JP2015510776A - Modified RNase H enzyme and use thereof - Google Patents

Abstract

The present invention provides an improvement over assays using RNase H cleavage for biological applications related to nucleic acid growth and detection, wherein the RNase H is reversibly inactivated.

Description

  The present invention relates to a method of chemically modifying a ribonuclease H (RNase H) enzyme with an acid anhydride for thermoreversible inactivation and uses using the modified enzyme.

  Polymerase chain reaction (PCR) is a ubiquitous method that exponentially amplifies single or minority copies of DNA. Although this technology has been around for 30 years, its use continues to grow and is now integrated into sequencing, functional genomics, diagnostics, forensics and gene expression methods.

  There are currently a number of variants of PCR, many of which are several basic steps: denaturing at high temperature to break hydrogen bonds between complementary bases to form single-stranded target DNA; The step of annealing the primer at a low temperature; and the step of extending the primer using a thermostable polymerase enzyme having optimal activity at 60 ° C. or higher; thereby amplifying the target DNA. PCR is not a perfect system and typically non-specific amplification occurs at low temperatures where thermostable enzymes still have little activity. This shortcoming has prevented many uses of PCR, and a “hot start PCR” method has been devised to reduce or eliminate non-specific growth.

  “Hot start PCR” refers to the use of a method that prevents the initiation of the polymerase chain reaction until the reaction is heated to an elevated temperature, typically 95 ° C. or around, and cooled to a primer annealing temperature, usually 60 ° C. or around. The first hot start PCR method used a physical barrier that could be broken by heating to remove the physical barrier between reaction components. In one early embodiment of this approach, nucleic acids (target DNA, primers, deoxynucleotides and buffer) are separated from the DNA polymerase by a wax seal. When the reaction is heated to 95 ° C., the wax melts and the DNA polymerase and other reaction components can mix, and PCR begins when the reaction is sufficiently cooled to cause primer binding. Currently, hot start PCR usually involves performing with a homogeneous reaction mix that inactivates DNA polymerase that can be reversed by heating in several ways. Examples include chemical modifications (eg, anhydride modification schemes used in the present invention to reversibly inactivate RNase H2), antibodies that bind to DNA polymerase, or aptamers that bind to DNA polymerase Is included. In either case, the substance that limits the DNA polymerase activity is denatured or decomposed by heating.

Hot start Taq DNA polymerases that are reversibly inactivated are typically 5 to 10 times more expensive than unmodified native Taq polymerases. Despite the high cost, hot start PCR is currently used almost exclusively in PCR applications. Using the hot start method, PCR results are improved in two directions:
1) Improved specificity. In the absence of a hot start method, primers can bind to sites in complex nucleic acid samples with incomplete sequence matches at low temperatures and initiate DNA synthesis, which can cause unwanted products to grow. Hot start methods can suppress or prevent this type of mispriming.
2) The reaction can be kept inert at room temperature before PCR cycling begins. For high-throughput screening methods, it is common to set up a large number of PCR plates (including thousands of individual reactions) that are held at room temperature for later loading into a PCR thermocycler by robotic lab equipment. . In the absence of a hot start method, once PCR is finally started, reagents are consumed, and side reactions that depend on DNA polymerase and primers reduce the quality of the PCR.

  Variations on PCR have been developed that utilize other enzymes that are essentially inert at low temperatures and thus limit undesired non-specific growth. One example described in Walder et al. (US patent application 2009/0325169) uses primers that contain a protecting group at or near the 3 'end. This primer cannot extend until the protecting group is cleaved by an RNase H enzyme that has no or little activity at low temperatures.

  RNase H is an endoribonuclease that cleaves phosphodiester bonds in the RNA strand when it is part of an RNA: DNA duplex. This enzyme does not cleave DNA or unhybridized single stranded RNA. This feature makes RNase H useful in biological applications such as cDNA synthesis that degrades the RNA template once the desired complementary DNA is synthesized by reverse transcription.

Anhydride modifications are most commonly used for thermoreversible inactivation when applied to thermostable DNA polymerases such as the general DNA polymerase from Thermus aquaticus (Taq). Widely used to modify proteins (see Birch et al., US Pat. No. 5,773,258). These anhydrides have the formula 1

（式中、Ｒ_１及びＲ_２は水素、または置換もしくは未置換のアルキルまたはアリール基であり、或いはＲ_１及びＲ_２は環状基を形成する）
に示す一般構造を有している。

(Wherein R ₁ and R ₂ are hydrogen, or a substituted or unsubstituted alkyl or aryl group, or R ₁ and R ₂ form a cyclic group)
The general structure shown in FIG.

  Examples of preferred anhydrides include, but are not limited to, citraconic anhydride and 3,4,5,6-tetrahydrophthalic anhydride. Reacting these reagents with RNase H2 protein resulted in reversible inactivation. Anhydrides modify the terminal amine of lysine and the N-terminus of the protein, changing the charge and possibly affecting the protein's conformation (Figure 1). Depending on the type of anhydride used, the removal kinetics will vary (see Walder et al., Mol. Pharmacol, 1977, 13 (3): 407-414), but these protein modifications are sensitive to high temperatures and low pH. Are known to be highly sensitive (see Dixon and Perham, Biochem. J, 1968, 109 (2): 312-314).

  The present invention also provides an improvement over assays that use RNase H cleavage for biological applications related to nucleic acid growth and detection, wherein the RNase H is reversibly inactivated. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

US Patent Application Publication No. 2009/0325169 US Pat. No. 5,773,258

Dixon and Perham, Biochem. J, 1968, 109 (2): 312-314. Walder et al., Mol Pharmacol, 1977, 13 (3): 407-414.

  The present invention also provides an improvement over assays that use RNase H cleavage for biological applications related to nucleic acid growth and detection, wherein the RNase H is reversibly inactivated.

  The use of RNase H, in particular the thermophilic RNase H enzyme, is also implicated in numerous other biological assays (see Walder et al., US Patent Application No. 2009/0325169, which is incorporated herein by reference in its entirety). Is done. The thermophilic RNase H enzyme enables a hot start protocol in nucleic acid proliferation and detection assays, which include a thermostable RNase H or other nicking enzyme that acquires activity at the high temperatures at which the hot start component is used in the reaction. These include, but are not limited to, PCR, OLA (oligonucleotide ligation assay), LCR (ligation chain reaction), polynomial growth and DNA sequencing. The assay uses a modified oligonucleotide of the invention that does not participate in the reaction until it hybridizes to a complementary nucleic acid sequence and is cleaved to yield a functional 5 'or 3' end. The specificity is greatly enhanced compared to the corresponding assay using standard unmodified DNA oligonucleotides. Furthermore, the requirement for reversibly inactivated DNA polymerase or DNA ligase is removed.

There are several alternatives to hot start RNase H: 1) heat resistance with essentially no or little activity at low temperatures as in the case of Pyrococcus abysii RNase H2 2) a thermostable RNase H that is reversibly inactivated by chemical modification; and 3) a thermostable RNase H that is reversibly inactivated by a blocking antibody. In addition, mutant versions of RNase H can be synthesized by means known in the art, such as random mutagenesis, which can further improve the desired RNase H trait in the assays of the invention. Alternatively, mutants of other enzymes that have desirable properties for the present invention may be used. The method of the present invention is mainly directed to the second selection.

