WO2013142364A1 - Enzymes rnase h modifiées et leurs utilisations - Google Patents

Enzymes rnase h modifiées et leurs utilisations Download PDF

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WO2013142364A1
WO2013142364A1 PCT/US2013/032372 US2013032372W WO2013142364A1 WO 2013142364 A1 WO2013142364 A1 WO 2013142364A1 US 2013032372 W US2013032372 W US 2013032372W WO 2013142364 A1 WO2013142364 A1 WO 2013142364A1
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rnase
primer
modified
enzyme
protein
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PCT/US2013/032372
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Joseph Alan Walder
Mark Aaron Behlke
Scott D. Rose
Joseph Dobosy
Susan Marie RUPP
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Integrated Dna Technologies, Inc.
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Priority to CA2867435A priority Critical patent/CA2867435A1/fr
Priority to EP13713666.9A priority patent/EP2828403A1/fr
Priority to JP2015501817A priority patent/JP2015510776A/ja
Priority to AU2013203590A priority patent/AU2013203590A1/en
Publication of WO2013142364A1 publication Critical patent/WO2013142364A1/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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/26—Endoribonucleases producing 5'-phosphomonoesters (3.1.26)
    • C12Y301/26004—Ribonuclease H (3.1.26.4)

Definitions

  • This invention pertains to methods of chemically modifying a Ribonuclease H (RNase H) enzyme with acid anhydrides for heat-reversible inactivation, as well as applications utilizing such modified enzymes.
  • RNase H Ribonuclease H
  • PCR Polymerase chain reaction
  • PCR is not a perfect system, and typically non-specific amplification occurs at lower temperatures where the thermostable enzymes still have slight activity. Many applications of PCR are hindered by this limitation, and "Hot start PCR" methods have been devised to reduce or eliminate non-specific amplification.
  • Hot start PCR refers to the use of methods that prevent initiation of the polymerase chain reaction until the reaction has been heated to a high temperature, usually at or around 95°C, and cooled to the primer annealing temperature, usually at or around 60°C.
  • the first hot start PCR methods employed physical barriers that could be disrupted by heating to remove the barriers between the reaction components.
  • the nucleic acids target DNA, primers, deoxynucleotides, and buffer
  • the wax melts when the reaction is heated to 95°C, permitting mixing of the DNA polymerase with the other reaction components, and PCR commences once the reaction cools sufficiently for primer binding to occur.
  • hot start PCR is usually performed using a homogenous reaction mix wherein the DNA polymerase is inactivated by some method that can be reversed by heating.
  • examples include chemical modification (such as the anhydride modification schemes used in the present invention to reversibly inactivate RNase H2), antibodies that bind the DNA polymerase, or aptamers that bind the DNA polymerase.
  • the agent limiting DNA polymerase activity is reversed, denatured, or degraded by heating.
  • a reversibly-inactivated hot-start Taq DNA polymerase typically costs 5-10 fold more than unmodified native Taq polymerase.
  • hot start PCR is almost exclusively used in PCR applications today. Use of hot start methods improves the outcome of PCR in two ways:
  • primers can bind at low temperatures at sites in a complex nucleic acid sample having an imperfect sequence match and initiate DNA synthesis, leading to amplification of undesired products. Hot start methods can reduce or prevent mis-priming of this type.
  • Variations of PCR have been developed that utilize other enzymes that are inherently inactive at lower temperatures, thereby limiting undesired non-specific amplification.
  • One example described by Walder et al., (U.S. Patent Application 2009/0325169 ), uses a primer containing a blocking group at or near the 3'-end. The primer cannot extend until the blocking group is cleaved by an RNase H enzyme that has little to no activity at lower temperatures.
  • RNase H is an endoribonuclease that cleaves the phosphodiester bond in an RNA strand when it is part of an RNA:DNA duplex. The enzyme does not cleave DNA or unhybridized single-stranded RNA. This characteristic makes RNase H useful in biological applications, such as in cDNA synthesis wherein the RNA template is destroyed once the desired complementary DNA is synthesized by reverse transcription.
  • Examples of the preferred anhydrides include but are not limited to citraconic anhydride and 3,4,5,6-Tetrahydrophthalic anhydride. These reagents were reacted with the RNase H2 protein to generate the reversible inactivation.
  • the anhydrides modify the terminal amines of lysines and the N-terminus of the protein, altering the charge and likely affecting the conformation of the protein ( Figure 1). These protein modifications are known to be highly sensitive to high temperature and low pH (see Dixon and Perham, Biochem J 1968, 109(2):312-314), with different removal kinetics dependent on the nature of the anhydride utilized (see Walder et al, Mol Pharmacol 1977, 13(3):407- 414).
  • the current invention also provides improvements to assays that employ RNase H cleavage for biological applications related to nucleic acid amplification and detection, where the RNase H has been reversibly inactivated.
  • the invention provides a provides improvements to assays that employ RNase H cleavage for biological applications related to nucleic acid amplification and detection, where the RNase H has been reversibly inactivated.
  • thermophilic RNase H enzymes also extends to a number of other biological assays (see Walder et al, U.S. Application Number 2009/0325169, incorporated herein in its entirety).
  • Thermophilic RNase H enzymes can enable hot start protocols in nucleic acid amplification and detection assays including but not limited to PCR, OLA (oligonucleotide ligation assays), LCR (ligation chain reaction), polynomial amplification and DNA sequencing, wherein the hot start component is a thermostable RNase H or other nicking enzyme that gains activity at the elevated temperatures employed in the reaction.
  • Such assays employ a modified oligonucleotide of the invention that is unable to participate in the reaction until it hybridizes to a complementary nucleic acid sequence and is cleaved to generate a functional 5'- or 3 '-end. Compared to the corresponding assays in which standard unmodified DNA oligonucleotides are used, the specificity is greatly enhanced.
  • thermostable RNase H enzyme that has intrinsically little or no activity at reduced temperatures as in the case of Pyrococcus abysii RNase H2; 2) a thermostable RNase H reversibly inactivated by chemical modification; and 3) a thermostable RNase H reversibly inactivated by a blocking antibody.
  • mutant versions of RNase H can be synthesized that can further improve the traits of RNase H that are desirable in the assays of the present invention.
  • mutant strains of other enzymes that share the characteristics desirable for the present invention could be used.
  • the methods of the present invention are primarily directed to the second alternative.
  • Figure 1 contains structures of various anhydride compounds useful for protein modification. The chemical structures of cis-aconitic anhydride, citraconic anhydride, and 3,4,5,6-tetrahydrophthalic anhydride are shown.
  • Figure 2 shows a scheme for reaction of a lysine residue with 3,4,5,6- tetrahydrophthalic anhydride and removal of the anhydride by heat treatment.
  • the reaction scheme for coupling 3,4,5,6-tetrahydrophthalic anhydride to a lysine residue is shown (top), which results in inactivation of a modified enzyme.
  • Treatment of this structure with heat or low pH reverses the reaction (bottom), which results in reactivation of the now unmodified enzyme.
  • FIG. 3 shows a FQ-Reporter oligonucleotide assay for RNase H2 activity.
  • a fluorescence-quenched hairpin probe assay for RNase H2 activity is shown.
  • DNA bases are uppercase, RNA bases are lower case, FAM is 6-carboxyfluorescein, and FQ is Iowa Black ® FQ dark quencher.
  • the probe forms a hairpin which aligns the FAM reporter dye with the Iowa Black dark quencher. In this configuration, the probe is "dark”.
  • Cleavage of the probe by RNase H2 occurs at the 5 '-side of the ribo-C residue.
  • the cleaved fragment dissociates, separating the reporter dye from the quencher. In this state the probe is "bright” and a positive signal is detected at 520 nm FAM emission.
