REACTIONS

BACKGROUND

Non-specific primer extension prior to reaction initiation in thermocycling DNA amplification reactions such as polymerase chain reaction (PCR), or isothermal DNA amplification reactions such as loop- mediated isothermal amplification (LAMP) may inhibit specific product formation, and lead to non-specific amplification and reaction

irreproducibility. It is, therefore, desirable to block the activity of the polymerase, and hence primer extension, prior to reaction initiation. This has been achieved using antibodies (Kellogg, et al., Biotechniques, 16(6) : 1134-7 (1994)), affybodies (Affibody AB, Stockholm, Sweden), aptamers (Dang, et al., Journal of Molecular Biology, 264(2) : 268-78 (1996)), and chemical modification of the polymerase (US Patent No. 6,183,998). Although each of these techniques can be effective, they each have unique limitations. For example, preparation of antibodies requires use of animal systems, affybodies and aptamers require screening libraries of molecular variants, and chemical modifications require extra heat incubation steps to reverse the inactivating

modification. It would be desirable to have a generalizable approach to rapidly and effectively create hot-start inhibitors targeted towards DNA polymerases. SUMMARY

In general, in one aspect, a preparation is provided that

includes: a synthetic single-strand nucleic acid having a 3' end and a 5' end, capable of forming a double-stranded region that extends from the 3' end and a single-stranded region having a 5' single-strand extension containing at least one uracil or inosine; and a buffer. An example of a synthetic single-strand nucleic acid is shown in Figure 1.

Embodiments may include one or more of the following features: the at least one double-strand region has a melting temperature (Tm) of at least 10°C less than a Tm for a target DNA in an amplification reaction, for example, below 90°C, 89°C , 88°C , 87°C , 86°C, 85°C, 75°C, 65°C, 55°C, 45°C or 35°C; a uracil or inosine is positioned at the fourth position in the 5' single-strand extension numbered from the 3' end; the synthetic nucleic acid is capable of forming a plurality of single-strand regions; a second single-strand region is a spacer; a third single-strand region forms a single-stranded loop at an internal location in the synthetic nucleic acid; the buffer may contain at least one of a polymerase, dNTPs, or primers; the spacer comprises hexa- ethylene glycol, a 3 carbon molecule or a l',2'-dideoxyribose; the synthetic nucleic acid contains a derivative nucleotide and/or

nucleotide linkage in a nucleic acid sequence at the 3' end where the derivative nucleotide may be selected from one or more inverted nucleotides, di-deoxynucleotides or amino-modified nucleotides; for example, the nucleotide linkage may be a phosphorothioate linkage;

In an embodiment, the preparation may additionally include one or more polymerases for example, one or more thermostable

polymerases, for example at least one archaeal polymerase; a bacterial polymerase, and/or a variant of a wild type archaeal or bacterial polymerase. The synthetic nucleic acid and the polymerase may be present in a molar ratio of between 0.5 : 1 to 10 : 1. In general in one aspect, a variant of a wild type polymerase includes at least 93% sequence identity to SEQ ID NO : 25 and further includes at least one mutation at an amino acid position

corresponding to 278, 307, and/or 402 in SEQ ID NO: 25. In another aspects, mutations at 278, 307 and/or 402 may be inserted into any of the Bst polymerase variants described in U.S. application serial number 13/823,811.

Embodiments may include one or more of the following features of the preparation : fusion of variant polymerase to a DNA binding domain such as Sso7d; and/or the variant polymerase optionally having an amino acid at one or more of the positions corresponding to 278, 307, and/or 402 that is not a histidine; for example where one or more mutations may be selected from a group of mutations

corresponding to H278Q, H307R, H402Q.

In general in one aspect, a method is provided for inhibiting a polymerase extension reaction; that includes adding a preparation described above to a mixture containing a polymerase, a target DNA and dNTPs; and maintaining for a period of time prior to extension or amplification of the target DNA, the mixture at a temperature below the Tm of the double-stranded portion of the synthetic nucleic acid.

Embodiments may include one or more of the following features: the at least one double-strand region has a Tm of at least 10°C less than a Tm for a target DNA in an amplification reaction, for example, below 90°C, 89°C , 88°C, 87°C, 86°C, 85°C, 75°C, 65°C, 55°C, 45°C or 35°C; a uracil or inosine is positioned at the fourth position in the 5' single-strand extension numbered from the 3'end; the synthetic nucleic acid may include additional single-stranded nucleic acid regions such as a second single-strand region is a spacer; where for example, the spacer may include a hexa-ethylene glycol, a 3 carbon molecule or a l',2'-dideoxyribose; and/or a third single-strand region forms a single-stranded loop at an internal location in the synthetic nucleic acid.

