 2 demonstrates a lack of MIP arm displacement by mutant RT polymerase. Y axis = CT score for qPCR. Higher CT score = lower amount of product. White bar indicates capture region amplicon. Black bar indicates overextension region. Samples using Y64A polymerase show much less

overextension product (strand displacement) than WT and control reverse transcriptase enzymes.

FIGs. 3A and 3B summarize data for MIP reactions using Taq DNA Ligase to detection RNA. This experiment demonstrates optimal dNTP and MgCl₂ concentrations for molecular inversion probe reactions performed using Tth Reverse Transcriptase and Taq DNA ligase. 10⁶ (3 A) and 10⁸ (3B) input copies of RNA template are present in the respective panels. Efficiency is calculated by qPCR detection of the number of molecules of molecular inversion probe generated, divided by number of template molecules present in the reaction, expressed as a percentage.

FIGs. 4A and 4B summarize data for reactions using T4 DNA Ligase to detection RNA. As for FIG. 3, this experiment demonstrates optimal dNTP and MgCl₂ concentrations for molecular inversion probe reactions performed using Tth Reverse Transcriptase and T4 DNA ligase. 4 A and 4B represent at 10⁶ and 10⁸ input copies of RNA template, respectively.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The invention is based, at least in part, on the discovery of methods for direct capture of RNA sequences using MIPs. Leading to this aspect of the invention, Applicants:

• Identified RNase H negative RTs to preserve RNA integrity and

enable multiple cycles of interrogation.

Identified non-strand displacing RT mutant that does not displace 5 ' binding arm, which is a key requirement for molecular inversion probe technique.

For existing protocols, any RNA detection by molecular inversion probes requires an RT step, followed by an amplification step using a DNA polymerase, while Applicants identified Mn2+ switching DNA polymerase (Tth) that enables PCR requires addition of a second enzyme, thus providing time, labor and reagent efficiency savings for this protocol.

Ligation of DNA to DNA when hybridized to an RNA template is poorly catalysed by known DNA ligases. Applicants optimized the reaction conditions to enable highest affinity molecular inversion probe circularization, and sequencing of R A present at 10⁶ copies per reaction.

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• DNA ligases are highly sensitive to changes in Mg when ligating

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DNA:R A hybrids. Applicants developed a protocol that uses Mg dependent reverse transcriptase to enable use of lower concentrations

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of Mg for the combined gap-filling and ligation step.

Molecular Inversion Probes (also known as Padlock Probes or MIPs) generate a circular DNA molecule from a single-stranded, unimolecular probe based on the composition of a template nucleic acid strand. MIPs for use in the present invention are described extensively in, e.g. WO2011/156795 and

PCT/US2012/054901 , both of which are herein incorporated by reference in their entirety.

Probes of the invention

A "circularizing capture oligonucleotide probe" or simply "probe" refers to a linear, unbranched polynucleic acid comprising two homologous probe sequences separated by a backbone sequence, where the first homologous probe sequence is at a first terminus of the nucleic acid and the second homologous probe sequence is at the second terminus to the nucleic acid, and where the probe is capable of circularizing capture of a region of interest of at least 2 nucleotides. "Circularizing capture" refers to a probe becoming circularized by incorporating the sequence complementary to a region of interest. A circularizing capture oligonucleotide probe that has captured the region of interest and been circularized is a "circularized capture oligonucleotide probe." Basic design principles for circularizing probes, such as simple molecular inversion probes (MIPs) as well as related capture probes are known in the art and described in, for example, Nilsson et al, Science,

265:2085-88 (1994), Hardenbol et al, Genome Res., 15:269-75 (2005), Akharas et al, PLOS One, 9:e915 (2007), Porecca et al, Nature Methods, 4:931-36 (2007); Deng et al, Nat. BiotechnoL, 27(4):353-60 (2009), U.S. Patent Nos. 7,700,323 and 6,858,412.