  • Figure 4 shows an assay of inactivation and heat reactivation of 3,4,5,6- tetrahydrophthalic anhydride-modified P. a. RNase H2 using 2.6 mU enzyme.
  • the relative activity of unmodified and 3,4,5,6-tetrahydrophthalic anhydride-modified P. a. RNase H2 was characterized using the FQ-reporter oligonucleotide assay. Assays were run in 10 reactions using 0.9 nM enzyme (2.6 mU for the unmodified enzyme) at 60°C. Fluorescence measurements were collected every 11 seconds during the 10 minute incubation. Enzyme was either added directly to the reactions (top panel) or following 10 minutes incubation at 95°C to reverse the anhydride modification and reactivate enzyme activity (bottom panel).
  • RFUs are relative fluorescence units.
  • Figure 5 shows an assay of inactivation and heat reactivation of 3,4,5,6- tetrahydrophthalic anhydride-modified P. a. RNase H2 using 200 mU enzyme.
  • the relative activity of unmodified and 3,4,5,6-tetrahydrophthalic anhydride-modified P. a. RNase H2 was characterized using the FQ-reporter oligonucleotide assay.
  • Assays were run in 10 reactions using 67 nM enzyme (200 mU for the unmodified enzyme) at 60°C. Fluorescence measurements were collected every 11 seconds during the 10 minute incubation. Enzyme was either added directly to the reactions (top panel) or following 10 minutes incubation at 95°C to reverse the anhydride modification and reactivate enzyme activity (bottom panel).
  • RFUs are relative fluorescence units.
  • Figure 6 shows an assay of inactivation and heat reactivation of cz ' s-aconitic anhydride-modified P. a. RNase H2 using 2.6 mU enzyme.
  • the relative activity of unmodified and cz ' s-aconitic anhydride -modified P. a. RNase H2 was characterized using the FQ-reporter oligonucleotide assay. Assays were run in 10 reactions using 0.9 nM enzyme (2.6 mU for the unmodified enzyme) at 60°C. Fluorescence measurements were collected every 11 seconds during the 10 minute incubation. Enzyme was either added directly to the reactions (top panel) or following 10 minutes incubation at 95°C to reverse the anhydride modification and reactivate enzyme activity (bottom panel).
  • RFUs are relative fluorescence units.
  • Figure 7 shows an assay of inactivation and heat reactivation of cz ' s-aconitic anhydride-modified P. a. RNase H2 using 200 mU enzyme.
  • the relative activity of unmodified and cz ' s-aconitic anhydride -modified P. a. RNase H2 was characterized using the FQ-reporter oligonucleotide assay.
  • Assays were run in 10 reactions using 67 nM enzyme (200 mU for the unmodified enzyme) at 60°C. Fluorescence measurements were collected every 11 seconds during the 10 minute incubation. Enzyme was either added directly to the reactions (top panel) or following 10 minutes incubation at 95°C to reverse the anhydride modification and reactivate enzyme activity (bottom panel).
  • RFUs are relative fluorescence units.
  • Figure 8 shows an assay of inactivation and heat reactivation of citraconic anhydride-modified P. a. RNase H2 using 2.6 mU enzyme.
  • the relative activity of unmodified and citraconic anhydride-modified P. a. RNase H2 was characterized using the FQ-reporter oligonucleotide assay. Assays were run in 10 reactions using 0.9 nM enzyme (2.6 mU for the unmodified enzyme) at 60°C. Fluorescence measurements were collected every 11 seconds during the 10 minute incubation. Enzyme was either added directly to the reactions (top panel) or following 10 minutes incubation at 95°C to reverse the anhydride modification and reactivate enzyme activity (bottom panel). RFUs are relative fluorescence units.
  • Figure 9 shows an assay of inactivation and heat reactivation of citraconic anhydride-modified P. a. RNase H2 using 200 mU enzyme.
  • the relative activity of unmodified and citraconic anhydride-modified P. a. RNase H2 was characterized using the FQ-reporter oligonucleotide assay. Assays were run in 10 reactions using 67 nM enzyme (200 mU for the unmodified enzyme) at 60°C. Fluorescence measurements were collected every 11 seconds during the 10 minute incubation. Enzyme was either added directly to the reactions (top panel) or following 10 minutes incubation at 95°C to reverse the anhydride modification and reactivate enzyme activity (bottom panel). RFUs are relative fluorescence units.
  • FIG 10 shows the ESI-MS spectra of unmodified recombinant Pyrococcus abyssi RNase H2.
  • FIG 11 shows ESI-MS spectra of recombinant Pyrococcus abyssi RNase H2 modified with 3,4,5,6-tetrahydrophthalic anhydride.
  • ESI-MS electrospray ionization mass spectrometry
  • Figure 12 shows ESI-MS spectra of recombinant Pyrococcus abyssi RNase H2 modified with 3,4,5,6-tetrahydrophthalic anhydride followed by heat treatment.
  • P. a RNase H2 was reacted with a total of 3-fold molar excess of 3,4,5,6-tetrahydrophthalic anhydride and the modified protein was heated at 95°C for 10 minutes to reverse the modification reaction. The final product was examined by electrospray ionization mass spectrometry (ESI-MS). A deconvolution trace of the mass spectra is shown and the molecular weights (Daltons) of the primary peaks are indicated.
  • ESI-MS electrospray ionization mass spectrometry
  • Figure 13 shows amplification plots of qPCR done after overnight incubation at room temperature using a hot-start DNA polymerase.
  • Amplification reactions were performed using a hot start DNA polymerase (iTaq). All reaction components were mixed together and reaction plates were incubated overnight at room temperature.
  • Use of unmodified primers resulted in efficient amplification reactions and no difference was seen between addition of native P.a. RNase H2 (top left) and 3,4,5,6- tetrahydrophthalic anhydride-modified hot start P.a. RNase H2 (bottom left).
  • Use of blocked-cleavable primers (right panels) resulted in efficient amplification reactions and no difference was seen between addition of native P.a. RNase H2 (top right) and 3,4,5,6- tetrahydrophthalic anhydride-modified hot start P.a. RNase H2 (bottom right).
  • Figure 14 shows amplification plots of qPCR done after overnight incubation at room temperature using native (non-hot start) Taq DNA polymerase. Amplification reactions were performed using native Taq DNA polymerase (not hot start). All reaction components were mixed together and reaction plates were incubated overnight at room temperature. Use of unmodified primers (left panels) resulted in no detectable amplification of the target nucleic acid sequence; reactions were run with native P.a. RNase H2 (top left) and 3,4,5,6-tetrahydrophthalic anhydride-modified hot start P.a. RNase H2 (bottom left).
  • Figure 15 contains amplification plots of RT-qPCR detecting the human SFRS9 gene using high temperature RT with unmodified primers and native P.a. RNase H2. Reactions were performed using the HawkZ05TM Fast One-Step RT-PCR Master mix in a single-tube format with unmodified Forward and Reverse PCR primers. The Reverse PCR primer also functioned as the RT primer.
  • the reverse transcription (RT) reaction was done using 20 ng of HeLa cell RNA and proceeded in a stepwise fashion with incubations of 5 minutes at 55°C, 5 minutes at 60°C, and 5 minutes at 65°C followed by a 10 minute denaturation step at 95°C, after which 45 cycles of PCR was performed. Reactions were done without RNase H2 or with 2.6 mU, 25 mU, or 200 mU of native P.a. RNase H2 as indicated.
  • Figure 16 contains amplification plots of RT-qPCR detecting the human SFRS9 gene using high temperature RT with unmodified primers and anhydride- modified HS-P.a. RNase H2. Reactions were performed using the HawkZ05TM Fast One-Step RT-PCR Master mix in a single-tube format with unmodified Forward and Reverse PCR primers. The Reverse PCR primer also functioned as the RT primer.