Includes the structure of various anhydride compounds useful for protein modification. Figure 2 shows the chemical structure of cis-acotinic anhydride, citraconic anhydride and 3,4,5,6-tetrahydrophthalic anhydride. 2 shows a scheme of reaction of a lysine residue with 3,4,5,6-tetrahydrophthalic anhydride and removal of the anhydride by heat treatment. A reaction scheme for coupling 3,4,5,6-tetrahydrophthalic anhydride to lysine residues resulting in inactivation of the modified enzyme is shown (top). Treatment of this structure with heat or low pH (bottom) reverses the reaction, at which point the unmodified enzyme is reactivated. FIG. 6 shows FQ reporter oligonucleotide assay for RNase H2 activity. Figure 3 shows a fluorescence-quenched hairpin probe assay for RNase H2 activity. DNA bases are upper case, RNA bases are lower case, FAM is 6-carboxyfluorescein, and FQ is Iowa Black® FQ dark quencher. In the intact state, the probe forms a hairpin in which the FAM reporter dye is aligned with the Iowa black dark quencher. With this arrangement, the probe is “dark”. Cleavage of the probe by RNase H2 occurs 5 'to the ribo-C residue. At high reaction temperatures, the cleavage fragment dissociates and the reporter dye separates from the quencher. In this state, the probe is “prite” and a positive signal is detected with 520 nm FAM excitation. 3,4,5,6-tetrahydrophthalic acid modified P.A. using 2.6 mU enzyme a. Figure 3 shows an assay for RNase H2 inactivation and heat reactivation. Unmodified and 3,4,5,6-tetrahydrophthalic acid modified P.I. a. The relative activity of RNase H2 was characterized using the FQ reporter oligonucleotide assay. The assay was performed in a 10 μL reaction with 0.9 nM enzyme (2.6 mU for unmodified enzyme) at 60 ° C. Fluorescence measurements were collected every 11 seconds during a 10 minute incubation. Enzyme is added directly to the reaction (upper panel) or after 10 minutes incubation at 95 ° C. (lower panel) to reverse anhydride modification and reactivate enzyme activity. RFU is a relative fluorescence unit. Modified 3,4,5,6-tetrahydrophthalic acid anhydride using 200 mU enzyme a. Figure 3 shows an assay for RNase H2 inactivation and heat reactivation. Unmodified and 3,4,5,6-tetrahydrophthalic acid modified P.I. a. The relative activity of RNase H2 was characterized using the FQ reporter oligonucleotide assay. The assay was performed in a 10 μL reaction using 67 nM enzyme (200 mU for unmodified enzyme) at 60 ° C. Fluorescence measurements were collected every 11 seconds during a 10 minute incubation. Enzyme is added directly to the reaction (upper panel) or after 10 minutes incubation at 95 ° C. (lower panel) to reverse anhydride modification and reactivate enzyme activity. RFU is a relative fluorescence unit. Anhydrous cis-acotinic acid modified P.A. using 2.6 mU enzyme. a. Figure 3 shows an assay for RNase H2 inactivation and heat reactivation. Unmodified and cis-acotinic acid modified P.I. a. The relative activity of RNase H2 was characterized using the FQ reporter oligonucleotide assay. The assay was performed in a 10 μL reaction with 0.9 nM enzyme (2.6 mU for unmodified enzyme) at 60 ° C. Fluorescence measurements were collected every 11 seconds during a 10 minute incubation. Enzyme is added directly to the reaction (upper panel) or after 10 minutes incubation at 95 ° C. (lower panel) to reverse anhydride modification and reactivate enzyme activity. RFU is a relative fluorescence unit. Anhydrous cis-acotinic acid modified p. a. Figure 3 shows an assay for RNase H2 inactivation and heat reactivation. Unmodified and cis-acotinic acid modified P.I. a. The relative activity of RNase H2 was characterized using the FQ reporter oligonucleotide assay. The assay was performed in a 10 μL reaction using 67 nM enzyme (200 mU for unmodified enzyme) at 60 ° C. Fluorescence measurements were collected every 11 seconds during a 10 minute incubation. Enzyme is added directly to the reaction (upper panel) or after 10 minutes incubation at 95 ° C. (lower panel) to reverse anhydride modification and reactivate enzyme activity. RFU is a relative fluorescence unit. Anhydrous cis-acotinic acid modified P.A. using 2.6 mU enzyme a. Figure 3 shows an assay for RNase H2 inactivation and heat reactivation. Unmodified and cis-acotinic acid modified P.I. a. The relative activity of RNase H2 was characterized using the FQ reporter oligonucleotide assay. The assay was performed in a 10 μL reaction with 0.9 nM enzyme (2.6 mU for unmodified enzyme) at 60 ° C. Fluorescence measurements were collected every 11 seconds during a 10 minute incubation. Enzyme is added directly to the reaction (upper panel) or after 10 minutes incubation at 95 ° C. (lower panel) to reverse anhydride modification and reactivate enzyme activity. RFU is a relative fluorescence unit. 200mU enzyme modified citraconic anhydride modified P.I. a. Figure 3 shows an assay for RNase H2 inactivation and heat reactivation. Unmodified and citraconic anhydride modified P.I. a. The relative activity of RNase H2 was characterized using the FQ reporter oligonucleotide assay. The assay was performed in a 10 μL reaction with 67 nM enzyme (200 mUmU for unmodified enzyme) at 60 ° C. Fluorescence measurements were collected every 11 seconds during a 10 minute incubation. Enzyme is added directly to the reaction (upper panel) or after 10 minutes incubation at 95 ° C. (lower panel) to reverse anhydride modification and reactivate enzyme activity. RFU is a relative fluorescence unit. Figure 2 shows an ESI-MS spectrum of unmodified recombinant Pyrococcus abyssi RNase H2. P. a. RNase H2 was tested by electrospray ionization mass spectrometry (ESI-MS). Shows the mass of the spectrum of the deconvolution trace and shows the molecular weight (Dalton) of the main peak Figure 2 shows an ESI-MS spectrum of recombinant Pyrococcus abyssi RNase H2 modified with 3,4,5,6-tetrahydrophthalic anhydride. P. a. RNase H2 was reacted with a total 3-fold molar excess of 3,4,5,6-tetrahydrophthalic acid and the modified protein was tested by electrospray ionization mass spectrometry (ESI-MS). A deconvolution trace of the mass spectrum is shown, showing the molecular weight (Dalton) of the main peak. Fig. 5 shows an ESI-MS spectrum of recombinant Pyrococcus abyssi RNase H2 modified with 3,4,5,6-tetrahydrophthalic anhydride and then heat-treated. P. a. RNase H2 was reacted with a total 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 tested by electrospray ionization mass spectrometry (ESI-MS). A deconvolution trace of the mass spectrum is shown, showing the molecular weight (Dalton) of the main peak. Shown is a growth plot of qPCR performed after overnight incubation at room temperature with hot start DNA polymerase. The growth reaction was performed using hot start DNA polymerase (iTaq). All reaction components were mixed together and the reaction plate was incubated overnight at room temperature. Using an unmodified primer (left panel) resulted in an efficient proliferative response and natural P. cerevisiae reaction. a. RNase H2 (upper left) and 3,4,5,6-tetrahydrophthalic acid modified hot start P.I. a. There was no difference between the addition of RNase H2 (bottom left). Use of a protected cleavable primer (right panel) resulted in an efficient growth reaction and natural P. cerevisiae reaction. a. RNase H2 (upper right) and 3,4,5,6-tetrahydrophthalic acid modified hot start P.I. a. No difference was seen during the addition of RNase H2 (bottom right). Shows a growth plot of qPCR performed after overnight incubation at room temperature with native (non-hot start) Taq DNA polymerase. Proliferation reactions were performed using native Taq DNA polymerase (non-hot start). All reaction components were mixed together and the reaction plate was incubated overnight at room temperature. Using unmodified primer (left panel) did not result in detectable growth of the target nucleic acid sequence; a. RNase H2 (upper left) and 3,4,5,6-tetrahydrophthalic acid modified hot start P.I. a. Performed with RNase H2 (bottom left). Using protected cleavable primers (right panel), 3,4,5,6-tetrahydrophthalic anhydride modified hot start P.I. a. When RNase H was used, efficient growth of the target nucleic acid occurred (lower right). a. No growth occurred when using RNase H2 (top right). Unmodified primer and natural P.I. a. Figure 6 shows a growth plot of RT-qPCR detecting human SFRS9 gene using high temperature RT using RNase H2. The reaction was performed using a HawkZ05 ™ Fast One-Step RT-PCR master mix in a single tube format with unmodified forward and reverse PCR primers. The reverse PCR primer also functions as an RT primer. A reverse transcription (RT) reaction was performed with 20 ng of HeLa cell RNA, followed by stepwise incubation at 55 ° C. for 5 minutes, 60 ° C. for 5 minutes and 65 ° C. for 5 minutes, followed by 95 ° C. for 10 minutes. A denaturation step followed by 45 cycles of PCR was performed. Reactions were performed without RNase H2 or 2.6 mU, 25 mU or 200 mU native P.I. as indicated. a. Performed with RNase H2. Unmodified primer and anhydride modified HS-P. a. Figure 6 shows a growth plot of RT-qPCR detecting human SFRS9 gene using high temperature RT using RNase H2. The reaction was performed using a HawkZ05 ™ Fast One-Step RT-PCR master mix in a single tube format with unmodified forward and reverse PCR primers. The reverse PCR primer also functions as an RT primer. A reverse transcription (RT) reaction was performed with 20 ng of HeLa cell RNA, followed by stepwise incubation at 55 ° C. for 5 minutes, 60 ° C. for 5 minutes and 65 ° C. for 5 minutes, followed by 95 ° C. for 10 minutes. Denaturation / RNase H2 activation step followed by 45 cycles of PCR. The reaction was carried out in the absence of RNase H2 or 2.6, 25 or 200 mU of 3,4,5,6-tetrahydrophthalic anhydride modified P. a. Performed with RNase H2. Protected cleavable For PCR primers and anhydride modified HS-P. a. Figure 6 shows a growth plot of RT-qPCR detecting human SFRS9 gene using high temperature RT using RNase H2. The reaction was performed using a HawkZ05 ™ Fast One-Step RT-PCR master mix in a single tube format with a protected cleavable forward PCR primer and an unmodified reverse PCR primer. The reverse PCR primer also functions as an RT primer. A reverse transcription (RT) reaction was performed with 20 ng of HeLa cell RNA, followed by stepwise incubation at 55 ° C. for 5 minutes, 60 ° C. for 5 minutes and 65 ° C. for 5 minutes, followed by 95 ° C. for 10 minutes. Denaturation / RNase H2 activation step followed by 45 cycles of PCR. The reaction was carried out in the absence of RNase H2 or 2.6, 25 or 200 mU of 3,4,5,6-tetrahydrophthalic anhydride modified P. a. Performed with RNase H2. 3,4,5,6-tetrahydrophthalic anhydride modified P.I. a. Shown is the growth product of the SFRS9 gene from high temperature RT-qPCR using RNase H and unmodified external RT primer. Reactions performed with HawkZ05 ™ Fast One-Step RT-PCR master mix in single tube format using unmodified (U) For and Rev PCR primers or protected cleavable (B) For and Rev rhPCR primers did. A reverse transcription (RT) reaction was performed using 10 ng of HeLa cell RNA, followed by stepwise incubation at 55 ° C. for 5 minutes, 60 ° C. for 5 minutes and 65 ° C. for 5 minutes, followed by 95 ° C. for 10 minutes. Denaturation / RNase H2 activation step followed by 45 cycles of PCR. The reaction was reacted with 10 mU of 3,4,5,6-tetrahydrophthalic acid modified P.I. a. Performed with RNase H2. Unmodified external RT primer was used at the indicated concentration. The position of the desired 145 bp amplicon (made from For and Rev PCR primers) is shown. The location of the undesired 170 bp amplicon (made from For PCR and RT primers) is indicated. 3,4,5,6-tetrahydrophthalic anhydride modified P.I. a. Shown is the growth product of the SFRS9 gene from high temperature RT-qPCR using a modified external RT primer containing RNase H and a central rC RNA residue. Reactions performed with HawkZ05 ™ Fast One-Step RT-PCR master mix in single tube format using unmodified (U) For and Rev PCR primers or protected cleavable (B) For and Rev rhPCR primers did. A reverse transcription (RT) reaction was performed using 10 ng of HeLa cell RNA, followed by stepwise incubation at 55 ° C. for 5 minutes, 60 ° C. for 5 minutes and 65 ° C. for 5 minutes, then at 95 ° C. for 10 minutes. Denaturation / RNase H2 activation step followed by 45 cycles of PCR. The reaction was reacted with 10 mU of 3,4,5,6-tetrahydrophthalic acid modified P.I. a. Performed with RNase H2. A modified external RT primer containing one centrally located rc RNA residue was used at the indicated concentration. The position of the desired 145 bp amplicon (made from For and Rev PCR primers) is shown. The location of the undesired 170 bp amplicon (made from For PCR and RT primers) is indicated. Control reactions were performed in the absence of external RT primer (0 nM). 3,4,5,6-tetrahydrophthalic anhydride modified P.I. a. FIG. 5 shows the growth product of the SFRS9 gene from high temperature RT-qPCR using a modified external RT primer containing RNase H and a central abasic naphthyl-azo modifier. Reactions performed with HawkZ05 ™ Fast One-Step RT-PCR master mix in single tube format using unmodified (U) For and Rev PCR primers or protected cleavable (B) For and Rev rhPCR primers did. A reverse transcription (RT) reaction was performed using 10 ng of HeLa cell RNA, followed by stepwise incubation at 55 ° C. for 5 minutes, 60 ° C. for 5 minutes and 65 ° C. for 5 minutes, followed by 95 ° C. for 10 minutes. Denaturation / RNase H2 activation step followed by 45 cycles of PCR. The reaction was reacted with 10 mU of 3,4,5,6-tetrahydrophthalic acid modified P.I. a. Performed with RNase H2. A modified external RT primer containing a centrally located abasic naphthyl-azo modifier was used at the indicated concentrations. The position of the desired 145 bp amplicon (made from For and Rev PCR primers) is shown. The location of the undesired 170 bp amplicon (made from For PCR and RT primers) is indicated.

  In one embodiment, the compositions and methods of the invention include modifications of the enzyme to reversibly inactivate the RNase H2 enzyme so that it is reactivated upon heating. RNase H2 is modified with an acid anhydride to produce a chemically modified hot start RNase H2 enzyme (HS-RNase H2). In a further embodiment, the RNase H2 enzyme is derived from the microorganism Pyrococcus abyssi (Pa). The methods described herein also describe the improved utility of HS-RNase H2 in PCR and reverse transcription PCR (RT-PCR) assays.