Probes provided by the invention include two homologous probe sequences, each of which specifically hybridizes to distinct target sequences in the target molecule, e.g., an RNA (to produce a "bimolecular hybrid DNA/RNA molecule"), adjacent to a region of interest comprising at least two nucleotides. The probes may further comprise a backbone sequence, which contains a detectable moiety and a primer, between the homologous probe sequences. Typically, the homologous probe sequence at the 3' end of the probe is termed HI (or the extension arm) and the homologous probe sequence at the 5' end of the probe is termed H2 (the ligation or anchor arm). Upon hybridization to the target sites in molecule of interest, the probe/target duplexes are suitable substrates for polymerase-dependent (e.g., an RNA-dependent DNA polymerase) incorporation of at least two nucleotides on the probe (on the extension arm), and subsequent ligase-dependent circularization of the probes. "Ligase" refers to DNA ligases that are able to catalyze the final ligation step to form a circularized capture oligonucleotide probe as described herein, e.g. , while hybridized to an RNA template.

A "homologous probe sequence" is a portion of a probe provided by the invention that specifically hybridizes to a target sequence present in the molecule of interest. "Specifically hybridizes" means a nucleic acid hybridizes to a target sequence with a T_m of not more than 14 °C below that of a perfect complement to the target sequence. "Hybridize" and "hybridization", and the like, refers to canonical Watson-Crick base-pairing. In some embodiments, a probe "specifically hybridizes" when it hybridizes to a target sequence under stringent hybridization conditions. "Stringent hybridization conditions" refers to hybridizing nucleic acids in 6xSSC and 1% SDS at 65°C, with a first wash for 10 minutes at about 42°C with about 20% (v/v) formamide in O. lxSSC, and a subsequent wash with 0.2xSSC and 0.1% SDS at 65°C. The terms "homologous probe sequence," "probe arm," "homer," and the like each refer to homologous probe sequences that may specifically hybridize to target genomic sequences, and are used interchangeably herein. "Target sequence" refers to a nucleic acid sequence on a single strand of nucleic acid in the molecule of interest. "Region of interest" refers to the sequence between the nearest termini of the two target sequences of the homologous probe sequences in a probe. As an illustrative example, in an exemplary 10 nucleotide nucleic acid, probe arms that bind to the first and last four nucleotides of the nucleic acid, respectively, are separated by a two nucleotide region of interest. In some embodiments, the homologous probe sequences in the probes are each at least 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, or more nucleotides in length. In particular embodiments, the homologous probe sequences are 18-50, 18-36, 20-32, or 22-28 nucleotides in length. In more particular embodiments, the homologous probe sequences are 22-28 nucleotides in length. In certain embodiments, the two homologous probe sequences in a probe are the same length; in other embodiments they are different lengths. In particular embodiments, the homologous probe sequences of a probe differ in length, but by less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides.

The invention is preferably employed with enzymes and enzyme mixtures comprising ligases with high affinity for DNA/R A hybrids as well as "non- displacing RNA-dependent DNA polymerases"— i.e. polymerases that displace the ligation arm of a MIP significantly less than displacing polymerases, e.g. 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or less displacement, e.g. 2, 3, 4, 5, 6, 78, 9, 10, 20, 50, 100, 500, or 1000-fold less displacement. Displacing polymerases, for example, include 3173 Pol described in Moser et al., PLOS ONE, 7:e38371 (2012); although non-displacing mutants of this polymerase may be used in the methods of the invention. Particularly useful enzymes for use in the methods provided by the invention are the commercially available Taq and T4 ligases (as well as mutants of these with increased DNA/RNA affinity and or activity, e.g. at least 5, 10, 20, 30, 40, 50% or 1, 2, 3, 4, 5, 10, 20, 30-fold or more increased affinity for DNA/RNA); as well as the Tth polymerase (available from PROMEGA™; see also U.S. Patent No. 5,618,711, incorporated by reference in its entirety) as well as the Y64A mutant M-MuLV fragment described in Paulson et al., Virology 366:361-76 (2007), which is incorporated by reference in its entirety. Fingers domain and 5 '-3' exonuclease domains of the above polymerases, or other polymerases can be mutated to reduced strand displacement activity.