  • RT reverse transcription
  • Reactions were done without RNase H2 or with 2.6 mU, 25 mU, or 200 mU of 3,4,5,6-tetrahydrophthalic anhydride- modified P.a. RNase H2 as indicated.
  • Figure 17 contains amplification plots of RT-qPCR detecting the human SFRS9 gene using high temperature RT with a blocked-cleavable For PCR primer and anhydride-modified HS-P.a. RNase H2. Reactions were performed using the
  • RT-PCR Master mix in a single-tube format with a blocked- cleavable Forward PCR primer and an unmodified Reverse PCR primer.
  • the Reverse PCR primer also functioned as the RT primer.
  • the reverse transcription (RT) reaction was done using 20 ng of HeLa cell RNA and proceeded in a stepwise fashion with incubations of 5 minutes at 55°C, 5 minutes at 60°C, and 5 minutes at 65°C followed by a 10 minute denaturation/RNase H2 activation step at 95°C, after which 45 cycles of PCR was performed. Reactions were done without RNase H2 or with 2.6 mU, 25 mU, or 200 mU of 3,4,5,6-tetrahydrophthalic anhydride-modified P.a. RNase H2 as indicated.
  • Figure 18 shows amplification products of the SFRS9 gene from high- temperature RT-qPCR using 3,4,5,6-tetrahydrophthalic anhydride-modified P.a. RNase H2 and an unmodified external RT primer. Reactions were performed using the
  • the reverse transcription (RT) reaction was done using 10 ng of HeLa cell RNA and proceeded in a stepwise fashion with incubations of 5 minutes at 55°C, 5 minutes at 60°C, and 5 minutes at 65°C followed by a 10 minute denaturation/RNase H2 activation step at 95°C, after which 45 cycles of PCR was performed. Reactions were done with 10 mU of 3,4,5,6-tetrahydrophthalic anhydride-modified P.a. RNase H2.
  • An unmodified external RT primer was employed at the concentrations indicated. Position of the desired 145 bp amplicon is indicated (made from the For and Rev PCR primers). Position of the undesired 170 bp amplicon is indicated (made from the For PCR primer and the RT primer).
  • Figure 19 shows amplification products of the SFRS9 gene from high- temperature RT-qPCR using 3,4,5,6-tetrahydrophthalic anhydride-modified P. a. RNase H2 and a modified external RT primer containing a central rC RNA residue. Reactions were performed using the HawkZ05TM Fast One-Step RT-PCR Master mix in a single- tube format with unmodified (U) For and Rev PCR primers or blocked-cleavable (B) For and Rev rhPCR primers. The reverse transcription (RT) reaction was done using 10 ng of HeLa cell RNA and proceeded in a stepwise fashion with incubations of 5 minutes at 55°C, 5 minutes at 60°C, and 5 minutes at 65°C followed by a 10 minute
  • Figure 20 shows amplification products of the SFRS9 gene from high- temperature RT-qPCR using 3,4,5,6-tetrahydrophthalic anhydride-modified P. a. RNase H2 and a modified external RT primer containing a central abasic napthyl-azo modifier. Reactions were performed using the HawkZ05TM Fast One-Step RT-PCR Master mix in a single-tube format with unmodified (U) For and Rev PCR primers or blocked- cleavable (B) For and Rev rhPCR primers.
  • the reverse transcription (RT) reaction was done using 10 ng of HeLa cell RNA and proceeded in a stepwise fashion with incubations of 5 minutes at 55°C, 5 minutes at 60°C, and 5 minutes at 65°C followed by a 10 minute denaturation/RNase H2 activation step at 95°C, after which 45 cycles of PCR was performed. Reactions were done with 10 mU of 3,4,5,6-tetrahydrophthalic anhydride-modified P. a. RNase H2.
  • a modified external RT primer containing a single centrally-positioned abasic napthyl-azo modifier was employed at the concentrations indicated. Position of the desired 145 bp amplicon is indicated (made from the For and Rev PCR primers). Position of the undesired 170 bp amplicon is indicated (made from the For PCR primer and the RT primer).
  • compositions and methods of the invention involve modification of an RNase H2 enzyme to make it reversibly inactivated, and become reactivated upon heating.
  • RNase H2 is modified with acid anhydrides to generate a chemically modified hot-start RNase H2 enzyme (HS-RNase H2).
  • the RNase H2 enzyme is from the organism Pyrococcus abyssi (P. a.).
  • RT-PCR reverse-transcription PCR
  • rhPCR PCR
  • DNA synthesis reactions that are dependent on primers cannot occur using blocked-cleavable primers until the primers have been activated by RNase H2 cleavage; certain RNase H2 enzymes, such as P.a. RNase H2, have minimal activity at room temperature. It is therefore possible that rhPCR may perform well using native Taq DNA polymerase, avoiding the need for a costly commercial hot start DNA polymerase; i.e., rhPCR may inherently display hot-start behavior.
  • references to HS-RNase H2 refer to non-native RNase H2.
  • the HS-RNase H2 also can be used in high temperature RT reactions. It may be beneficial to use high-specificity rhPCR (which employs blocked-cleavable primers and RNase H2) to quantify target gene levels in cDNA, which is made from RNA by reverse transcription (RT). RT reactions employ DNA
  • RNA:DNA heteroduplex RNA:DNA heteroduplex
  • RNase H2 activity in an RT reaction could degrade the target RNA, decreasing the efficiency of the reaction.
  • RT-qPCR is often done as a 2-step process, where the RT reaction is first done at low temperature (typically 37-42°C), for example using the avian myeloblastosis virus (AMV) RT enzyme or the Moloney murine leukemia virus (MMLV) RT enzyme.
  • AMV avian myeloblastosis virus
  • MMLV Moloney murine leukemia virus
  • PCR is performed at high temperature (typically 60-72°C). If these reactions are performed in separate tubes, the RNase H2 enzyme can be added after cDNA synthesis is complete. If RT and PCR steps are linked in a single tube, then the RNase H2 must be present during RT and may degrade the RNA target.
  • Example 5 demonstrates an additional advantage of the invention, whereby SNPs can be identified in RNA sequences using the HS-RNase H2 and rhPCR. This can be used in many diverse fields, anywhere that RNA must be analyzed for sequence changes.
  • Example 5 The ability of the HS-RNase H2 to perform in RT-qPCR single-nucleotide polymorphism (SNP) assays is demonstrated in Example 5, where a single nucleotide difference between two different RNA samples is detected using a one -tube RT-PCR system and a single blocked primer with the potential SNP placed opposite the RNA base.
  • SNP single-nucleotide polymorphism
  • the HS-RNase H2 also can be used in high temperature RT reactions, where the activity of the native enzyme would destroy the RNA before it could be reverse- transcribed.
  • This advantage is displayed in examples 9 and 10.
  • Example 9 demonstrates and additional advantage of the invention, whereby SNPs can be identified in RNA sequences using the HS-RNase H2 and rhPCR. This can be used in many diverse fields, anywhere that RNA must be analyzed for sequence changes.
  • the P. a RNase H2 has low activity at 25°C, but this may not be sufficient when long pre-incubation times occur before the rhPCR is performed(i.e. when large numbers of reactions are performed in batch with a robot).
  • the HS-RNase H2 allows for the reversible inactivation of the enzyme to occur, and allows for complete return to functionality when required. An example of this advantage is shown in example 11.
  • nucleic acid and “oligonucleotide,” as used herein, refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides
  • an oligonucleotide also can comprise nucleotide analogs in which the base, sugar or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.
  • Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al, 1979, Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al, 1981, Tetrahedron Lett. 22: 1859-1862; and the solid support method of U.S. Pat. No.
  • hybridization refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing.
  • Hybridization can occur between fully complementary nucleic acid strands or between "substantially complementary” nucleic acid strands that contain minor regions of mismatch.
  • Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”.
  • Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions.
  • nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al, 1989, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).