  The combined use of protected cleavable primers and RNase H2 improves the specificity of PCR (rhPCR). Furthermore, primer-dependent DNA synthesis reactions cannot occur with cleavable primers that are protected until the primer is activated by RNase H2 cleavage; a. Certain RNase H2 enzymes, such as RNase H2, have minimal activity at room temperature. Thus, rhPCR can be performed well with native Taq DNA polymerase without the need to use expensive commercial hot start DNA polymerases; that is, rhPCR can exhibit essentially hot start behavior. In this specification, unless otherwise stated, references to HS-RNase H2 refer to non-natural RNase H2.

  In one embodiment, HS-RNase H2 can also be used in high temperature RT reactions. It is advantageous to use high-specificity rhPCR (using protected cleavable primers and RNase H2) to quantify target gene levels in cDNA generated by reverse transcription (RT) from RNA possible. The RT reaction uses DNA oligonucleotides to prime the synthesis of cDNA from an RNA template. The priming complex forms an RNA: DNA heteroduplex, and the presence of RNase H2 activity in the RT reaction can degrade the target RNA and reduce the efficiency of the reaction. RT-qPCR often first performs the RT reaction at low temperatures (typically 37-42 ° C.) using, for example, avian myeloblastosis virus (AMV) RT enzyme or Moloney murine leukemia virus (MMLV) RT enzyme 2 It is done as a step process. After completion of the cDNA synthesis reaction, PCR is performed at an elevated 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 the RT and PCR steps are related in one tube, RNase H2 must be present in the RT and can degrade the RNA target. Example 5 demonstrates another effect of the present invention that SNPs in RNA sequences can be identified using HS-RNase H2 and rhPCR. This can be used in various fields where RNA must be analyzed for sequence changes.

  It is demonstrated in Example 4 that one-tube RT-PCR can be performed using protected primers and the HS-RNase H2 enzyme. In this example, one protected primer is used with an unprotected reverse primer that acts as both an RT primer and a reverse primer in subsequent PCR.

  It is demonstrated in Example 5 that HS-RNase H2 can function in RT-qPCR single nucleotide polymorphism (SNP) assays. In this example, a single nucleotide difference between two different RNA samples is detected using a one-tube RT-PCR system and one protected primer with a SNP that may be on the opposite side of the RNA base. ing.

  It is demonstrated in Example 11 below that HS-RNase H2 can function in an RT-qPCR assay containing two protected primers and an external unprotected reverse transcription primer.

  HS-RNase H2 can also be used in high temperature RT reactions where the activity of the natural enzyme degrades RNA before it is reverse transcribed. This effect is shown in Examples 9 and 10. Example 9 demonstrates the additional effect of the present invention that SNPs in RNA sequences can be identified using HS-RNase H2 and rhPCR. This can be used in many different fields where RNA must be analyzed for sequence changes.

  P. a. RNase H2 has a low activity at 25 ° C., but this is when there is a long preincubation time before performing the rhPCR (ie when multiple reactions are performed in batches using a robot). It may not be enough. HS-RNase H2 can cause reversible inactivation of the enzyme and can fully restore functionality when needed. An example of this effect is shown in Example 11.

Definitions To assist in understanding the present invention, several terms are defined below.

  As used herein, the terms “nucleic acid” and “oligonucleotide” refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyriboribonucleic acids (containing D-ribose). Refers to nucleotides and other types of polynucleotides that are N-glycosides of purine or pyrimidine bases. The terms “nucleic acid”, “oligonucleotide” and “polynucleotide” are not intended to be distinguished in terms of length, and these terms are used interchangeably. These terms refer only to the main structure of the molecule. Thus, these terms include double stranded and single stranded DNA, as well as double stranded and single stranded RNA. As used herein, oligonucleotides can include 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 made by any suitable method, including those described by Narang et al., 1979, Meth., Each incorporated herein by reference. Enzymol. 68: 90-99; Brown et al., 1979, Meth. Enzymol. , 68: 109-151; Beaucage et al., 1981, Tetrahedron Lett. , 22: 1859-1862; and U.S. Pat. Direct chemical synthesis by methods such as the solid auxiliary method of 4,458,066 is included. A review of methods for synthesizing oligonucleotides and modified nucleotide conjugates is described in Goodchild, 1990, Bioconjugate Chemistry, 1 (3): 165-187, incorporated herein by reference.

  The term “hybridization” as used herein refers to the formation of a duplex structure by two single stranded nucleic acids for complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands containing a minor region of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is highly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be formed under less stringent hybridization conditions; the degree of mismatch allowed can be controlled by appropriately adjusting the hybridization conditions. Those skilled in the art of nucleic acid technology will consider the guidelines provided by the industry (eg, by reference), taking into account a number of variables including, for example, oligonucleotide length and base pair composition, ionic strength and mismatched base pair frequency. Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Biol. 227-259; and Owezarzy et al., 2008, Biochemistry, 47: 5336-5353. It is possible to investigate the duplex stability empirically in accordance with).

  As used herein, the terms “target”, “target sequence”, “target region” and “target nucleic acid” are synonymous and refer to the region or sequence of a nucleic acid to be amplified, sequenced or detected. .

  As used herein, the term “primer” refers to an oligonucleotide that can act as a starting point for DNA synthesis under appropriate conditions. The conditions include the synthesis of a primer extension product complementary to the nucleic acid strand in the presence of four different nucleoside triphosphates and an extender (eg, DNA polymerase or reverse transcriptase) in a suitable buffer. Includes conditions that trigger on temperature. Primer extension can also be performed in the absence of one or more nucleotide triphosphates, in which case a limited length extension product is produced. As used herein, the term “primer” includes an oligonucleotide used in a ligation-mediated reaction in which one oligonucleotide is “elongated” by ligation to a second oligonucleotide that hybridizes at adjacent positions. It is intended. Thus, as used herein, the term “primer extension” refers to the polymerization of individual nucleoside triphosphates using a primer as a starting point for DNA synthesis and the ligation of two oligonucleotides to form an extension product. Refers to both.

  The primer is preferably single-stranded DNA. The appropriate length of the primer depends on the intended use of the primer, but is typically in the range of 6-50 nucleotides, preferably 15-35 nucleotides. Short primer molecules usually require lower temperatures to form a sufficiently stable hybrid complex 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 growth of a given target sequence is known in the art and is described in the literature cited herein.

  A primer may incorporate additional features that allow for detection or immobilization of the primer but do not alter the basic properties of the primer that act as a starting point for DNA synthesis. For example, the primer may not hybridize to the target nucleic acid but may include additional nucleic acid sequences at the 5 'end that facilitate cloning or detection of the amplified product. The region of the primer that is sufficiently complementary to the hybridizing template is referred to herein as the hybridizing region.

  The term “amplification reaction” refers to a chemical reaction that includes an enzymatic reaction that results in a greater copy of the template nucleic acid sequence or that results in transcription of the template nucleic acid. For growth reactions, see reverse transcription, real-time PCR (see US Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (edited by Innis et al., 1990)). Polymerase chain reaction (PCR), and ligase chain reaction (LCR) (see Barany et al., US Pat. No. 5,494,810). Exemplary “amplification reaction conditions” or “amplification conditions” typically comprise two or three step cycles. The two-step cycle includes a high temperature denaturation step followed by a hybridization / extension (or ligation) step. A three-step cycle includes a denaturation step, a hybridization step, and another extension or ligation step in sequence.

As used herein, “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. Usually, this enzyme initiates synthesis at the 3 'end of the primer annealed to the nucleic acid template sequence. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1), E. coli DNA polymerase I (Lecomte and Dobleday, 1983, Nucleic Acids Res., 11). : 7505), T7 DNA polymerase (Nordstrom et al., 1981, J. Biol. Chem., 256: 3112), Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand, 1991, Biochemistry 61:76). ), Bacillus stearothermophilus (Bacillus stearo hermophilus) DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta, 475: 32), hyperthermophilic also called archaea (Thermococcus litoralis) (Tli) DNA polymerase (Vent DNA polymerase, Cariello et al., 1991, Nucleic Acids Res., 19: 4193), Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998, Braz J. Med. Res., 31: 1239), Thermus aquatics ( Thermus aquat ( Thermus aquat ) Taq) DNA polymerase (Chien et al., 1976, J.Bacteoriol, 127:. 1550), hyperthermophilic archaeon (Pyrococcus kodakaraens) KOD DNA polymerase (Takagi et al., 1997, Appl.Environ.Microbiol, 63:. 4504), JDF-3 DNA polymerase (Patent application WO 0132887 ) And Pyrococcus GB-D (PGB-D) DNA polymerase (Juncosa-Ginsta et al., 1994, Biotechniques, 16: 820). The polymerase activity of the enzyme can be measured by means known in the art.

  As used herein, if a primer primarily hybridizes to a target nucleic acid when used in a growth reaction under sufficiently stringent conditions, the primer will be against the target sequence. “Specific”. Typically, a primer is relative to a target sequence if the primer-target duplex stability is higher than the duplex stability formed between the primer and other sequences present in the sample. And specific. Those skilled in the art will appreciate that various factors such as salt conditions and the basic composition of the primer and the location of the mismatch affect the specificity of the primer and that routine experimental confirmation of primer specificity is often necessary. It has recognized. Hybridization conditions can be selected that allow the primer to form a stable duplex only with the target sequence. Thus, when the target-specific primer is used under appropriately stringent growth conditions, the target sequence containing the target primer binding site can be selectively grown.

  As used herein, the term “non-specific growth” refers to the growth of nucleic acid sequences other than the target sequence as a result of the primer hybridizing to a sequence other than the target sequence and acting as a substrate for primer extension. To do. Hybridization of a primer to a non-target sequence is referred to as “non-specific hybridization”, particularly during lower temperatures, lower stringency, pre-growth conditions, or in the case of a single nucleotide polymorphism (SNP) in a sample. This is likely to occur if there are mutant alleles in the sample that have sequences that are very closely related to the true target.

  As used herein, the term “reaction mixture” refers to a solution containing the reagents necessary to carry out a given reaction. An “amplification reaction mixture”, which refers to a solution containing the reagents necessary to perform a growth reaction, typically contains oligonucleotide primers and DNA polymerase or ligase in a suitable buffer. A “PCR reaction mixture” typically contains oligonucleotide primers, DNA polymerase (most typically a thermostable DNA polymerase), dNTPs and a divalent metal cation in a suitable buffer. A reaction mixture is said to be complete if it contains all the necessary reagents to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. One skilled in the art will know that the reaction components are routinely stored as separate solutions, each containing a subset of all components, so that convenience, storage stability reasons, or component concentrations can be adjusted depending on the application. And combining the reaction components to create a complete reaction mixture prior to reaction. Further, those skilled in the art will recognize that the reaction components are packaged separately for commercialization, and that useful commercial kits may contain a subset of reaction components including the protected primers of the present invention. I understand good things.