The methods provided by the invention enable "high efficiency" synthesis of a complementary sequence of a target RNA molecule— i.e., high efficiency capture. "High efficiency" of the reaction means at least about 0.5, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2., 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2., 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2., 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5 %, or more, such as at least 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0 %, as measured by the number of molecules of circularized capture oligonucleotide probes generated, divided by number of template molecules present as assayed by qPCR (quantitative polymerase chain reaction).

Applications

In the earliest uses, the formation of the circular molecule depended solely on the template composition at a single nucleotide, with that nucleotide either enabling the circularization or with the single nucleotide copied from the template into the probe during circularization with the resulting circular molecules detected by microarray hybridization. More recent embodiments of this technology have copied longer segments of the template into each circularized molecule and then sequenced the captured region from each circular molecule.

While the previous descriptions of circular molecule generation through MIPs may have described the technology as working on a nucleic acid template, all previous disclosures have enabled the use of the technology on DNA templates no previous disclosure has enabled the use of the technology on an RNA template. Here, Applicants describe two embodiments of the technology that enable capture directly from RNA templates without a cDNA intermediate.

There are numerous applications of MIP technology in healthcare

diagnostics, environmental surveillance, food and water safety, agricultural plant and animal breeding, and human genetics that require capture from an RNA template:

RNA viruses: Infectious agents causing disease. RNA viruses are the etiological agents behind many common diseases that are important health concerns. Some commonly known RNA viruses include human immunodeficiency virus (HIV), the agent causing acquired immune deficiency syndrome (AIDS); rotavirus, an agent causing diarrhea; poliovirus, the causative agent of polio; hepatitis C virus (HCV), an agent causing hepatitis; the influenza virus, the agent causing influenza; and SARS coronavirus, the agent behind severe acute respiratory syndrome (SARS). RNA viruses: Causes of morbidity and mortality in cancer. It has been established that infectious agents, including viruses, can promote carcinogenesis. RNA viruses have been linked to several forms of cancer, including T-cell leukemia (Human T lymphotrophic virus type 1 (HTLV-1) and liver cancer (hepatitis C virus (HCV)).

Bacterial Gene expression: many healthcare or surveillance applictions look for the expression of drug resistance or toxicity genes to determine the clinical or safety significance of bacterial presence. While sequencing or detectiong of genomic DNA can reveal the presence of resistance or virulence genes, such genes might be turned off or hyper-expressed due to mutations elsewhere in the genome. Quantifying the transcript level of the gene provides a simpler and more accurate method of detecting the gene's expression level.

Pathogen detection: A pathogenic organism (generally a bacteria or fungus) may be clinically significant though present at only a few copies in a given clinical sample (e.g., in a severely infected patient, only a few hundred bacteria may be present in one mL of blood). This low copy count presents a challenge to a diagnostic method that detects the genomic DNA of the organism. By detecting the RNA transcripts of highly expressed genes, however, the system may have thousands of template molecules to work with, thus easing the technical challenges of detecting the organism at low levels.

Human Cancer: a similar logic applies to the detection of circulating tumor cells from a clinical sample. These cells, present in the blood at low levels, may reveal the presence and identity of a tumor elsewhere in the body. However, detecting such a cell at low levels is extremely difficult if one looks for genomic DNA signatures. By instead looking for highly expressed RNAs that are unique to tumor cells, Applicants can increase the sensitivity of the method. In some cases, it would be possible to use MIPs to detect both the presence of an infectious agent that promotes cancer, such as HCV, alongside mutations common in cancer, such as p53 mutations in liver cancer.

Human disease: Detecting the presence of mutations in human genes is important in the treatment of certain diseases (e.g. , Cystic Fibrosis, cancer as mentioned above). Trying to sequence a gene from genomic DNA may be difficult because the genome contains many short exons, requiring many probes. By capturing and sequencing from the RNA transcript, the capture process is easier because we're working with a single contiguous piece of sequence.

Human disease: Capture of RNA is also useful in human disease in that some diseases or conditions are characterized by the inclusion or exclusion of particular exons in the transcript. Thus, the condition cannot be detected by sequencing the DNA genome and must be detected by examining the RNA transcript.