  • target is synonymous and refer to a region or sequence of a nucleic acid which is to be amplified, sequenced or detected.
  • primer refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product
  • primer extension is intended to encompass the oligonucleotides used in ligation-mediated reactions, in which one oligonucleotide is "extended" by ligation to a second
  • primer extension refers to both the polymerization of individual nucleoside triphosphates using the primer as a point of initiation of DNA synthesis and to the ligation of two oligonucleotides to form an extended product.
  • a primer is preferably a single-stranded DNA.
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from 6 to 50 nucleotides, preferably from 15-35 nucleotides. 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 nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.
  • Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis.
  • primers may contain an additional nucleic acid sequence at the 5' end which does not hybridize to the target nucleic acid, but which facilitates cloning or detection of the amplified product.
  • the region of the primer which is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.
  • Amplification reaction refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid.
  • Amplification reactions include reverse transcription, the polymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al, eds, 1990)), and the ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810).
  • PCR polymerase chain reaction
  • Exemplary "amplification reactions conditions” or “amplification conditions” typically comprise either two or three step cycles. Two step cycles have a high temperature denaturation step followed by a
  • hybridization/elongation (or ligation) step Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation or ligation step.
  • a "polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. Generally, the enzyme will initiate synthesis at the 3'-end of the primer annealed to a nucleic acid template sequence.
  • DNA polymerase catalyzes the polymerization of deoxyribonucleotides.
  • Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al, 1991, Gene, 108: 1), E. coli DNA polymerase I (Lecomte and Doubleday, 1983, Nucleic Acids Res. 11 :7505), T7 DNA polymerase (Nordstrom et al, 1981, J. Biol. Chem.
  • Thermus thermophilus (Tth) DNA polymerase Myers and Gelfand 1991, Biochemistry 30:7661
  • Bacillus stearothermophilus DNA polymerase Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32)
  • Thermococcus litoralis (Tli) DNA polymerase also referred to as Vent DNA polymerase, Cariello et al., 1991, Nucleic Acids Res, 19: 4193
  • Thermotoga maritima (Tma) DNA polymerase Diaz and Sabino, 1998 Braz J. Med.
  • a primer is "specific," for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to the target nucleic acid.
  • a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample.
  • salt conditions such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in many cases.
  • Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence.
  • the use of target-specific primers under suitably stringent amplification conditions enables the selective amplification of those target sequences which contain the target primer binding sites.
  • non-specific amplification refers to the amplification of nucleic acid sequences other than the target sequence which results from primers hybridizing to sequences other than the target sequence and then serving as a substrate for primer extension.
  • the hybridization of a primer to a non-target sequence is referred to as “non-specific hybridization” and is apt to occur especially during the lower temperature, reduced stringency, pre-amplification conditions, or in situations where there is a variant allele in the sample having a very closely related sequence to the true target as in the case of a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • reaction mixture refers to a solution containing reagents necessary to carry out a given reaction.
  • An “amplification reaction mixture” which refers to a solution containing reagents necessary to carry out an amplification reaction, typically contains oligonucleotide primers and a DNA polymerase or ligase in a suitable buffer.
  • a “PCR reaction mixture” typically contains oligonucleotide primers, a DNA polymerase (most typically a thermostable DNA polymerase), dNTP's, and a divalent metal cation in a suitable buffer.
  • reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents.
  • reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture.
  • reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components which includes the blocked primers of the invention.
  • cleavage domain or "cleaving domain,” as used herein, are synonymous and refer to a region located between the 5 ' and 3 ' end of a primer or other oligonucleotide that is recognized by a cleavage compound, for example a cleavage enzyme, that will cleave the primer or other oligonucleotide.
  • a cleavage compound for example a cleavage enzyme
  • the cleavage domain is designed such that the primer or other oligonucleotide is cleaved only when it is hybridized to a complementary nucleic acid sequence, but will not be cleaved when it is single-stranded.
  • the cleavage domain or sequences flanking it may include a moiety that a) prevents or inhibits the extension or ligation of a primer or other oligonucleotide by a polymerase or a ligase, b) enhances discrimination to detect variant alleles, or c) suppresses undesired cleavage reactions.
  • One or more such moieties may be included in the cleavage domain or the sequences flanking it.
  • the term "RNase H cleavage domain,” as used herein, is a type of cleavage domain that contains one or more ribonucleic acid residue or an alternative analog which provides a substrate for an RNase H.
  • An RNase H cleavage domain can be located anywhere within a primer or oligonucleotide, and is preferably located at or near the 3'- end or the 5 '-end of the molecule.
  • RNase HI cleavage domain generally contains at least three residues.
  • An "RNase H2 cleavage domain” may contain one RNA residue, a sequence of contiguously linked RNA residues or RNA residues separated by DNA residues or other chemical groups.
  • the RNase H2 cleavage domain is a 2'- fluoronucleoside residue.
  • the RNase H2 cleavable domain is two adjacent 2'-fluoro residues.
  • cleavage compound refers to any compound that can recognize a cleavage domain within a primer or other oligonucleotide, and selectively cleave the oligonucleotide based on the presence of the cleavage domain.
  • the cleavage compounds utilized in the invention selectively cleave the primer or other oligonucleotide comprising the cleavage domain only when it is hybridized to a substantially complementary nucleic acid sequence, but will not cleave the primer or other oligonucleotide when it is single stranded.
  • the cleavage compound cleaves the primer or other oligonucleotide within or adjacent to the cleavage domain.
  • adjacent means that the cleavage compound cleaves the primer or other oligonucleotide at either the 5 '-end or the 3' end of the cleavage domain. Cleavage reactions preferred in the invention yield a 5 '-phosphate group and a 3' -OH group.
  • the cleavage compound is a "cleaving enzyme.”
  • a cleaving enzyme is a protein or a ribozyme that is capable of recognizing the cleaving domain when a primer or other nucleotide is hybridized to a substantially
  • cleaving enzyme will also not cleave the primer or other oligonucleotide comprising the cleavage domain when it is single stranded.
  • cleaving enzymes are RNase H enzymes and other nicking enzymes.
  • blocking group refers to a chemical moiety that is bound to the primer or other oligonucleotide such that an amplification reaction does not occur. For example, primer extension and/or DNA ligation does not occur.
  • the oligonucleotide is capable of participating in the assay for which it was designed (PCR, ligation, sequencing, etc).
  • the "blocking group” can be any chemical moiety that inhibits recognition by a polymerase or DNA ligase.
  • the blocking group may be incorporated into the cleavage domain but is generally located on either the 5'- or 3 '-side of the cleavage domain.
  • the blocking group can be comprised of more than one chemical moiety.
  • the "blocking group” is typically removed after hybridization of the oligonucleotide to its target sequence.
  • fluorescent generation probe refers either to a) an oligonucleotide having an attached fluorophore and quencher, and optionally a minor groove binder or to b) a DNA binding reagent such as SYBR ® Green dye.
  • fluorescent label refers to compounds with a fluorescent emission maximum between about 350 and 900 nm.
  • fluorophores can be used, including but not limited to: 5-FAM (also called 5- carboxyfluorescein; also called Spiro(isobenzofuran-l(3H), 9'-(9H)xanthene)-5- carboxylic acid,3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6- Hexachloro-Fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-6-carboxylic acid]); 6- Hexachloro
  • quencher refers to a molecule or part of a compound, which is capable of reducing the emission from a fluorescent donor when attached to or in proximity to the donor. Quenching may occur by any of several mechanisms including fluorescence resonance energy transfer, photo-induced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and exciton coupling such as the formation of dark complexes. Fluorescence is
  • quencher when the fluorescence emitted by the fluorophore is reduced as compared with the fluorescence in the absence of the quencher by at least 10%, for example, 15%>, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or more.