  As used herein, the terms “cleavage domain” or “cleaving domain” are synonymous and are recognized by a cleaving compound that cleaves a primer or other oligonucleotide, eg, a cleaving enzyme. Refers to the region located between the 5 'and 3' ends of a primer or other oligonucleotide. For the purposes of the present invention, the cleavage domain is designed to cleave only when a primer or other oligonucleotide hybridizes to a complementary nucleic acid sequence, but not when single stranded. The cleavage domain or sequence adjacent to it a) prevents or suppresses the extension or ligation of primers or other oligonucleotides by polymerase or ligase, b) enhances discrimination to detect mutant alleles, or c) It may contain moieties that suppress undesirable cleavage reactions. One or more of the portions may be included in the cleavage domain or sequences adjacent to it.

  The term “RNase H cleavage domain” as used herein is a type of cleavage domain that contains one or more ribonucleic acid residues or other analogs that provide a substrate for RNase H. The RNase H cleavage domain can be located anywhere within the primer or oligonucleotide, and is preferably located at or near the 3 'or 5' end of the molecule.

  An “RNase H1 cleavage domain” usually contains at least 3 residues. An “RNase H2 cleavage domain” may contain single RNA residues, RNA residues linked in series, or RNA residues separated by DNA residues or other chemical groups . In one embodiment, the RNase H2 cleavage domain is a 2'-fluoro nucleoside residue. In a more preferred embodiment, the RNase H2 cleavable domain is two adjacent 2'-fluoro residues.

  The term “cleaving compound” or “cleaving agent” as used herein recognizes a cleavage domain within a primer or other oligonucleotide and selectively cleaves the oligonucleotide based on the presence of the cleavage domain. Refers to the resulting compound. The cleavage compound used in the present invention selectively cleaves a primer or other oligonucleotide containing a cleavage domain only when hybridizing to a substantially complementary nucleic acid sequence, but when it is single stranded Or do not cleave other oligonucleotides. A cleavage compound cleaves a primer or other oligonucleotide within or adjacent to the cleavage domain. As used herein, the term “adjacent” means that the cleavage compound cleaves a primer or other oligonucleotide at either the 5 ′ end or 3 ′ end of the cleavage domain. A preferred cleavage reaction in the present invention yields a 5'-phosphate group and a 3'-OH group.

  In a preferred embodiment, the cleaving compound is a “cleaving enzyme”. A cleavage enzyme can recognize a cleavage domain when a primer or other nucleotide hybridizes to a substantially complementary nucleic acid sequence, but does not cleave the complementary nucleic acid sequence (ie, one in the duplex). A protein or ribozyme that cleaves this strand. Cleavage enzymes do not cleave primers or other oligonucleotides that contain a cleavage domain when single stranded. Examples of cleavage enzymes are RNase H enzyme and other nicking enzymes.

  As used herein, the term “protecting group” refers to a chemical moiety that is attached to a primer or other oligonucleotide such that a growth reaction does not occur. For example, primer extension and / or DNA ligation does not occur. Once the protecting group is removed from the primer or other oligonucleotide, the oligonucleotide can participate in the designed assay (PCR, ligation, sequencing, etc.). Thus, a “protecting group” can be a chemical moiety that prevents recognition by a polymerase or DNA ligase. The protecting group can be incorporated into the cleavage domain, but is usually located 5 'or 3' to the cleavage domain. A protecting group may contain more than one chemical moiety. In the present invention, the “protecting group” is typically removed after hybridizing the oligonucleotide to its target sequence.

  The term “fluorogenic probe” refers to any of a) an attached fluorescent group and quencher, optionally an oligonucleotide with a minor groove binder, or b) a DNA binding reagent such as SYBR® green dye. Point to.

  The term “fluorescent label” or “fluorescent group” refers to a compound having a maximum fluorescence emission of about 350-900 nm. A wide range of fluorescent groups can be used, among which 5-FAM (also referred to as 5-carboxyfluorescein; 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'-pivaloyl-fluoresceinyl) -6-carboxylic acid]); 6-hexachloro-fluorescein; ([4,7,2', 4 ', 5', 7'-hexachloro- (3 ', 6' -Dipivaloylfluoresceinyl) -5-carboxylic acid]); 5-tetrachloro-fluorescein; ([4,7,2 ', 7'-tetrachloro- (3', 6'-dipivalo Ilfluo Seynyl) -5-carboxylic acid]); 6-tetrachloro-fluorescein; ([4,7,2 ′, 7′-tetrachloro- (3 ′, 6′-dipivaloylfluoresceinyl) -6 -Carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine); xanthylium, 9- (2,4-dicarboxyphenyl) -3,6-bis (dimethyl-amino); 6-TAMRA (6-carboxyl) Tetramethylrhodamine); 9- (2,5-dicarboxyphenyl) -3,6-bis (dimethylamino); EDANS (5-((2-aminoethyl) amino) naphthalene-1-sulfonic acid); 5-IAEDANS (5-((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid); Cy5 (indodicarbocyanine-5); y3 (Indo-dicarbocyanine-3); and BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propion Acid); quasar®-670 dye (Biosearch Technologies); Calflow® orange dye (Biosearch Technologies); Rox dye; Max dye (Integrated DNA Technologies), and suitable derivatives thereof, It is not limited to.

  As used herein, the term “quencher” refers to a molecule or portion of a compound that is capable of reducing light emission from a donor when bound to or near a fluorescent donor. Quenching can be by any of several mechanisms including exciton coupling, such as fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and dark complex formation. Can occur. Fluorescence is at least 10%, for example 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, as compared to fluorescence in the absence of a quencher. %, 90%, 95%, 98%, 99%, 99.9% or more are “quenched”. Numerous commercial quenchers are known in the art, including DABCYL, Black HoleTM quenchers (BHQ-1, BHQ-2 and BHQ-3), Iowa Black® FQ and Iowa Black® RQ However, it is not limited to these. These are so-called dark quenchers. They have no natural fluorescence and almost eliminate the background problems seen with other quenchers that are intrinsically fluorescent, such as TAMRA.

  The invention is further illustrated by the following examples, which should, of course, not be construed as limiting the scope of the invention.

[Example 1]
This example demonstrates reversible modification / inactivation of Pyrococcus abysii (Pa) RNase H2 by reaction with various anhydrides.

  P. a. RNase H2 was chemically modified with 3,4,5,6-tetrahydrophthalic anhydride, citraconic anhydride or cis-acotinic anhydride and the chemical modification was removed by heat treatment. The chemical structures of the three anhydrides used in this study are shown in FIG. A chemical reaction in which 3,4,5,6-tetrahydrophthalic anhydride modifies a primary amine on a polypeptide and a reversible reaction catalyzed by heating are shown in FIG. The relative enzyme activity of the unmodified enzyme was compared with that before and after heat treatment of the chemically modified enzyme.

Method:
P. a. The 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, ll: e80; Walder et al., US 2009 / 0325169A1). Recombinant P.P. a. E. coli harboring the RN2 expression plasmid was grown in a 10 L fermentation reactor by University of Iowa Center for Biocatalysis and Bioprocessing (Coralville, Illinois, USA). The resulting cell paste was stored at -80 ° C. Dissolve a portion of the cell paste (~ 50 grams) a. The RNase H2 enzyme was purified to near homogeneity by Enzymatics (Beverly, Mass., USA). The enzyme stock solution was stored in buffer F (20 mM Tris (pH 8.4), 0.1 mM EDTA, 100 mM KCl, 0.1% Triton X-100 and 50% glycerol) at -20 ° C.

  Wild type P.I. a. The sequence of RNase H2 is shown below as SEQ ID NO: 1. The lysine residue (K) is shown in bold and underlined.

  SEQ ID NO: 1 a. RNase H2,224 amino acids, 25394.18 daltons

  The recombinant protein contains several additional amino acids introduced by the expression vector and is separated from the natural enzyme by a vertical line (|). Recombinant P.P. a. The sequence of the RNase H2 enzyme is shown below.

  SEQ ID NO: 2: Recombinant P.I. a. RNase H2,246 amino acids, 27573.70 daltons

  Recombinant P.P. a. The RNase H2 protein contains 28 lysine residues. Thus, a total of 29 free amine groups, including the amino terminus, are available for modification by chemical reaction with any of the three anhydrides. Therefore, P.I. a. Due to the chemical treatment with RNase H2 anhydride, an anhydride: protein molar ratio of 29: 1 corresponds to a 1: 1 ratio of anhydride to total reactive amine. In order to make it easier to understand, P. a. "1: 1 treatment" with RNase H2 anhydride indicates the use of a 29: 1 anhydride: protein molar ratio and is sufficient to react with all free amine groups assuming 100% efficiency. Indicates that the reagent was used.

P. a. Modification of RNase H2 with 3,4,5,6-tetrahydrophthalic anhydride, citraconic anhydride or cis-acotinic anhydride:
By adding 77 μg (864 units) of concentrated stock recombinant enzyme to a buffer containing 74 μL of 150 mM Na borate (pH 9.0) and 0.1% Triton X-100 to a final concentration of 38 μΜ, The same P.I. a. RNase H2 solution was prepared. Note that the reaction was carried out in borate buffer avoiding the Tris containing solution because the anhydride could react with the free amine in Tris to quench the reaction. Fresh 3,4,5,6-tetrahydrophthalic anhydride, citraconic anhydride or cis-acotinic anhydride was dissolved in DMF at 40 mM. 1.0 μL of 3,4,5,6-tetrahydrophthalic anhydride is added to the first RNase H2 aliquot, 1.0 μL of citraconic anhydride is added to the second RNase H2 aliquot, and 1.0 μL of anhydrous cis- Acotinic acid was added to the third RNase H2 aliquot. The three treatments correspond to the addition of a 0.5: 1 molar ratio of 14.5: 1 anhydride: enzyme, ie anhydride to total amine present in the protein. Samples were vortexed and incubated on ice for 30 minutes. After incubation, 1 μL aliquots are removed from each reaction mix and individually 57 μL of Buffer D (20 mM Tris HCl, 0.1 mM EDTA, 100 mM KCl, 0.1% Triton X-100, 10% glycerol, pH 8.4). And stored on ice for subsequent analysis (final concentration 0.67 μΜ). The above procedure is repeated five more times for each anhydride, and the final product is reacted 6 × in a molar ratio of 0.5: anhydride to primary amine to give a molar ratio of anhydride to primary amine of 3 P. causing the accumulation process. a. There were 3 samples of RNase H2. After the 6th anhydride treatment cycle, the reaction was quenched and 18.5 μL of 100 mM Tris HCl (pH 8.4) in 3 samples of modified RNase H2 protein to prevent further chemical modification from occurring. Was added (resulting in a final concentration of 20 mM Tris).

  Bulk anhydride-treated samples were prepared as D-Tube ™ Dialyzer Mini 'with a molecular weight cut-off of 6-8 kDa against a buffer containing 20 mM Tris (pH 8.4), 0.1 mM EDTA and 100 mM KCl. Dialysis was performed using s (EMD Chemicals Inc., San Diego, CA). Dialysis was performed at 4 ° C. at 3 × 200 mL every 2 hours, then overnight at 1 × 200 mL, changing with fresh buffer each time. After dialysis, protein concentration was visualized using a 4-20% SDS PAGE gel stained with Coomassie Brilliant Blue and confirmed by comparison with the band intensity of modified RNase H2 against BSA standards ranging from 100-600 ng. Gel images were quantified using ImageJ band densitometry. The modified enzyme was stored at -20 ° C.