Gene expression: In many applications, investigators wish to detect the level (either absolute or relative to other genes) of expression of one or more genes that indicate the phenotype of the cell or cells in the sample. The phenotype may be bacterial drug resistance or toxin production, human cancer or tumor signature, human drug metabolism phenotype that indicates the appropriate dosage of a drug, human pathogen response phenotype, where the gene expression signature indicates the presence of a pathogen or class of pathogens, human drug response signature, where the gene expression profile indicates the body's response to a therapy.

Traditional uses of MIPs for these applications, and most existing assays that detect, sequence, or attempt to quantify RNA from a sample, begin with a reverse transcription step that converts the RNA into a full-length cDNA— i.e., the capture/detection is indirect, relying on two distinct polymerase steps: reverse transcription, followed by polymerase based capture of a full-length cDNA sequence by hybridizing a probe (i.e., MIP) to the cDNA. This step may be general, using random primers or a poly-T primer, or it may use a target-specific primer to convert only specific regions of RNA. Therefore, in the present invention where the capture/ synthesis of a complementary sequence of a target RNA molecule is "without generating an intervening full length cDNA molecule of the target RNA molecule" and the like, inter alia, distinguishes the instant methods from existing RNA capture methods that rely two polymerase steps and an intervening full-length cDNA.

In contrast, the methods enabled by this application do not require a distinct reverse-transcription step in order to capture or synthesize (and subsequently, e.g. , detect, sequence, and quantify) a target sequence in an RNA. Instead, the reverse- transcription generates the circular DNA molecules themselves without any intermediate full-length cDNA. This direct capture reduces the assay's cost, time to result, and labor requirements. The circular DNA molecules produced by these methods may be used in any of the same ways as would circular DNA molecules produced from a DNA template, including, but not limited to: the molecules may be hybridized to a microarray or the molecules may be amplified by rolling circle amplification. In this use, the probe(s) will typically contain a backbone between the two probe arms and the backbone will include a binding site for a primer used in the RCA.

The circularized capture oligonucleotide comprising the complementary sequence of a target RNA molecule may be amplified by PCR. In this use, the probes will typically contain a backbone between the two probe arms and the backbone will include binding sites for two primers oriented in opposite directions and suitable to amplify the section of the circularized molecules containing the probe arms and capture region. The circularized capture oligonucleotide may be cleaved if the probes contain a backbone between the probe arms where the backbone contains a cleavage site. Cleavage of the circularized probes is typically performed after the circularized molecules are purified from the input reaction mixture (e.g., by exonucleasing the linear template molecules) so as to make PCR amplification or hybridization easier or more efficient.

The circularized capture oligonucleotide may be sequenced directly if the backbone contains suitable sequencing adapters.

The circularized capture oligonucleotide may be quantified by qPCR using primers against any of the backbone, probe arms, or capture sequence.

EXEMPLIFICATION

Protocols

Method #1 Capture with T4 Ligase

Applicants have demonstrated that they can perform RNA capture with MIPs utilizing Tth polymerase, T4 ligase and Exonuclease I and III.

RTMIP T4 PROGRAM

Temp (C) Time (min) NOTES

70 1

60 RAMP

60 20 15 HOLD Add LIGASE MIX

37 20

94 2

37 HOLD Add EXO MIX

37 30

94 15

15 HOLD END

Hybridization Master Mix - total 15ul volume, added in sequential order

MM Tth Final concentration in 15ul

20x Tth buffer lx Tth buffer

25mM MgC12 0, 2, 3mM MgC12

25mM MnS04 0.783mM

MIP probes 3nM 0.2nM

H20 nuclease free Qs to 4.02ul

TOTAL 4.02

Final concentration in 15ul

RNA template Copy # variable dNTP mix Final concentration in 15ul lOmM dNTP 125, 200, 300uM H20 nuclease free Qs to 7.98 Tth (5U/ul) 0.9375U TOTAL 7.98

STEP B: Ligation

T4 Ligase Master Mix (15ul per reaction)

Volume (ul)

T4 Ligase 400U/ul 0.88

PNK Buffer 3

H20 11.12

TOTAL 15

STEP C: Exonuclease

Exo Master Mix

Volume (ul)

Exol 0.5

ExoIII 0.5

TOTAL 1

Method #2 Capture with Taq Ligase

Applicants have demonstrated that they can perform RNA capture with MIPs utilizing Tth polymerase, Taq ligase and Exonuclease I and III.

