WO2013119827A1 - Capture directe d'arn par des sondes d'inversion moléculaire - Google Patents

Capture directe d'arn par des sondes d'inversion moléculaire Download PDF

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Publication number
WO2013119827A1
WO2013119827A1 PCT/US2013/025167 US2013025167W WO2013119827A1 WO 2013119827 A1 WO2013119827 A1 WO 2013119827A1 US 2013025167 W US2013025167 W US 2013025167W WO 2013119827 A1 WO2013119827 A1 WO 2013119827A1
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Prior art keywords
rna molecule
probe
rna
capture
dna
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PCT/US2013/025167
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English (en)
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Thomas Clarke
Alexander SHEH
Sarah GRUSZKA
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Pathogenica, Inc.
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Publication of WO2013119827A1 publication Critical patent/WO2013119827A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • RNA molecules Direct capture of RNA molecules, in the absence of generating an intermediate cDNA, for, e.g., sequencing or quantifying particular species
  • 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.
  • MIPs molecular inversion probes
  • 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.
  • MIPs molecular inversion probes
  • the invention also provides kits and compositions for performing these methods.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • White bar indicates capture region amplicon. Black bar indicates overextension region. Samples using Y64A polymerase show much less
  • overextension product strand displacement
  • WT overextension product
  • reverse transcriptase enzymes strand displacement
  • FIGs. 3A and 3B summarize data for MIP reactions using Taq DNA Ligase to detection RNA.
  • This experiment demonstrates optimal dNTP and MgCl 2 concentrations for molecular inversion probe reactions performed using Tth Reverse Transcriptase and Taq DNA ligase. 10 6 (3 A) and 10 8 (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.
  • this experiment demonstrates optimal dNTP and MgCl 2 concentrations for molecular inversion probe reactions performed using Tth Reverse Transcriptase and T4 DNA ligase.
  • 4 A and 4B represent at 10 6 and 10 8 input copies of RNA template, respectively.
  • 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:
  • 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.
  • DNA ligases are highly sensitive to changes in Mg when ligating
  • DNA:R A hybrids DNA:R A hybrids. Applicants developed a protocol that uses Mg dependent reverse transcriptase to enable use of lower concentrations
  • MIPs Molecular Inversion Probes
  • 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,
  • 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.
  • HI or the extension arm
  • H2 the homologous probe sequence at the 5' end of the probe
  • 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.
  • polymerase-dependent e.g., an RNA-dependent DNA polymerase
  • 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.
  • 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.
  • Regular of interest refers to the sequence between the nearest termini of the two target sequences of the homologous probe sequences in a probe.
  • 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.
  • 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.
  • 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 PROMEGATM; see also U.S. Patent No.
  • 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
  • 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.
  • RNA template 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)).
  • HCV human immunodeficiency virus
  • HCV hepatitis C virus
  • SARS coronavirus the agent
  • 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.
  • a pathogenic organism generally a bacteria or fungus
  • a pathogenic organism 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.
  • 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.
  • an infectious agent that promotes cancer such as HCV
  • 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.
  • diseases e.g. , Cystic Fibrosis, cancer as mentioned above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA capture with MIPs utilizing Tth polymerase, T4 ligase and Exonuclease I and III.
  • Hybridization Master Mix total 15ul volume, added in sequential order
  • 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
  • RNA capture with MIPs utilizing Tth polymerase, Taq ligase and Exonuclease I and III.
  • 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
  • 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.
  • 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.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • 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.

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Abstract

L'invention concerne des procédés de capture directe de séquences d'ARN à l'aide de sondes oligonucléotidiques de capture à circularisation (également connues comme sondes d'inversion moléculaire - MIP) sans avoir besoin de générer une molécule d'ADNc, de type intron, de longueur totale. L'invention concerne également des kits et des compositions destinés à la réalisation de ces procédés.
PCT/US2013/025167 2012-02-07 2013-02-07 Capture directe d'arn par des sondes d'inversion moléculaire WO2013119827A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015154028A1 (fr) * 2014-04-04 2015-10-08 Affymetrix, Inc. Compositions et procédés améliorés pour analyses au moyen de sondes d'inversion moléculaire
CN114277447A (zh) * 2021-12-21 2022-04-05 翌圣生物科技(上海)股份有限公司 靶序列随机sgRNA全覆盖组的制备方法

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US6221603B1 (en) * 2000-02-04 2001-04-24 Molecular Dynamics, Inc. Rolling circle amplification assay for nucleic acid analysis
US20100015602A1 (en) * 2005-04-01 2010-01-21 Qiagen Gmbh Reverse transcription and amplification of rna with simultaneous degradation of dna
US7989166B2 (en) * 2005-04-12 2011-08-02 In Situ Rcp A/S Circle probes and their use in the identification of biomolecules

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015154028A1 (fr) * 2014-04-04 2015-10-08 Affymetrix, Inc. Compositions et procédés améliorés pour analyses au moyen de sondes d'inversion moléculaire
CN114277447A (zh) * 2021-12-21 2022-04-05 翌圣生物科技(上海)股份有限公司 靶序列随机sgRNA全覆盖组的制备方法

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