Analysis of modified RNase H2 enzyme activity:
Unmodified and chemically modified P.I. a. The enzymatic activity of RNase H2 was examined using a synthetic fluorescence quenched oligonucleotide reporter assay (FQ reporter assay). The sequence of the synthetic reporter is shown below.

  SEQ ID NO: 3: RNH2 rC FAM reporter

  SEQ ID NO: 4: RNH2 rC competitor

  DNA bases are upper case, RNA bases are lower case, FAM is 6-carboxyfluorescein, and FQ is Iowa Black® FQ dark quencher.

  The reporter is a self-complementary sequence that forms a hairpin / loop structure with a 19-base stem domain and a 4-base loop domain. This molecule contains a FAM fluorescent dye at the 5 'end and a dark quencher at the 3' end so that the dye and quencher are contacted during hairpin formation. In this configuration, the fluorescent dye is quenched and the reporter is “dark”. One ribonucleotide (rC) residue is located at position 11 from the 5 'end of the molecule containing the RNase H2 cleavage site. After cleavage of the reporter molecule with RNase H2, the 10 base 5 'terminal fragment of the molecule dissociates and the fluorescent dye is separated from the quencher. In this arrangement, the dye is not quenched and the reporter is “bright”. A schematic of the RNase H2 activity assay is shown in FIG.

  Using the FQ reporter assay, unmodified P.I. a. The activity of RNase H2 was compared to an aliquot already “taken out for later analysis” taken from the bulk enzyme modification reaction above. Anhydrous modified P.I. a. The reactivation of RNase H2 aliquots was similarly tested. After each cycle of anhydride modification, 1 μl of each reaction was diluted with Buffer D to a final concentration of 667 nM enzyme (equal to 200 mU / μL activity for the unmodified enzyme). These stocks were used at this concentration or further diluted with buffer D to a 9 nM concentration (equal to 2.6 mU / μL activity for the unmodified enzyme). Aliquots of unmodified enzyme and modified enzyme (at 667 nM and 9 nM concentrations) were studied without further treatment or heated at 95 ° C. for 10 minutes prior to activity testing. The reaction was set up as follows: The FQ reporter assay was performed in a final volume of 10 μL with 1 μL of unmodified and modified enzyme dilutions; in the case of unmodified enzyme, the amount of enzyme used was 200 mU or 2. Corresponds to 6 mU enzyme. The components of the FQ reporter assay are listed below in Table 1.

  10 μL of the reaction was incubated with Roche LightCycler® 480 (Roche Applied Science, Indianapolis, Ind.) For 10 minutes in a 384 well plate at 60 ° C., and fluorescence measurements were performed once every 11 seconds. The assay is unmodified P.I. a. Using RNase H2 for all anhydride treated samples (0.5 ×, 1.0 ×, 1.5 ×, 2.0 ×, 2.5 × and 3 × modified) for every three anhydrides To reverse the modification, it was performed before and after the heat treatment at 95 ° C. for 10 minutes. Anhydrous 3,4,5,6-tetrahydrophthalic acid modified P.I. a. The results of the 2.6 mU assay for RNase H2 are shown in FIG. There was a significant loss of activity after the first treatment (0.5 × modification), but no activity was detected in the 1.0 × to 3.0 × treatment. Even in the most highly modified sample (3.0 × modified), complete recovery of enzyme activity was seen after heat treatment at 95 ° C. for 10 minutes. Anhydrous 3,4,5,6-tetrahydrophthalic acid modified P.I. a. The results of the 200 mU assay for RNase H2 are shown in FIG. With this very high concentration of enzyme, 0.5 ×, l. Residual activity was seen in the 0x and 1.5x treated samples, but no activity was detected in the 2.0x, 2.5x or 3.0x treated samples. Similar to the results obtained with 2.6 mU enzyme, even the most highly modified sample (3.0 × modified) showed complete recovery of enzyme activity after heat treatment at 95 ° C. for 10 minutes. .

  Anhydrous cis-acotinic acid modified P.I. a. The results of the 2.6 mU assay for RNase H2 are shown in FIG. There was a loss of activity after the first treatment (0.5 × modification), but no complete inactivation of the enzyme occurred until the 2.0 × level of modification. Unlike the results obtained with 3,4,5,6-tetrahydrophthalic anhydride, even in the case of 0.5 × treated samples, full activity was not recovered after heat treatment at 95 ° C. for 10 minutes. Extending the heat treatment to 15 minutes did not improve the results. Anhydrous cis-acotinic acid modified P.I. a. The results of the 200 mU assay for RNase H2 are shown in FIG. Using this very high concentration of enzyme, enzyme activity was seen in all of the treated samples, indicating that complete inactivation of the enzyme was not achieved using this treatment protocol. As seen in the 2.6 mU assay, none of the 200 mU assay samples recovered to full activity after heat treatment.

  Citraconic anhydride modified P.I. a. The results of the 2.6 mU assay for RNase H2 are shown in FIG. Loss of activity is seen after the first treatment (0.5 × modification) and complete inactivation of the enzyme Achieved with 0 × level of modification. Similar to the results obtained using cis-Acotinic anhydride, complete activity was not recovered after heat treatment at 95 ° C. for 10 minutes even in the case of 0.5 × treated samples. Extending the heat treatment for 15 minutes did not improve the results. Citraconic anhydride modified P.I. a. The results of the 200 mU assay for RNase H2 are shown in FIG. With this very high concentration of enzyme, the enzyme activity is 0.5 ×, l. Although detected in 0x and 1.5x treated samples, complete inactivation was observed for 2.0x to 3.0x treated samples. For the 200 mU assay sample, enzyme activity recovered almost completely after heat treatment, but a slightly slower substrate cleavage was seen at the start of the reaction.

  P. using 3,4,5,6-tetrahydrophthalic anhydride, citraconic anhydride or cis-acotinic anhydride. a. Treatment of RNase H2 reduces enzyme activity, and partial or complete recovery of activity can be achieved with short term incubation at 95 ° C. This enzyme is very thermostable and can be incubated for 30 minutes without significant loss of activity, and an anhydride-based inactivation / reactivation method is suitable for making hot start RNase H2 enzymes Provide an approach. Of the various treatments tested, 3,4,5,6-tetrahydrophthalic anhydride exhibits the most favorable properties, and treatment of the enzyme with a 2-fold molar excess of anhydride relative to the free primary amine in the protein. Totally inactivated the enzyme activity. Furthermore, the chemical modification was reversible upon heat treatment at 95 ° C. for 10 minutes.

[Example 2]
This example shows a spectrophotometric analysis of chemically modified Pyrococcus abysii RNase H2.

  P. from Example 1 above. a. Electrospray ionization mass spectrometry (ESI-MS) to determine the molecular weight of RNase H2 samples to determine the efficiency of chemical modification (inactivation) and the ability to remove modifying groups by heat treatment (reactivation) Was studied. Only enzyme samples modified 6 times with a 0.5 × molar ratio of 3,4,5,6-tetrahydrophthalic anhydride (final 3 × molar ratio) were studied.

Modified P.I. a. Mass spectrometric evaluation of RNase H2:
Recombinant P.P. a. Three samples of RNase H2 were made for mass spectrometry in dialysis buffer, 20 mM Tris (pH 8.4), 0.1 mM EDTA and 100 mM KCl:
1. Unmodified P.I. a. RNase H2 (control)
2. Modification (3 × anhydride treatment) a. RNase H2 (inactive)
3. Modification incubated at 95 ° C. for 10 minutes (3 × anhydride treatment) a. RNase H2 (activity).

  Three samples were tested using electrospray ionization mass spectrometry (ESI-MS) in Novatia, LLC (Princeton, NJ, USA). When each primary amine group in the protein is reacted with 3,4,5,6-tetrahydrophthalic anhydride, the molecular weight increases by 152 Daltons. a. When all 29 amine groups in RNase H2 have reacted, the mass should increase by 4408 Daltons. The expected molecular weights of natural and modified enzymes are shown in Table 2 below.

  Unmodified recombinant P. aeruginosa a. The deconvolved ESI-MS spectrum obtained for RNase H2 is shown in FIG. a. For RNase H2, the heat-treated (reversed) 3 × anhydride-treated P.P. a. RNase H2 is shown in FIG. The mass values of the major spectral peaks identified are summarized in Table 3 below. The unmodified enzyme showed a primary mass of 27,571 Da. The 3x modified enzyme shows 10 mass peaks corresponding to protein species with 19-28 primary amines modified with 3,4,5,6-tetrahydrophthalic anhydride, the most prevalent species being It has 22 modified amines. The heat-treated 3x modified enzyme shows 6 mass peaks corresponding to protein species with 0-5 primary amines modified with 3,4,5,6-tetrahydrophthalic anhydride, the most dominant One species has three modified amines.

  Heat treated anhydride modified P.I. a. RNase H2 shows an approximately 50% reduction in the cleavage rate of the FQ reporter oligonucleotide substrate compared to the unmodified enzyme, which retains 0-5 modified groups on the amine at this sample mass vector. Is related to.

  The unmodified RNase H2 sample exhibited the expected mass. The RNase H2 sample modified with 3,4,5,6-tetrahydrophthalic anhydride to a final 3 × molar ratio showed a 1000-fold reduction in activity, which correlates with the modification of most of the primary amine groups of the enzyme. doing. The heat treatment effectively reactivated the enzyme and almost complete activity was seen after 10 minutes incubation at 95 ° C. This treatment did not completely remove all of the modified amine groups. However, the reactivated enzyme functioned effectively in all biochemical performance tests performed.

[Example 3]
This example shows that even if the reaction is left overnight at room temperature before cycling, the anhydride modified hot start P.P. a. It demonstrates that rhPCR using RNase H2 works well with native Taq DNA polymerase (non-hot start DNA polymerase) and eliminates the need for expensive hot start DNA polymerase. This assay consists of unmodified versus protected cleavable primers, natural versus hot start Taq DNA polymerase, and natural versus hot start P.I. a. Compare PCR with RNase H2.