  • a number of commercially available quenchers are known in the art, and include but are not limited to DABCYL, Black HoleTM Quenchers (BHQ-1, BHQ-2, and BHQ-3), Iowa Black ® FQ and Iowa Black ® RQ. These are so-called dark quenchers. They have no native fluorescence, virtually eliminating background problems seen with other quenchers such as TAMRA which is intrinsically fluorescent.
  • RNase H2 was chemically modified using 3,4,5,6-tetrahydrophthalic anhydride, citraconic anhydride, or czs-aconitic anhydride, and the chemical
  • the P.a. rnb gene was codon optimized for expression in E. coli and cloned into an expression vector as previously described (Dobosy et al, BMC Biotechnology 2011, l l :e80; Walder et al, US 2009/0325169A1).
  • E. coli bearing the recombinant P.a. RN2 expression plasmid was grown in a 10L fermentation reactor by the University of Iowa Center for Biocatalysis and Bioprocessing (Coralville, IA, USA). The resulting cell paste was stored at -80°C. A fraction of the cell paste (-50 grams) was lysed and the recombinant P.a.
  • RNase H2 enzyme was purified to near homogeneity by Enzymatics (Beverly, MA, USA). Stock solutions of the enzyme were stored in Buffer F (20 mM Tris pH 8.4, 0.1 mM EDTA, 100 mM KC1, 0.1% Triton X-100, and 50% glycerol) at -20°C.
  • SEQ ID NO. 1 Native P.a. RNase H2, 224 amino acids, 25394.18 Daltons
  • the recombinant protein includes some additional amino acids introduced by the expression vector, which are separated from the native enzyme by a vertical bar (
  • the sequence of the recombinant P.a. RNase H2 enzyme is shown below.
  • SEQ ID NO. 2 Recombinant P.a. RNase H2, 246 amino acids, 27573.70 Daltons
  • the recombinant P.a. RNase H2 protein contains 28 lysine residues.
  • a total of 29 free amine groups are therefore available to be modified by chemical reaction with any of the 3 anhydrides.
  • a molar ratio of 29:1 of anhydride: protein therefore represents a 1 : 1 ratio of anhydride to total reactive amines.
  • a "1 : 1 treatment" of P.a. RNase H2 with an anhydride will indicate use of a molar ratio of 29: 1 of anhydride: protein, indicating that sufficient reagent was employed to react with every free amine group, assuming 100% efficiency.
  • Fresh 3,4,5,6-tetrahydrophthalic anhydride, citraconic anhydride, or cz ' s-aconitic anhydride were dissolved in DMF at 40 mM. 1.0 ⁇ , of the 3,4,5,6-tetrahydrophthalic anhydride was added to the first RNase H2 aliquot, 1.0 of the citraconic anhydride was added to the second RNase H2 aliquot, and 1.0 ⁇ ⁇ of the cz ' s-aconitic anhydride was added to the third RNase H2 aliquot. These treatments represent addition of 14.5:1 anhydride to enzyme, or a 0.5:1 molar ratio of anhydride to total amines present in the protein.
  • the bulk anhydride -treated sample was dialyzed into a buffer containing 20 mM Tris pH 8.4, 0.1 mM EDTA and 100 mM KCl using D-TubeTM Dialyzer Mini's (EMD Chemicals Inc., San Diego, CA) with a molecular weight cut-off of 6-8 kDa. Dialysis was performed at 4° C, 3 x 200 mL for 2 hours each, then 1 x 200 mL overnight, replacing with fresh buffer each time. After dialysis, protein concentration was verified by visualization on 4-20% SDS PAGE gels stained with Coomassie
  • DNA bases are uppercase, RNA bases are lowercase, FAM is 6-carboxyfluorescein, and FQ is Iowa Black® FQ dark quencher.
  • the reporter is a self-complementary sequence which forms a hairpin/loop structure with a 19-base stem domain and a 4 base loop domain.
  • the molecule contains a FAM fluorescent dye at the 5 '-end and a dark quencher at the 3 '-end such that dye and quencher are brought into contact upon hairpin formation. In this configuration the fluorescent dye is quenched and the reporter is "dark".
  • a single ribonucleotide (rC) residue is positioned at position 11 from the 5 '-end of the molecule, comprising an RNase H2 cleavage site.
  • the FQ reporter assay was used to compare activity of unmodified P. a. RNase H2 with aliquots previously "removed for later analysis" taken from the bulk enzyme modification reaction above. Reactivation of the anhydride-modified P. a.
  • Reactions were set up as follows: the FQ-reporter assays were done in 10 final volumes using 1 ⁇ of the unmodified and modified enzyme dilutions; for the unmodified enzyme, the amount of enzyme employed corresponds to 200 mU or 2.6 mU of enzyme, respectively.
  • Results for the 2.6 mU assay of cz ' s-aconitic anhydride-modified P.a. RNase H2 are shown in Figure 6. Loss of activity was seen after the first treatment (0.5x modified) however complete inactivation of the enzyme was not achieved until the 2. Ox level of modification. Unlike the results obtained using 3,4,5,6-tetrahydrophthalic anhydride, full activity did not return after heat treatment for 10 minutes at 95°C, even for the 0.5x treated sample. Extending the heat treatment to 15 minutes did not improve results. Results for the 200 mU assay of cz ' s-aconitic anhydride-modified P.a. RNase H2 are shown in Figure 7.
  • Results for the 2.6 mU assay of citraconic anhydride-modified P.a. RNase H2 are shown in Figure 8. Loss of activity was seen after the first treatment (0.5x modified) and complete inactivation of the enzyme was achieved by the l .Ox level of modification. Like the results obtained using czs-aconitic anhydride, full activity did not return after heat treatment for 10 minutes at 95°C, even for the 0.5x treated sample. Extending the heat treatment to 15 minutes did not improve results. Results for the 200 mU assay of citraconic anhydride -modified P.a. RNase H2 are shown in Figure 9.
  • 3,4,5,6-tetrahydrophthalic anhydride showed the most favorable properties and treatment of the enzyme with a 2-fold molar excess of anhydride to free primary amines in the protein totally inactivated enzymatic activity. Further, the chemical modification was reversible with heat treatment at 95°C for 10 minutes.
  • This example illustrates the spectrometry analysis of chemically-modified Pyrococcus abysii RNase H2.
  • the P.a. RNase H2 samples from Example 1 above were studied using electrospray ionization mass spectrometry (ESI-MS) to determine their molecular weights to determine the efficiency of chemical modification (inactivation) and the ability to remove the modifying groups by heat treatment (reactivation). Only the enzyme sample modified 6 times with a 0.5x molar ratio of 3,4,5,6-tetrahydrophthalic anhydride (final 3x molar ratio) was studied.
  • ESI-MS electrospray ionization mass spectrometry
  • Mass spectrometry evaluation of modified P.a. RNase H2 Three samples of recombinant P.a. RNase H2 were prepared for mass spectrometry analysis in dialysis buffer, 20 mM Tris pH 8.4, 0.1 mM EDTA and 100 mM KC1:
  • Table 3 Summary of ESI-MS mass values obtained for P. a. RNase.
  • the unmodified RNase H2 sample displayed the expected mass.
  • the RNase H2 sample modified to a final 3x molar ratio with 3,4,5,6-tetrahydrophthalic anhydride showed a 1000-fold reduction in activity which correlated with modification of a large fraction of the enzyme's primary amine groups.
  • Heat treatment effectively reactivated the enzyme and near full activity was seen following 10 minutes incubation at 95°C. This treatment did not entirely remove all of the modified amine groups; however the reactivated enzyme functioned effectively in all biochemical performance tests performed.
  • the present example demonstrates that rhPCR using the anhydride -modified hot- start P. a. RNase H2 of the present invention performs well using native Taq DNA polymerase (non-hot-start DNA polymerase), even when reactions sit overnight at room temperature prior to commencing cycling, thus eliminating the need for use of a costly hot start DNA polymerase.