RTMIP T4 PROGRAM

Temp (C) Time (min) NOTES 70 1

60 RAMP

60 20

15 HOLD Add LIGASE MIX

45 20

94 2

37 HOLD Add EXO MIX

37 30

94 15

15 HOLD END

Hybridization Master Mix

MM Tth Final concentration in 15ul

20x Tth buffer lx Tth buffer

25mM MgC12 0, 2, 3mM MgC12

25mM MnS04 0.783mM

MIP probes 3nM 0.2nM

H20 nuclease free Qs to 4.02ul

TOTAL 4.02

Final concentration in 15ul

STEP B: Ligation

Taq Ligase Master Mix (15ul per reaction):

Volume (ul)

Taq ligase 40U/ul 1

Taq buffer 1 OX pH 7.6 3

H20 11

TOTAL 15

STEP C: Exonuclease

Exo Master Mix

Volume (ul)

Exol 0.5

ExoIII 0.5 TOTAL 1

Results

We have tested several protocols using Tth Polymerase/Reverse

Transcriptase with Taq or T4 ligase to generate circularized DNA probes directly from the R A template. In each experiment, a single probe was circularized on the RNA template and a pair of qPCR primers was used to determine the amount of circularized product by having one primer hybridize to a primer site in the backbone of the circularized probe and the other primer hybridize to the capture region. Thus, the primers generate a product only in the presence of a probe that correctly captured the target region. Since the input material contained only RNA (the samples are DNAsed before use), any correctly circularized probe came from circularization on the RNA target.

To compute the efficiency of the reactions, Applicants compare the signal from the circularized probes to the signal produced by a construct that also contains the two primer sites. This construct is amplified from the same probe, circularized on a DNA target. This product of this first amplification is quantified on a

NanoDrop machine and then used as a template for the standard curve in the qPCR reaction.

The RNA template was a segment of the GFP gene generated with T7 transcriptase from a PCR amplicon where one of the PCR primers included a 5' T7 promoter and a 3' GFP-specific region.

RNA Capture with Tth and T4 Ligase

This experiment examined the effect of Mg++ and dNTP concentration on the formation of circularized products from the RNA template. Varying quantities of RNA template were also tested to ensure that the observations generalized to different input samples.

It should be understood that for all numerical bounds describing some parameter in this application, such as "about," "at least," "less than," and "more than," the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description at least 1, 2, 3, 4, or 5 also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.

For all patents, applications, or other reference cited herein, such as non- patent literature and reference sequence information, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited. Where any conflict exits between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GenelDs, Unigene IDs, or HomoloGene ID, or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mR A (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures) are hereby incorporated by reference in their entirety.

Headings used in this application are for convenience only and do not affect the interpretation of this application.

Preferred features of each of the aspects provided by the invention are applicable to all of the other aspects of the invention mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass

combinations and permutations of individual features {e.g. elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the invention including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed invention piecemeal without departing from the invention. For example, for material Is that are disclosed, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, elements of a composition of matter and steps of method of making or using the compositions.

The forgoing aspects of the invention, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art— thus to the extent an element is described in one or more references known to the person having ordinary skill in the art, they may be excluded from the claimed invention by, inter alia, a negative proviso or disclaimer of the feature or combination of features.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of high efficiency synthesis of a complementary sequence of a target RNA molecule, without generating an intervening full length cDNA molecule of the target RNA molecule, comprising

a) contacting an RNA-containing sample with a circularizing capture oligonucleotide probe that specifically hybridizes with the target RNA molecule, under conditions to form a bimolecular hybrid DNA/RNA molecule and wherein the circularizing capture oligonucleotide probe comprises two homologous probe sequences at the 5' and 3' termini of the probe, respectively, wherein the two homologous probe sequences specifically bind to the target RNA molecule at respective target sequences of the target RNA molecule, wherein the two target sequences in the target RNA molecule are separated by at least 2 nucleotides, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175,

200, 225, 250, 275, 300 nucleotides, or more; and

b) incubating the bimolecular hybrid DNA/RNA molecule in the

presence of a non-displacing RNA-dependent DNA polymerase and a ligase under conditions suitable for forming a circularized capture oligonucleotide probe comprising the complementary sequence of the target RNA molecule.