Method:
Quantitative real-time PCR (qPCR) was performed using the human SMAD7 gene (rs4939827, NM). 005904) was performed with 2 ng of human genomic DNA (GM18562, Coriell Institute for Medical Research, Camden, NJ, USA) using probes and primers specific for the sites in FIG. The reaction used either 0.4 U hot start Taq DNA polymerase (iTaq ™, Bio-Rad, Hercules, Calif., USA) or native Taq DNA polymerase (Enzymes, Inc., Beverly, Mass., USA). Reactions include 3 mM MgCl ₂ , 200 nM of each primer, 200 nM 5′-nuclease assay probe (SEQ ID NO: 9), 2 U SUPERaseln ™ RNase inhibitor (Life Technologies, Carlsbad, Calif., USA) and 5 fmoles P.I. a. ITaq ™ buffer containing RNase H2 (0.5 nM final concentration in 10 μL reaction, or 2.6 mU unmodified enzyme) was included. Protectable cleavable primer (protected SMAD7 For rC, SEQ ID NO: 8 and protected SMAD7 Rev rG, SEQ ID NO: 6) or unmodified primer (SMAD7 For, SEQ ID NO: 7 and SMAD7 Rev, SEQ ID NO: 5) was used. All oligonucleotides used in this study are listed below in Table 4. Reactions were set up and run immediately or set up and incubated overnight at room temperature before starting PCR cycling. Cycling was performed using a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, Ind.) As follows: 95 ° C. for 10 minutes, 95 ° C. for 10 seconds and 60 ° C. for 30 seconds. 45 cycles. All reactions were performed three times. Initial incubation at 95 ° C. for 10 minutes before initiating thermocycling resulted in hot start DNA polymerase (iTaq) and hot start (anhydride treatment) a. The RNase H2 enzyme can be reactivated. Natural Taq DNA polymerase and unmodified P.I. a. The RNase H2 enzyme does not require this activation step, but all reactions were nevertheless performed using the same cycling program.

result:
When PCR is performed using hot start DNA polymerase, the growth reaction can be performed with the same efficiency whether it is performed immediately after setting up the reaction (not shown) or incubated overnight at room temperature before thermocycling. Progressed. FIG. 13 shows a growth plot obtained with hot start DNA polymerase iTaq after overnight incubation at room temperature. When an unmodified primer (left panel) was used, an efficient growth reaction occurred, and natural P. cerevisiae reaction occurred. a. RNase H2 (upper left) and anhydride modified hot start P.I. a. There was no difference between the addition of RNase H2 (bottom left). Using a protected cleavable primer (right panel) also resulted in an efficient proliferative response, and natural P. cerevisiae reaction. a. RNase H2 (upper right) and anhydride modified hot start P.I. a. There was no difference between the addition of RNase H2 (bottom right). If RNase H2 was not added to the reaction, growth did not occur using protected cleavable primers (not shown).

  FIG. 14 shows a growth plot obtained using native Taq DNA polymerase. When the reaction was run immediately after mixing all reaction components, growth occurred with the expected efficiency and the resulting plot was similar to that obtained with hot start DNA polymerase (not shown). In contrast, when the reaction plate was incubated overnight at room temperature before thermocycling, unmodified primer (left panel) resulted in undetectable growth of the target nucleic acid sequence. In this case, active DNA polymerase is present together with unprotected primers, and undesirable side reactions occur at room temperature, consuming reagents and reducing the quality of subsequent desired growth reactions. Using protected cleavable primers (right panel), anhydride modified hot start P.P. a. When RNase H2 was used, efficient growth of the target nucleic acid occurred (lower right). a. This was not the case when RNase H2 was used (upper right). Therefore, P.I. a. Even though RNase H2 has little activity at room temperature, sufficient activity is maintained to require a modified hot start version of the enzyme when rhPCR is performed under these conditions.

  In a growth reaction at room temperature, undesirable reactions occur that depend on the presence of active DNA polymerase and primers in the reaction. These reactions consume reaction components and reduce the efficiency and quality of the desired growth reaction. The use of expensive hot start DNA polymerase can eliminate these artifacts. Alternatively, the use of rhPCR and protected cleavable primers and anhydride modified hot start RNase H2 enzyme can result in an efficient and specific growth reaction.

[Example 4]
The following examples show 3,4,5,6-tetrahydrophthalic anhydride modified hot start P.A. in a single tube high temperature RT-qPCR reaction. a. The use of RNase H2 will be described.

P. a. RNase H2 has minimal activity at low temperatures (eg, 25-45 ° C.), and a reduction in enzyme activity at the conditions used in the low temperature RT reaction may be sufficient to allow the enzyme to be present in RT. A two-step cold RT reaction was performed using an internal gene specific primer, a random hexamer primer or an oligo dT primer. a. Performed with or without RNase H2. After cDNA synthesis, qPCR was converted to human tumor necrosis factor receptor superfamily member 1A (TNFRSF1A, NM 001065) in order to amplify the 157 bp region. The primers, probes, and target nucleic acid sequences used are shown in Table 5 below. Note that the PCR assay places 1509 bases 5 'to the poly A chain site of this gene.

  The For (forward) and Rev (reverse) priming sites in the TNFRSF1A target nucleic acid sequence are underlined. Primer binding sites for gene-specific RT primers are shown in italics and are also underlined. FAM = 6-carboxyfluorescein and IBFQ = Iowa Black® FQ fluorescent quencher.

Reverse transcription was performed using 1 × 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 dNTP, 30 U Superscript-II RT, 5U SUPERase-In ™ ) In a 15 μL reaction containing RNase inhibitor and 1.3 μM TNFRSF1A 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) At 150 ng of HeLa cell total RNA. The reaction was performed with 2.6 mU of unmodified recombinant P. p. a. An RT enzyme inactivation step was performed at 70 ° C. for 15 minutes after 60 minutes at 42 ° C. with or without the addition of RNase H2.

An amplification reaction was performed using 2 μL of each of the RT reaction products (for example, cDNA prepared from 20 ng of total cellular RNA). The reaction was in a final 10 μL reaction volume in 1 × Imolease reaction buffer (16 mM (NH ₄ ) ₂ SO ₄ , 100 mM Tris HCl (pH 8.3) and 0.01% Tween-20), 0.4 U Imorase DNA polymerase (Bioline , Taunton, Mass., USA), 3 mM MgCl ₂ , 800 μM dNTP, 200 nM forward and reverse primers (SEQ ID NOs: 11 and 12) and 200 nM probe (SEQ ID NO: 13). The PCR cycling conditions used were 45 cycles of 2-step PCR with 95 ° C. for 5 minutes followed by 95 ° C. for 15 seconds and 60 ° C. for 60 seconds. The reaction was performed using a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN) thermocycler. All reactions were performed three times. The quantification cycle value (Cq) was determined using the absolute quantification / second derivative method.

  P. at low temperature RT. a. The results of PCR propagation of the TNFRSF1A gene from cDNA made with or without RNase H2 are shown in Table 6 below. In the absence of RNase H2, all three RT-primer variations produced similar results with Cq values in the 25-26 cycle range. In the presence of RNase H2, the target levels detected in the RT reaction primed with gene specific primers or random hexamers were nearly identical to the control reaction “without RNase H2”; however, oligo dT The RT reaction primed with 2 showed a two cycle delay, indicating that only a slightly lower level of target was present in this RT reaction. Thus, during the RT reaction carried out at 42 ° C. a. The presence of RNase H2 did not adversely affect the level of target cDNA created when the RT primer was placed close to the PCR assay site (gene specific primer and random hexamer), but the RT primer was When placed at 1509 bases from the PCR assay site (oligo dT), a less efficient RT reaction occurred. Perhaps this is due to the partial degradation of the RNA in the DNA: DNA heteroduplex present during cDNA synthesis that affected the sensitivity of the reaction when long cDNA extension was required. The reaction a. If performed at higher temperatures (eg, 55-65 ° C.) where RNase H2 has higher activity, degradation of the RNA template will increase.

RT can be performed at high temperature using thermostable reverse transcriptase. The high temperature RT method allows more faithful cDNA synthesis from RNA templates that have complex and stable secondary structures that interfere with DNA polymerase processing capacity at low temperatures. An example of this approach uses the HawkZ05 ™ RT enzyme (Roche Applied Science, Indianapolis, IN, USA). When manganese is used instead of magnesium as the divalent cation, this enzyme functions as both a thermostable reverse transcriptase and a DNA polymerase that can assist in both steps of RT-qPCR. P. a. Note that RNase H2 works well in the presence of either Mn ⁺⁺ or Mg ⁺⁺ cations and has good catalytic activity in the reaction conditions used in this example. The reaction is typically performed in a closed tube format in which both RT and PCR steps are performed sequentially in a single tube. This approach limits the airborne transmission of the reaction product that inevitably occurs when the reaction tube is opened to move the product, thereby preventing cross-contamination and false positive reactions, which are particularly important features for molecular diagnostic applications. To reduce.

Human SFRS9 gene (NM [003769) The amplification efficiency at the site in FIG. a. RNase H2 or 3,4,5,6-tetrahydrophthalic acid modified P.I. a. RNase H2 (see Example 1 above) was added and studied using high temperature RT-qPCR. Anhydrous modification a. RNase H2 is also referred to as “hot start RNase H2” or “HS-RNase H2”. Standard methods for single tube high temperature RT-qPCR use unmodified primers and a fluorescence quenched 5′-nuclease reporter probe; the RT reaction is primed with reverse PCR primers. This experiment was performed using unmodified primers (SFRS9 For and Rev, SEQ ID NO: 17 and 18), or unmodified Rev primer (SEQ ID NO: 18) and protected cleavable forward primer (protected SFRS9 For, sequence No. 19) was used. Note that the protected cleavable For primer requires activation by RNase H2 to function in PCR. Using protected cleavable forward primers instead of unmodified For primers increases reaction specificity and can be used to selectively amplify one allele if SNP discrimination is desired. (See Example 5). Note that the forward primer does not function during the RT phase of the reaction and does not need to be cleaved (activated) by RNase H2 until the PCR phase of the reaction begins. The oligonucleotide sequences are shown in Table 7 below.

RT-qPCR was performed at 20 ng per 10 μL of reaction containing HawkZ05 ™ Fast One-Step RT-PCR master mix (Roche Applied Science, Indianapolis, Ind.), 1.5 mM Mn (OAc) ₂ , 200 nM primer and 200 nM probe. Of HeLa cell RNA. 2.6, 25 or 200 mU unmodified P.I. a. RNase H2 or novel HS-P. a. RNase H2 was added; a control reaction without RNase H2 was also performed. Reactions in 5′-nuclease assay format with unmodified primers (SFRS9 For and SFRS9 Rev, SEQ ID NOs: 17 and 18), or protected forward and unmodified reverse primers (protected SFRS9 For and SFRS9 Rev) , SEQ ID NO: 19 and 18) were used with the SFRS9 probe (SEQ ID NO: 20).

  The RT phase of the reaction proceeded during the first 15 minutes of the incubation which was performed stepwise at 55 ° C for 5 minutes, 60 ° C for 5 minutes and 65 ° C for 5 minutes. Then, after incubating at 95 ° C. for 10 minutes to denature the target nucleic acid, PCR was performed for 45 cycles of 92 ° C. for 5 seconds, 60 ° C. for 40 seconds and 72 ° C. for 1 second. The reaction was performed using a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN) thermocycler. All reactions were performed three times. Incubation at 95 ° C was also performed using HS-P. a. Note the activation of the RNase H2 enzyme.