  • the assays offer a comparison of PCR using unmodified vs. blocked-cleavable primers, native vs. hot-start Taq DNA polymerase, and native vs. hot- start P.a. RNase H2.
  • Quantitative real-time PCR was performed with 2 ng of human genomic DNA (GM18562, Coriell Institute for Medical Research, Camden, NJ, USA) using primers and a probe specific for a site in the human SMAD7 gene
  • Reactions used either 0.4 U of a hot-start Taq DNA polymerase (iTaqTM, Bio-Rad, Hercules, CA, USA) or native Taq DNA polymerase (Enzymatics, Inc., Beverly, MA, USA). Reactions contained iTaqTM buffer with 3 mM MgCl 2 , 200 nM of each primer, 200 nM of a 5 '-nuclease assay probe (SEQ ID NO. 9), 2 U of SUPERaselnTM RNase inhibitor (Life Technologies, Carlsbad, CA, USA), and 5 fmoles of P.a.
  • RNase H2 (final concentration of 0.5 nM in a 10 reaction, or 2.6 mU of the unmodified enzyme).
  • Either blocked-cleavable primers (SMAD7 For rC blocked, SEQ ID NO. 8 and SMAD7 Rev rG blocked, SEQ ID # 6) or unmodified primers (SMAD7 For, SEQ ID NO. 7 and SMAD7 Rev, SEQ ID # 5) were used. All oligonucleotides used in this study are shown below in Table 4. Reactions were either set up and run immediately or were set up and allowed to incubate at room temperature overnight before PCR cycling was started. Cycling was performed on a Roche
  • LightCycler ® 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 95°C for 10 minutes followed by 45 cycles of 95°C for 10 seconds and 60°C for 30 seconds. All reactions were performed in triplicate. The initial 10 minute incubation at 95°C before thermocycling commences allows for reactivation of the hot-start DNA polymerase (iTaq) and the hot-start (anhydride-treated) P.a. RNase H2 enzymes. The native Taq DNA polymerase and the unmodified P.a. RNase H2 enzymes do not require this activation step, but all reactions were nevertheless run using the same cycling program.
  • RNase H2 top right and anhydride -modified hot start P. a. RNase H2 (bottom right). No amplification occurred when using blocked-cleavable primers if RNase H2 was not added to the reactions (not shown).
  • Figure 14 shows amplification plots obtained using native Taq DNA polymerase.
  • amplification occurred with the expected efficiency and the plots obtained were similar to those seen using a hot start DNA polymerase (not shown).
  • the reaction plates were incubated overnight at room temperature prior to thermocycling, use of unmodified primers (left panels) resulted in no detectable amplification of the target nucleic acid sequence.
  • active DNA polymerase was present with unblocked primers and undesired side reactions occurred at room temperature, consuming reagents and compromising the quality of the subsequent desired amplification reaction.
  • RNase H2 has minimal activity at low temperatures (e.g., 25-45°C) and the reduction in enzyme activity in the conditions used in low temperature RT reactions may be sufficient to allow this enzyme to be present during RT.
  • a 2-step low temperatures e.g. 25-45°C
  • Reverse transcription was performed using 150 ng HeLa cell total RNA in a 15 ⁇ ⁇ reaction with lx first- strand buffer (50 mM Tris-HCl, pH 8.3 at room temperature; 75 mM KCl; 3 mM MgCl 2 ), 0.01 mM DTT, 1 mM dNTPs, 30 U Superscript-II RT, 5 U SUPERase-InTM RNase inhibitor and either 1.3 ⁇ of the TNFRSF 1 A-specific RT primer (SEQ ID NO. 14), 250 ng oligo-dT primer (SEQ ID NO. 16), or 250 ng random hexamer primer (SEQ ID NO. 15).
  • Reactions were run either with or without the addition of 2.6 mU of unmodified recombinant P. a. RNase H2 at 42°C for 60 minutes, followed by a 15 minute RT enzyme inactivation step at 70°C. [0096] Amplification reactions were run using 2 ⁇ ⁇ of each of the above RT reactions (e.g., cDNA made from 20 ng of total cellular RNA).
  • Reactions comprised lx Immolase reaction buffer (16 mM (NH 4 ) 2 S0 4 , 100 mM Tris-HCL pH 8.3, and 0.01% Tween-20), 0.4 U Immolase DNA polymerase (Bioline, Taunton, MA, USA), 3 mM MgCl 2 , 800 ⁇ dNTPs, 200 nM forward and reverse primers (SEQ ID NOs. 11 & 12), and 200 nM probe (SEQ IN NO. 13) in a final 10 ⁇ reaction volume.
  • PCR cycling conditions employed were: 95°C for 5 minutes followed by 45 cycles of 2-step PCR with 95°C for 15 seconds and 60°C for 60 seconds. Reactions were run on a Roche
  • Results of PCR amplification of the TNFRSF1A gene from cDNA made using low temperature RT with and without P. a. RNase H2 are shown in Table 6 below. In the absence of RNase H2, all three RT-primer variations yielded similar results, having Cq values in the 25-26 cycle range. In the presence of RNase H2, the target levels detected in the RT reactions primed using the gene specific primer or random hexamers were nearly identical to the "minus RNase H2" control reactions; however, the RT reaction primed using oligo-dT showed a 2 cycle delay, indicating slightly lower levels of target were present in this RT reaction. Thus the presence of P. a.
  • RNase H2 in an RT reaction performed at 42°C did not adversely affect the level of target cDNA made when the RT primers were located near the PCR assay site (gene specific primer and random hexamers) but did result in a less efficient RT reaction when the RT primer was located 1509 bases from the site of the PCR assay (oligo-dT). Presumably this is due to partial degradation of the RNA in the RNA:DNA heteroduplex present during cDNA synthesis which only impacted the sensitivity of the reaction when long cDNA extension was required. Degradation of the RNA template would increase if the reaction was performed at a higher temperature where the P. a. RNase H2 has higher activity (e.g., 55-65°C).
  • Table 6 Amplification of a cDNA target made using low temperature RT with or without P. a. RNase H2 present.
  • RT can be performed at elevated temperatures using a thermostable reverse transcriptase.
  • High temperature RT methods allow for higher fidelity cDNA synthesis from RNA templates that have complex, stable secondary structures that interfere with the processivity of the DNA polymerase at lower temperatures.
  • One example of this approach employs the HawkZ05TM RT enzyme (Roche Applied Science, Indianapolis, IN, USA).
  • manganese as the divalent cation instead of magnesium
  • this enzyme functions as both a thermostable reverse transcriptase and a DNA polymerase which can support both steps of RT-qPCR.
  • P.a. RNase H2 functions well in the presence of either Mn++ or Mg++ cations and will have good catalytic activity in the reaction conditions employed in this example.
  • Reactions are typically done in a closed- tube format where both the RT and PCR steps are sequentially performed in a single tube. This approach limits the aerosol spread of reaction products that inevitably occurs when reaction tubes are opened to transfer products, thereby reducing the risk of cross- contamination and false-positive reactions, a particularly important feature for molecular diagnostic applications.
  • Amplification efficiency at a site in the human SFRS9 gene was studied using high temperature RT-qPCR without addition of RNase H2, with the addition of native P.a. RNase H2, or with the addition of 3,4,5,6-tetrahydrophthalic anhydride-modified P.a. RNase H2 (see Example 1 above).
  • the anhydride -modified P.a. RNase H2 will also be referred to as the "hot-start RNase H2" or the "HS-RNase H2".
  • Standard methods for single-tube high temperature RT-qPCR employ unmodified primers with a fluorescence-quenched 5 '-nuclease reporter probe; the RT reaction is primed by the reverse PCR primer.