2. The method of Claim 1, wherein the sample is a biological sample.

3. The method of Claim 2, wherein the biological sample is an environmental sample.

4. The method of Claim 2, wherein the biological sample is obtained from a mammalian subject.

5. The method of Claim 4, wherein the mammalian subject is a human subject.

6. The method of Claim 1 , wherein the RNA molecule is an mRNA molecule.

7. The method of Claim 1 , wherein the RNA molecule is a viral RNA.

8. The method of Claim 7, wherein the virus is a Group IV, V, VI or VII virus under the Baltimore virus classification system.

9. The method of any one of the preceding claims, wherein the non-displacing polymerase is selected from Tth polymerase and Y64A mutant in the fingers subdomain of M-MuLV reverse transcriptase.

10. The method of any one of the preceding claims, wherein the ligase is

selected from Taq, T4, or other suitable DNA/R A hybrid ligases, such as mixtures of wild type and mutant forms of these enzymes, such as K159L and K159A T4 mutants.

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11. The method of any one of the preceding claims, wherein the Mg

concentration is about 0.0 mM to about 3.0 mM, e.g., about 0.5-3.0 mM, 1.0-

3.0 mM, 1.5-2.5 mM, e.g. about 2.0 mM.

12. The method of any one of the preceding claims, wherein the ATP

concentration is about 50μΜ to about 3 mM, e.g., 0.250-3.0 mM, 0.50-2.0 mM, 0.75-1.5mM, 0.75- 1.25 mM, e.g. about 1.0 mM.

13. The method of any one of the preceding claims, wherein the dNTPs

concentration is about 50 to about 500 μΜ, e.g. 100-500, 150-450, 200-400, 250-350 μΜ, e.g. about 300 μΜ.

14. The method of any one of the preceding claims, wherein the efficiency of the reaction is at least about 0.5, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2., 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2., 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,

4.2., 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.

5 %, or more, such as at least 6.0,

6.5, 7.0,

7.5, 8.0,

8.5, 9.0,

9.5, 10.0,

10.5, 11.0,

11.5, 12.0,

12.5, 13.0,

13.5, 14.0,

14.5, 15.0, 15.5, 16.0 %, as measured by the number of molecules of circularized capture oligonucleotide probes generated, divided by number of template molecules present as assayed by qPCR (quantitative polymerase chain reaction).

15. The method of any one of the preceding claims, further comprising

contacting the sample with RNAse and/or exonuclease to degrade linear nucleic acids in the sample.

16. The method of any one of the preceding claims, further comprising the step of contacting the circularized capture oligonucleotide with one or more additional oligonucleotides that are not circularizing capture

oligonucleotides.

17. The method of Claim 16, wherein the one or more additional

oligonucleotides is a universal sequencing primer.

18. The method of Claim 16, or 17, further comprising a primer that hybridizes with the captured complementary sequence of the target RNA molecule.

19. The method of any one of the preceding claims, further comprising

determining the amount of the target RNA molecule present in the sample, based on the amount of the circularized capture oligonucleotide.

20. The method of any one of the preceding claims, further comprising

determining the sequence of the captured complementary sequence of the target RNA molecule.

21. The method of any one of the preceding claims, wherein the target RNA molecule comprises a polymorphic region.

22. The method of any one of the preceding claims, wherein the sensitivity of

8 8 RNA detection is less than about 10 copies of the RNA, e.g. less than 10 ,

5xl0⁷, 10⁷, 5xl0⁶, 10⁶, 5xl0⁵, 10⁵, 5xl0⁴, 10⁴ copies.

23. A kit suitable for performing the method of any one of the preceding claims, when used in conjunction with an isolated RNA containing sample.

24. A composition comprising the reagents of any one of the preceding claims.