  The results are shown in FIGS. In the case of the reaction performed using the unmodified primer, the single tube high temperature RT-qPCR reaction was sufficiently performed without RNase H2. However, natural P.I. a. The addition of RNase H2 has a detrimental effect on the reaction (Figure 15). The addition of 2.6 mU enzyme changed the Cq value by -10 cycles, the addition of 25 mU enzyme changed the Cq value by -14 cycles, and reactions performed with 200 mU enzyme showed no apparent growth. At the reaction temperature used for the RT reaction (55-65 ° C.). a. Is very active and probably degrades the RNA template during the initial phase of cDNA synthesis. In contrast, anhydride-modified HS-P. a. Reactions performed with RNase H2 showed SFRS9 target proliferation with similar efficiency as reactions performed in the absence of RNase H2 (FIG. 16). In this case, there was no difference between the reaction performed without RNase H2 and the reaction performed with 2.6-200 mU RNase H2, even when the modified RNase enzyme was performed at 55-65 ° C. It was confirmed that it was fully inactivated and did not degrade the RNA target during cDNA synthesis. The results of a reaction performed with protected cleavable For PCR primers and unmodified Rev PCR primers are shown in FIG. Consistent with the need for cleavage / activation of the protected primer, no growth was seen in the absence of RNase H2. 3,4,5,6-tetrahydrophthalic anhydride modified HS-P. a. Using RNase H2, the reaction with 25 mU or 200 mU of enzyme showed the same growth efficiency as that seen with the unmodified primer. Reactions performed with 2.6 mU enzyme showed a delay of approximately 4 cycles, indicating that the reaction conditions did not result in complete primer cleavage.

  In a high temperature RT-qPCR reaction, the a. Even in the presence of RNase H2, RNA is degraded during cDNA synthesis and growth is prevented. The novel anhydride modified P.I. a. Using RNase H2, the enzyme can be present in an inactive state during the RT reaction, thus cDNA synthesis proceeds normally. A heat denaturation step performed after cDNA synthesis activates the modified RNase H2, and then rhPCR can be performed with a cleavable primer that is protected. Thus, the higher specificity of rhPCR can be adapted to high temperature single tube RT-qPCR.

[Example 5]
The following examples show 3,4,5,6-tetrahydrophthalic anhydride modified P.D. in RT-qPCR single nucleotide polymorphism (SNP) assay. a. The usefulness of RNase H2 will be described. The assay of this example demonstrates the discrimination of KRAS SNP by single tube RT-rhPCR.

Human KRAS gene (NM The G / T SNP site in Fig. 004985) was added to Example 4 above using rhPCR and high temperature single tube RT-qPCR HawkZ05 ™ fast one-step RT-PCR master mix (Roche Applied Science, Indianapolis, Ind.). Studies were performed using methods similar to those described.

RT-qPCR was performed using HawkZ05 ™ Fast One Step RT-PCR master mix, 50 ng HCT-15 (G / G) or SW480 (T) per 10 μL reaction containing 1 mM Mn (OAc) ₂ , 200 nM primer and 200 nM probe. / T) Performed with total cellular RNA. Before performing RT and PCR, each reaction contained 200 mU of HS-p. a. RNase H2 was added. All reactions used the same unmodified KRAS Rev primer (SEQ ID NO: 21), which served as both the RT primer and the reverse PCR primer. In some reactions, the KRAS Rev primer was paired with an unmodified promiscuous KRAS For primer (SEQ ID NO: 22). In other reactions, the KRAS Rev primer was cleavable primer (SEQ ID NO: 23) protected with G-SNP-discriminating KRAS rG For or cleavable primer (SEQ ID NO: 24) protected with T-SNP-discriminating KRAS rU For. ). The same fluorescence quenched probe (SEQ ID NO: 25) was used as a 5′-nuclease assay reporter in all assays. The primers and probes used in Example 5 are shown in Table 8 below.

  The RT phase of the reaction proceeded during the first 15 minutes incubation, stepped at 55 ° C for 5 minutes, 60 ° C for 5 minutes and 65 ° C for 5 minutes. Then, after incubating at 95 ° C for 10 minutes to denature the target nucleic acid, PCR was performed for 45 cycles of 92 ° C for 5 seconds, 60 ° C for 40 seconds and 72 ° C for 1 second. The reaction was performed using a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, IN) thermocycler. All reactions were performed three times. Incubation at 95 ° C was also performed using HS-P. a. Note that it activates the RNase H2 enzyme.

  The results are shown in Table 9 below. Reactions performed with unmodified, promiscuous KRAS For primer showed similar Cq values for both cell lines. The protected cleavable KRAS rG primer showed a Cq of 25.9 using HCT-15 DNA (G / G), while 12.3 cycles were 38. 3 using SW480 DNA (T / T). Delayed to 2. Conversely, the protected cleavable KRAS rU primer shows 36.8 delayed Cq with HCT-15 DNA (G / G) and 25.9 Cq with SW480 DNA (T / T). It was.

  Anhydride modified HS-P. a. With RNase H2, a very accurate SNP rhPCR assay can be performed in a single tube high temperature RT-qPCR format, demonstrating the utility of the modified enzyme used in the method of the invention.

[Example 6]
This example demonstrates how to use two protected PCR primers in conjunction with an external RT primer in a one-tube RT-qPCR. In order to eliminate the possibility that growth products are derived from RT primers, RT primers retain the ability to prime cDNA synthesis but have been modified to not aid PCR.

  Examples 4 and 5 are anhydride modified HS-P. a. It has been demonstrated that RNase H2 can be present during high temperature RT reactions and that the inactivating enzyme does not degrade the RNA template during cDNA synthesis. This example further demonstrates that the enzyme can be reactivated by incubation at 95 ° C. for 10 minutes, after which rhPCR can be performed using protected cleavable primers. In these examples, the reverse PCR primer was not modified and also functioned as a gene-specific RT primer. If additional specificity is desired using a protected cleavable reverse primer (instead of an unmodified reverse primer), the PCR reverse primer is protected at this point, so as an RT primer It is necessary to add a third primer oligonucleotide to the reaction in order to function. A new RT primer is placed 3 'to the PCR reverse primer. However, because it is not modified, this primer can participate in the PCR reaction, eliminating the improved specificity gained by the use of a protected cleavable reverse primer. Thus, although it is possible to prime cDNA synthesis in an RT reaction, it is desirable to modify the RT primer so that it does not participate in subsequent growth reactions. One approach is to create an RT primer that has a lower melting point (Tm) than the PCR primer so that the RT reaction can proceed at, for example, 60 ° C., but the growth reaction proceeds at 70 ° C. That is, PCR is performed at a temperature well above the RT primer Tm, which primer no longer anneals to the template. However, the high temperature RT protocol used in commercial high temperature RT methods typically includes incubation at up to 65 ° C to disrupt RNA secondary structure, so that primer annealing occurs at or near 60 ° C. The PCR reaction is designed. Although different Tm can be used for RT vs. PCR primers. This method must be redesigned to operate PCR primers and reactions at high temperatures. This example demonstrates the use of modified RT primers at standard reaction temperatures, where RT primers are capable of priming cDNA synthesis but are not involved in subsequent growth reactions.

  Two modification strategies have been described that achieve the same objective. First, a cleavable linkage is contained within the RT primer. The RT reaction (cDNA synthesis) is performed with a primer intact, after which the RT primer is cleaved with a linkage that is susceptible to cleavage of chemical or enzymatic events. The remaining primer fragments no longer have sufficient binding affinity to prime further DNA synthesis reactions at the reaction temperatures normally used in PCR. Various approaches known to those skilled in the art can be used to introduce a cleavage site into the primer, such as linkages susceptible to chemical cleavage, restriction enzyme sites, and the like. In this example, a single RNA base is placed at or around the center of the RT primer. Anhydride modified HS-P. a. When RNase H2 is used, RNase H2 is inactive during cDNA synthesis and the primers function normally. After cDNA synthesis, the reaction was heated at 95 ° C. for about 10 minutes, and HS-P. a. Reactivate RNase H2. When the reaction is returned to 50-70 ° C. during PCR, the RT primer itself becomes a substrate for RNase H2 attachment. The RT fragment is cleaved and the resulting short fragment has a low Tm at this point and cannot participate in the growth reaction in the range of 50-70 ° C. Thus, RT primers help to prime cDNA synthesis but are not involved in PCR.

  Second, the modifying group is placed at or near the center of the RT primer that does not affect the ability of the oligonucleotide to prime DNA synthesis, but does affect the ability to function as a template for DNA synthesis. Thus, a linear primer extension reaction (eg, cDNA synthesis) is assisted but an exponential growth reaction is prevented; during subsequent growth cycles, the extension product stops prematurely at the primer protecting group site, resulting in The final growth product is shortened and does not contain a primer binding site long enough for the RT primer to bind at reaction temperatures in the range of 50-70 ° C. Various protecting groups that can serve this purpose, such as 2'-modified RNA residues (eg 2'-O-methyl RNA), abasic residues (aliphatic spacer, d-spacer), unnatural bases (eg 5 -Nitroindole) (see Behlke et al., U.S. Patent Nos. 7,112,406 and 7,629,152) are known to those skilled in the art. This example serves as a protecting group a non-nucleotide naphthyl-azo modifier that has the advantage of blocking template function (ie, inducing a chain termination reaction) but not destabilizing the hybridization of the modified primer to the target nucleic acid (See Laikter et al., US Pat. No. 8,084,588 and Rose et al., US Patent Application 2011/0236898). Many of the modifying groups that break the template function, such as aliphatic spacers, d-spacers, etc. also affect duplex formation (eg, lower Tm of primers).

Method:
RT-qPCR was performed using HawkZ05 ™ Fast One-Step RT-PCR Master Mix (Roche Applied Science, Indianapolis, Ind.), 1.5 mM Mn (OAc) ₂ , 200 nM PCR primer and 200 nM probe (SFRS9 probe, SEQ ID NO: 20) was performed using 10 ng of HeLa cell total RNA per 10 μL of the reaction. External RT primers were used at 200 nM, 100 nM, 50 nM, 10 nM or 0 nM. No RNase H2 was added to each reaction or 10 mU 3,4,5,6-tetrahydrophthalic acid modified HS-P. a. RNase H2 was added. Growth is performed using either unmodified PCR primers (SFRS9 For and Rev, SEQ ID NOs: 17 and 18) or protected cleavable rhPCR primers (SFRS9 For rG and SFRS9 Rev rA, SEQ ID NOs: 27 and 28). did. RT phase of reaction without external RT primer or unmodified primer (SFRS9-RT, SEQ ID NO: 31), modified primer with internal lRNA residue (SFRS9-RT-rC, SEQ ID NO: 30), or internal non-nucleotide naphthyl -Performed using a modified primer with an azo modifier (SFRS9-RT-ZEN, SEQ ID NO: 29). The sequence is shown in Table 10 below.

  The RT phase of the reaction proceeded during the first 15 minutes of the incubation which was performed stepwise at 55 ° C for 5 minutes, 60 ° C for 5 minutes and 65 ° C for 5 minutes. The target nucleic acid was then denatured by incubating at 95 ° C. for 10 minutes, after which PCR was performed for 45 cycles of 92 ° C. for 5 seconds, 60 ° C. for 40 seconds and 72 ° C. for 1 second. The reaction was performed using a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, Ind.) Thermocycler. All reactions were performed three times. Incubation at 95 ° C was also performed using HS-P. a. Note that it activates the RNase H2 enzyme. After growth, samples were removed, separated using polyacrylamide gel electrophoresis on an 8% non-denaturing gel, and stained for 10 minutes with 1 × GelStar® Nucleic Acid Stain (Lonza, Rockland, Maine, USA). . The product was visualized by fluorescence with UV excitation.