  • the present experiment was done using either unmodified primers (SFRS9 For and Rev, SEQ ID NOs. 17 &18) or with a blocked- cleavable forward primer (SFRS9 For blocked, SEQ ID NO. 19) paired with the unmodified Rev primer (SEQ ID NO. 18).
  • the blocked-cleavable For primer requires activation by RNase H2 to function in PCR.
  • Use of the blocked-cleavable forward primer in place of an unmodified For primer will increase reaction specificity and could be used to selectively amplify one allele if SNP discrimination was desired (see Example 5).
  • the forward primer has no function during the RT phase of the reaction and so does not need to be cleaved (activated) by RNase H2 until the PCR phase of the reaction begins. Oligonucleotide sequences are shown in Table 7 below.
  • RT-qPCR was performed using 20 ng of HeLa cell RNA per 10 reaction with the HawkZ05TM Fast One-Step RT-PCR Master Mix (Roche Applied Science, Indianapolis, IN, USA), 1.5 mM Mn(OAc) 2 , 200 nM primers, and 200 nM probe. 2.6, 25, or 200 mU of unmodified P.a. RNase H2 or the new HS-P.a.
  • the RT phase of the reaction proceeded during the first 15 minutes of incubation which was done stepwise at 55°C for 5 minutes, 60°C for 5 minutes, and 65°C for 5 minutes.
  • the target nucleic acids were then denatured with incubation at 95°C for 10 minutes after which PCR was run for 45 cycles of 92°C for 5 seconds, 60°C for 40 seconds, and 72°C for 1 second.
  • Reactions were run on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) thermocycler. All reactions were performed in triplicate. Note that the 95°C incubation also activates the HS-P.a. RNase H2 enzyme.
  • Results are shown in Figures 15-17.
  • the single-tube high temperature RT-qPCR reaction performed well without RNase H2 present.
  • Addition of even small amounts of native P.a. RNase H2 had a deleterious impact on the reaction ( Figure 15).
  • Addition of 2.6 mU of enzyme shifted the Cq value by -10 cycles, addition of 25 mU of enzyme shifted the Cq value by -14 cycles, and reactions done with 200 mU of enzyme showed no appreciable amplification.
  • P.a. is highly active and most likely degraded the RNA template during the early phase of cDNA synthesis.
  • RNase H2 of the present invention allows for the enzyme to be present during the RT reaction in an inactive state, so cDNA synthesis proceeds normally.
  • the heat denaturation step done after cDNA synthesis activates the modified RNase H2, after which rhPCR can be performed using blocked-cleavable primers. Therefore the higher specificity of rhPCR can be adapted to high-temperature, single-tube RT-qPCR.
  • the following example illustrates the utility of 3,4,5,6-tetrahydrophthalic anhydride-modified P. a. RNase H2 in a RT-qPCR single-nucleotide polymorphism (SNP) assay.
  • SNP single-nucleotide polymorphism
  • a G/T SNP site in the human KRAS gene (NM 004985) was studied using rhPCR and the high-temperature single-tube RT-qPCR HawkZ05TM Fast One-Step RT- PCR Master Mix (Roche Applied Science, Indianapolis, IN, USA) using methods similar to those described in Example 4 above.
  • RT-qPCR was performed using 50 ng HCT- 15 (G/G) or SW480 (T/T) total cellular RNA per 10 ⁇ reaction with HawkZ05TM Fast One-Step RT-PCR Master Mix, 1 mM Mn(OAc) 2 , 200 nM primers, and 200 nM probe. 200 mU of HS- .a. RNase H2 was added to each reaction before RT and PCR were performed. All reactions employed the same unmodified KRAS Rev primer (SEQ ID NO. 21), which served as both the RT primer and the reverse PCR primer. Some reactions paired the KRAS Rev primer with an unmodified non-discriminatory KRAS For primer (SEQ ID NO. 22).
  • KRAS rU For AACTTGTGGTAGTTGGAGCTGuTxxC SEQ ID NO. 24
  • KRAS Probe FAM-AGAGTGCCTTGACGATACAGC- IBFQ SEQ ID NO. 25
  • the RT phase of the reaction proceeded during the first 15 minutes of incubation which was done stepwise at 55°C for 5 minutes, 60°C for 5 minutes, and 65°C for 5 minutes.
  • the target nucleic acids were then denatured with incubation at 95°C for 10 minutes after which PCR was run for 45 cycles of 92°C for 5 seconds, 60°C for 40 seconds, and 72°C for 1 second.
  • Reactions were run on a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA) thermocycler. All reactions were performed in triplicate. Note that the 95°C incubation also activates the HS-P.a. RNase H2 enzyme.
  • Results are shown in Table 9 below. Reactions performed using the unmodified non-discriminatory KRAS For primer showed similar Cq values for both cell lines.
  • the blocked-cleavable KRAS rG primer showed a Cq of 25.9 using HCT-15 DNA (G/G) but was delayed 12.3 cycles to 38.2 using SW480 DNA (T/T).
  • the blocked-cleavable KRAS rU primer showed a delayed Cq of 36.8 using HCT-15 DNA (G/G) and a Cq of 25.9 using SW480 DNA (T/T).
  • This example demonstrates a method to utilize two blocked PCR primers with an external RT primer in one-tube RT-qPCR.
  • the RT primer is modified such that it retains the capacity to prime cDNA synthesis but does not support PCR.
  • Examples 4 and 5 demonstrate that anhydride -modified HS- .a. RNase H2 can be present in high temperature RT reactions and that the inactivated enzyme does not degrade the RNA template during cDNA synthesis.
  • the examples further demonstrate that the enzyme is reactivated with incubation at 95°C for 10 minutes, after which rhPCR can be performed using blocked-cleavable primers.
  • the reverse PCR primer was unmodified and also functioned as a gene-specific RT primer. If additional specificity is desired through use of a blocked-cleavable reverse primer (in place of the unmodified reverse primer), it becomes necessary to add a third primer oligonucleotide to the reaction to function as the RT primer, since the PCR reverse primer is now blocked.
  • the new RT primer is placed 3 '-to the PCR reverse primer.
  • this primer can participate in the PCR reaction, eliminating any specificity improvements gained from use of the blocked-cleavable reverse primer. It is therefore desirable to modify the RT primer so that it can prime cDNA synthesis in the RT reaction but does not participate in subsequent amplification reactions.
  • RT primer having a lower melting temperature (Tm) than the PCR primers so that the RT reaction could, for example, proceed at 60°C while the amplification reaction proceeds at 70°C, i.e., PCR is run at a temperature sufficiently above the Tm of the RT primer that this primer no longer anneals to template.
  • Tm melting temperature
  • the high temperature RT protocol in use in commercial high-temperature RT methods typically involves incubation up to 65°C to disrupt RNA secondary structure, and most PCR reactions are designed with primer annealing to occur at or around 60°C.
  • Tm melting temperature
  • a cleavable linkage is included internally within the RT primer.
  • the RT reaction (cDNA synthesis) is performed with the primer intact, after which a chemical or enzymatic event cleaves the RT primer at the scissile linkage. The remaining primer fragments no longer have sufficient binding affinity to primer further DNA synthesis reactions at the reaction temperatures commonly used in PCR.
  • a variety of approaches can be used to introduce a cleavage site in the primer, which are well known to those with skill in the art, such as linkages susceptible to chemical cleavage, restriction enzyme sites, and the like.
  • a single RNA base is placed at or around the middle of the RT primer.
  • the RNase H2 When using anhydride-modified HS-P.a. RNase H2, the RNase H2 is inactive during cDNA synthesis and the primer functions normally. After cDNA synthesis, the reaction is heated at 95°C for around 10 minutes and the HS- P.a. RNase H2 is reactivated. When the reaction returns to 50-70°C during PCR, the RT primer itself becomes a substrate for RNase H2 attach. The RT primer is cleaved, and the resulting short fragments now have a lowered Tm and cannot participate in amplification reactions in the 50-70°C range. Thus the RT primer serves to prime cDNA synthesis but does not participate in PCR.