  The cycle thresholds for the qPCR 5'-nuclease assay are shown in Table 11 below. As expected, reactions performed with protected primers did not amplify in the absence of RNase H2. All other growth reactions showed relatively similar Cq values, but amplification products varied greatly between reactions depending on the RT primer used, as can be seen in the gel images of FIGS.

  Proliferation reactions were performed using the desired 145 bp amplicon made from For and Rev PCR primers (SEQ ID NOs: 17 and 18, or 27 and 28), or For PCR primers (SEQ ID NOs: 17 and 27) and RT primers (SEQ ID NOs: 29, 30). Or an undesired 170 bp amplicon made from 31). Using For and Rev PCR primers without an external RT primer produced only the expected 145 bp amplicon (FIG. 19, “0 nM RT primer” lane).

  The unmodified RT primer (SEQ ID NO: 31) participated in the PCR reaction and different amounts of undesirable 170 bp product were formed (FIG. 18). The amount of this product was reduced using low concentrations of RT primer; however, a large amount of product remained even when using only 10 nM unmodified RT primer. In contrast, the use of a modified RT primer (SEQ ID NO: 30) with a central rCRNA residue produced exclusively the desired 145 bp amplicon (FIG. 19). This oligonucleotide primes the RT reaction, but after heat reactivation a. It is degraded by RNase H2, and thus cannot participate in PCR growth. The use of low RT concentrations (50 nM and 10 nM) gave the most robust yield of the desired product with all external primer designs. The use of a modified RT primer (SEQ ID NO: 29) containing a central abasic naphthyl-azo modifier (SEQ ID NO: 29) (FIG. 20) also produced the desired 145 bp amplicon. This oligonucleotide primes the RT reaction and remains competent to prime DNA synthesis during PCR, but is defective in template function, and the final growth product does not contain a complete primer binding site, so linear Can maintain growth and cannot support exponential growth. Using this primer at low concentrations (50 nM and 10 nM) showed the most robust reaction.

  3,4,5,6-tetrahydrophthalic anhydride modified HS-P. a. With RNase H2, rhPCR can be performed using For and Rev primers protected in a single tube high temperature RT-qPCR format. The use of unmodified RT primers results in undesirably longer growth products, but RT can be primed, but the use of modified RT primers that cannot participate in PCR produces the desired amplicon with high specificity.

  All references, including application publications, patent applications and patent specifications, cited in this specification are expressly incorporated by reference, with each reference individually and specifically incorporated by reference. Incorporated by reference to the same extent as described in the document.

  The terms “a”, “an”, “the” and similar uses in the context of describing the present invention (especially in the context of the following claims) are not specifically indicated in the specification. Unless the context clearly indicates otherwise, it should be construed to include both the singular and plural forms. The terms “comprising”, “having”, “inclusion” and “containing” are unconstrained terms (ie, but not limited to “including”) unless otherwise stated. . The description of a range of values herein is merely intended to be used as a shorthand notation for individually referring to each separate value falling within the range, unless otherwise indicated herein. Are incorporated into the specification as individually described herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples or exemplary terms (e.g., "such as") described herein are for the purpose of clarifying the invention only, and unless stated otherwise. The scope of the invention is not limited. No language in the specification should be construed as requiring any unclaimed element as essential to the practice of the invention.

  Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Modifications of these preferred embodiments will be apparent to those of skill in the art upon reading the above description. The inventors expect that those skilled in the art will use the above modifications as necessary, and that the inventors have practiced the invention in other ways than those specifically described herein. It is intended. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, all possible combinations of the above-described elements are encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims (37)

a) treating the sample in a reaction mixture comprising polymerase, deoxyribonucleoside triphosphate, buffer, reversibly inactive RNase H enzyme, first primer;
b) increasing the temperature to activate the RNase H enzyme;
c) hybridizing the first primer with the target RNA at an appropriate temperature;
d) treating the double-stranded product with said RNase H to remove the protecting group;
e) polymerizing the first primer to form a cDNA complementary to the target RNA;
Wherein the first primer is protected with a protecting group that can be removed with the RNase H enzyme at or near its 3 ′ end, The method, wherein the first primer is complementary to the target RNA so as to hybridize and form a double-stranded product.   The reaction mixture further includes a second primer complementary to the cDNA, and the method further hybridizes the second primer to the cDNA, polymerizes the second primer and is complementary to the cDNA. 2. The method of claim 1, wherein the extension product is formed.   The method of claim 1, wherein the RNase H enzyme is an RNase H2 enzyme. 4. The method of claim 3, wherein the RNase H2 enzyme is a Pyrococcus abyssi RNase H2 enzyme.   The method of claim 1, wherein the polymerase is not a hot start polymerase. At least one structure I:

Wherein R ¹ and R ² are independently selected from the group consisting of lower alkyl, lower cycloalkyl, lower alkenyl, lower aryl, lower arylalkyl, lower alkoxy, acyl or carboalkoxy groups, or together lower carbon A ring or lower heterocycle, each of R ¹ and R ² is independently optionally substituted with halogen, alkoxy, amino, acyl, carboxy, carboalkoxy or carbamyl)
A modified Pyrococcus abyssi RNase H2 protein comprising a modified lysine residue of One of R ¹ and ^{R 2} are H, modified Pyrococcus Abishi RN RNase H2 protein according to claim 6 the other of ^{R 1} and ^{R 2} is CH _3. One of R ¹ and ^{R 2} are H, modified Pyrococcus Abishi RN RNase H2 protein according to claim 6 the other of ^{R 1} and ^{R 2} are _{CH 2} CO 2 H. Modified Pyrococcus Abishi RN RNase H2 proteins of R ¹ and ^{R 2} according to claim 6 is CH _3. The modified Pyrococcus abyssi RNase H2 protein according to claim 6, wherein R ¹ and R ² are together butane-1,4-diyl.   The modified Pyrococcus abyss RNase H2 protein according to claim 6, wherein the lysine residue is a conserved lysine residue.   7. The modified Pyrococcus abyssi RNase H2 protein of claim 6, wherein about 25 lysine residues are modified.   7. The modified Pyrococcus abyssi RNase H2 protein of claim 6, wherein about 22 to about 28 lysine residues are modified.   The modified Pyrococcus abyss RNase H2 protein according to claim 6, wherein the activity at (temp) is less than about (low temperature activity). At least one structure I:

Wherein R ¹ and R ² are independently selected from the group consisting of lower alkyl, lower cycloalkyl, lower alkenyl, lower aryl, lower arylalkyl, lower alkoxy, acyl or carboalkoxy groups, or together lower carbon A ring or heterocycle, each of R ¹ and R ² is independently optionally substituted with halogen, alkoxy, amino, acyl, carboxy, carboalkoxy or carbamyl)
A modified Pyrococcus abyssi RNase H2 protein comprising a modified lysine residue; and at least one DNA polymerase or DNA ligase.   16. The kit according to claim 15, further comprising an oligonucleotide comprising an RNase H2 cleavage domain. Pyrococcus abyssi RNase H2 protein is represented by the formula II:

Wherein R ¹ and R ² are independently selected from the group consisting of lower alkyl, lower cycloalkyl, lower alkenyl, lower aryl, lower arylalkyl, lower alkoxy, acyl or carboalkoxy groups, or together lower carbon A ring or lower heterocycle, each of R ¹ and R ² is independently optionally substituted with halogen, alkoxy, amino, acyl, carboxy, carboalkoxy or carbamyl)
A method of modifying a Pyrococcus abyssi RNase H2 protein comprising contacting with a compound of:   18. The method of claim 17, further comprising repeating contacting the Pyrococcus abyssi RNase H2 protein with a compound of formula II.   The method of claim 18, wherein the repeated contacts include at least a total of three contacts.   The method of claim 18, wherein the repeated contact comprises a total of at least 5 contacts.   The method of claim 18, wherein the repeated contact comprises a total of at least 10 contacts.   Contact with the compound of formula II includes contact with a compound selected from the group consisting of maleic anhydride, citraconic anhydride, cis-acotinic anhydride and 3,4,5,6-tetrahydrophthalic anhydride. The method of claim 18. At least one structure I:

Wherein R ¹ and R ² are independently selected from the group consisting of lower alkyl, lower cycloalkyl, lower alkenyl, lower aryl, lower arylalkyl, lower alkoxy, acyl or carboalkoxy groups, or together lower carbon A ring or lower heterocycle, each of R ¹ and R ² is independently optionally substituted with halogen, alkoxy, amino, acyl, carboxy, carboalkoxy or carbamyl)
A method of reactivating a modified Pyrococcus abyssi RNase H2 protein comprising a modified lysine residue comprising heating the modified Pyrococcus abyssi RNase H2 protein.   24. The method of claim 23, wherein the heating comprises heating to a temperature of at least about 95 ° C. A method of cleaving an oligonucleotide with an RNase H2 cleavage domain comprising:
An oligonucleotide comprising an RNase H2 cleavage domain is converted to at least one structure I:

Wherein R ¹ and R ² are independently selected from the group consisting of lower alkyl, lower cycloalkyl, lower alkenyl, lower aryl, lower arylalkyl, lower alkoxy, acyl or carboalkoxy groups, or together lower carbon A ring or lower heterocycle, each of R ¹ and R ² is independently optionally substituted with halogen, alkoxy, amino, acyl, carboxy, carboalkoxy or carbamyl)
Contacting the modified Pyrococcus abyssi RNase H2 protein comprising a modified lysine residue with a temperature sufficient to reactivate the modified Pyrococcus abyssi RNase H2 protein, thereby cleaving said oligonucleotide Including methods.   26. The method of claim 25, wherein the method is a hot start method.   26. The method of claim 25, wherein the method is a single tube method.   The methods include nucleic acid proliferation assay, nucleic acid detection assay, oligonucleotide ligation assay (OLA), primer probe assay, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse transcriptase polymerase chain reaction (RT-PCR), 26. The method of claim 25, wherein the method is a step in at least one of a ligase chain reaction (LCR), a polynomial propagation method, a DNA sequencing method, or a method comprising primer extension.   26. The method of claim 25, wherein contacting an oligonucleotide comprising an RNase H2 cleavage domain comprises contacting an oligonucleotide comprising an RNA base that is substituted with a single RNA residue, or at least one alternative nucleoside.   26. The method of claim 25, wherein contacting the oligonucleotide comprises contacting a double stranded oligonucleotide.   26. The method of claim 25, wherein contacting the oligonucleotide comprises contacting a primer for DNA replication.   The method according to claim 2, for use in discriminating single nucleotide polymorphisms (SNPs) in a DNA molecule in which the first and second primers are discriminating primers.   The method according to claim 2, further comprising a third primer, wherein the third primer is modified so as not to participate in a subsequent proliferation reaction.   34. The method of claim 33, wherein the modification forms a cleavable linkage.   35. The method of claim 34, wherein the cleavable linkage is susceptible to chemical cleavage or restriction enzymes.   34. The method of claim 33, wherein the modification comprises a protecting group.   38. The method of claim 36, wherein the protecting group comprises a 2'-modified RNA residue, an abasic residue, an unnatural base, or a non-nucleotide naphthyl-azo modifier.

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