  • modifying group is placed at or around the center of the RT primer which does not affect the ability of the oligonucleotide to prime DNA synthesis but which impairs its ability to function as a template for DNA synthesis.
  • linear primer extension reactions are supported (such as cDNA synthesis), but exponential
  • RNA residues e.g., 2'-0-methyl RNA
  • abasic residues aliphatic spaces, d-spacer
  • unnatural bases e.g., 5-nitroindole
  • the present example employs a non-nucleotide napthyl-azo modifier as the blocking group (see Laikhter et al, U.S. Patent No. 8,084,588 and Rose et al, U.S. Patent Application 201 1/0236898), which has the advantage of blocking template function (i.e., inducing chain termination) while not destabilizing hybridization of the modified primer to the target nucleic acid.
  • blocking template function i.e., inducing chain termination
  • Many of the modifying groups which disrupt template function such as aliphatic spacers, d-spacers, and the like, also impair duplex formation (e.g., lower Tm of the primer).
  • RT-qPCR was performed using 10 ng HeLa cell total RNA per 10 ⁇ . reaction with HawkZ05TM Fast One-Step RT-PCR Master Mix (Roche Applied Science, Indianapolis, IN, USA), 1.5 mM Mn(OAc) 2 , 200 nM PCR primers, and 200 nM probe (SFRS9 probe, SEQ ID NO. 20). External RT primers were used at 200 nM, 100 nM, 50 nM, 10 nM, or 0 nM. Either no RNase H2 or 10 mU of 3,4,5,6- tetrahydrophthalic anhydride-modified HS-P.a. RNase H2 was added to each reaction.
  • Amplification was performed using either unmodified PCR primers (SFRS9 For and Rev, SEQ ID NOs. 17 and 18) or blocked-cleavable rhPCR primers (SFRS9 For rG and SFRS9 Rev rA, SEQ ID NOs. 27 and 28).
  • the RT phase of the reaction was performed using either no external RT primer, an unmodified primer (SFRS9-RT, SEQ ID NO. 31), a modified primer having an internal RNA residue (SFRS9-RT-rC, SEQ ID NO. 30), or a modified primer having an internal non-nucleotide napthyl-azo modifier (SFRS9-RT- ZEN, SEQ ID NO. 29). Sequences are shown in Table 10 below.
  • the RT phase of the reaction proceeded during the first 15 minutes of incubation which was done stepwise at 55°C for 5 minutes, 60°C for 5 minutes, and 65°C for 5 minutes.
  • the target nucleic acids were then denatured with incubation at 95°C for 10 minutes after which PCR was run for 45 cycles of 92°C for 5 seconds, 60°C for 40 seconds, and 72°C for 1 second.
  • Reactions were run on a Roche LightCycler ® 480 (Roche Applied Science, Indianapolis, IN, USA) thermocycler. All reactions were performed in triplicate. Note that the 95°C incubation also activates the HS-P.a. RNase H2 enzyme.
  • samples were removed and separated using polyacrylamide gel electrophoresis with an 8% non-denaturing gel and were stained for 10 minutes with lx GelStar ® Nucleic Acid Stain (Lonza, Rockland, ME, USA).
  • Cycle threshold values of the qPCR 5 '-nuclease assay are shown in Table 1 1 below. As expected, reactions done using blocked primer did not amplify in the absence of RNase H2. All other amplification reactions showed relatively similar Cq values, however the amplified products varied significantly between reactions depending on the RT primer employed, as can be seen in the gel images in Figures 18-20.
  • Table 1 1 Cq values for RT-qPCR of a human SFRS9 amplicon comparing different designs for external RT primers Reaction No Hot-start
  • the amplification reactions produced either the desired 145 bp amplicon made from the For and Rev PCR primers (SEQ ID NOs. 17 & 18 or 27 & 28) or an undesired 170 bp amplicon made from the For PCR primer (SEQ ID NOs. 17 or 27) and the RT primer (SEQ ID NOs. 29, 30,or 31).
  • Use of the For and Rev PCR primers without an external RT primer produced only the expected 145 bp amplicon ( Figure 19, "0 nM RT Primer" lanes).
  • the 3,4,5,6-tetrahydrophthalic anhydride-modified HS- .a. RNase H2 permits rhPCR to be performed using blocked For and Rev primers in a single-tube high- temperature RT-qPCR format.
  • Use of an unmodified RT primer results in production of undesired, longer amplification products but use of modified RT primers that can prime RT but cannot participate in PCR results in production of the desired amplicon with high specificity.

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Abstract

Cette invention concerne des améliorations apportées à des dosages qui utilisent le clivage par la RNase H dans des applications biologiques concernant l'amplification et la détection de l'acide nucléique, la RNase H ayant été inactivée de manière réversible.
PCT/US2013/032372 2012-03-19 2013-03-15 Enzymes rnase h modifiées et leurs utilisations WO2013142364A1 (fr)

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JP2015501817A JP2015510776A (ja) 2012-03-19 2013-03-15 修飾ｒｎアーゼｈ酵素及びその使用
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Cited By (17)

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Publication number Priority date Publication date Assignee Title
US10227641B2 (en) 2008-04-30 2019-03-12 Integrated Dna Technologies, Inc. RNase H-based assays utilizing modified RNA monomers
EP2971072A1 (fr) * 2013-03-15 2016-01-20 Integrated DNA Technologies Inc. Essais à base d'arnase-h utilisant de monomères d'arn modifiés
DE102014210092A1 (de) * 2014-05-27 2015-12-03 Siemens Aktiengesellschaft Vorrichtung und Verfahren zur Detektion und Quantifizierung von einzelsträngigen Ziel-Nukleinsäuren
US10011861B2 (en) 2014-08-14 2018-07-03 Luminex Corporation Cleavable hairpin primers
US11371082B2 (en) 2014-08-14 2022-06-28 Luminex Corporation Cleavable hairpin primers
US10683535B2 (en) 2014-08-14 2020-06-16 Luminex Corporation Cleavable hairpin primers
US11926866B2 (en) 2015-11-25 2024-03-12 Integrated Dna Technologies, Inc. Method for detecting on-target and predicted off-target genome editing events
US10886006B2 (en) 2015-11-25 2021-01-05 Integrated Dna Technologies, Inc. Methods for variant detection
US11268117B2 (en) 2016-06-10 2022-03-08 Life Technologies Corporation Methods and compositions for nucleic acid amplification
EP3469078A4 (fr) * 2016-06-10 2020-03-04 Life Technologies Corporation Procédés et compositions d'amplification d'acide nucléique
EP3497227A4 (fr) * 2016-08-09 2020-10-07 Integrated DNA Technologies, Inc. Mutants de rnase h dans une émulsion
US10982273B2 (en) 2016-08-09 2021-04-20 Integrated Dna Technologies, Inc. RNase H mutants in an emulsion
WO2018031625A2 (fr) 2016-08-09 2018-02-15 Integrated Dna Technologies, Inc. Mutants de rnase h dans une émulsion
WO2018190894A1 (fr) * 2017-04-13 2018-10-18 Integrated Dna Technologies, Inc. Procédés de détection de variants
US11338296B2 (en) 2018-07-26 2022-05-24 Lex diagnostics Ltd. Variable temperature reactor, heater and control circuit for the same
CN113278717A (zh) * 2021-06-10 2021-08-20 广州赛哲生物科技股份有限公司 一种靶向测序法检测血流感染的引物池、试剂盒及方法
CN113278717B (zh) * 2021-06-10 2024-04-19 湖南赛哲智造科技有限公司 一种靶向测序法检测血流感染的引物池、试剂盒及方法

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