In an embodiment, the synthetic nucleic acid contains at least one derivative nucleotide and/or nucleotide linkage at the 3' end where the at least one derivative nucleotide may be selected from one or more inverted nucleotides, di-deoxynucleotides or amino-modified nucleotides; and for example, the at least one nucleotide linkage may be a phosphorothioate linkage.

In an embodiment, the one or more polymerases may include one or more thermostable polymerases, for example at least one archaeal polymerase; a bacterial polymerase, and/or a variant of a wild type archaeal or bacterial polymerase; and the synthetic nucleic acid and the polymerase may be present in a molar ratio of between 0.5 : 1 to 10 : 1.

In one embodiment, an additional step may be included of reversing the inhibition of the polymerase extension reaction by raising the reaction temperature above a Tm for the synthetic nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a synthetic nucleic acid in the form of a hairpin oligonucleotide containing a 5' overhang, a 3' blocked end to prevent DNA polymerase extension and exonuclease cleavage and at least one non-standard base. (1) is the optional spacer; (2) is the double- stranded region or "stem"; (3) is the 5' single-strand; and (4) is the blocked 3' end : N = rNMP, dNMP or non-standard base; X = base that is recognized by the DNA polymerase uracil binding pocket; * = 3' end modifications: phosphorothioate bonds and/or inverted base and/or dideoxynucleoside.

Figure 2 shows a gel of the PCR products obtained with an

Archaeal polymerase in the presence or absence of hairpin

oligonucleotide inhibitors. In the absence of the hairpin

oligonucleotide, the polymerase fails to amplify the expected 2kb product. In the presence of the oligonucleotides the 2kb product is amplified.

Lane 1 contains 2-log DNA ladder (New England Biolabs, Ipswich, MA), a MW marker for detection of 2Kb amplicon.

Lane 2 contains 5nM Archaeal Family B DNA polymerase without the synthetic nucleic acid present.

Lane 3 contains 5nM Archaeal Family B DNA polymerase and 5nM the synthetic nucleic acid, TM39U 1G-IS.

Lane 4 contains 5nM Archaeal Family B DNA polymerase and 5nM the synthetic nucleic acid, TM39U 1G-I*.

Lane 5 contains 5nM Archaeal Family B DNA polymerase and 5nM the synthetic nucleic acid, TM39U.

Lane 6 contains 5nM Archaeal Family B DNA polymerase and 5nM the synthetic nucleic acid, TM39LooplOT.

Lane 7 contains 5nM Archaeal Family B DNA polymerase and 5nM the synthetic nucleic acid, TM39U3-IS.

DETAILED DESCRIPTION OF THE EMBODIMENTS Synthetic nucleic acids are described that reversibly inhibit polymerase extension reactions. These synthetic nucleic acid

preferably contain at least one non-standard base (e.g. U or I) in a 5' single-strand overhang adjacent to a double-strand region. If the double-strand region is denatured into a single-strand or strands, the synthetic nucleic acid no longer blocks the polymerase from replicating substrate DNA. Preferably, inhibition of polymerase activity occurs at a first temperature that is at least 10°C lower than a second

temperature suitable for polymerase extension reactions. A

polymerase extension reaction refers to the extension of a first single- strand nucleic acid by a polymerase where the extension is

complementary to a second nucleic acid in association with the first strand. In an embodiment, a synthetic nucleic acid is engineered so that the double-strand region melts at a desired temperature which is selected to melt at about 15°C or 14°C or 13°C or 12°C or 11°C or 10°C or 9°C or 8°C below polymerization extension conditions. Polymerase extension conditions include conditions for isothermal amplification occurring at for example 65°C or a thermocycling amplification such as PCR which occurs at higher temperatures such as about 95°C. For example, the double-strand region in the synthetic nucleic acid may be designed to remain intact at a specific temperature in the range of - 80°C to 37°C but become denatured at a specific temperature in a range of 37°C to 100°C. The Tm of the synthetic nucleic acid can be modulated by one or more factors that include: changing the sequence or length of the double-strand region, changing the length of an internal single-strand region, adding mismatched or modified bases to the double-strand region, selecting a nucleotide composition having weaker base pairing properties such as an adenine, thymine or uracil rich sequence, or a sequence containing inosine, or abasic sites such as l',2' dideoxyribose in a polymerization reaction buffer with a selected salt type (for example magnesium) and concentration. An example of a buffer is Thermopol ^® Buffer (New England Biolabs,

Ipswich, MA).

In an embodiment of the invention, the design of a synthetic nucleic acid reversible inhibitor of polymerase extension reactions includes the following features: the synthetic nucleic acid can be DNA, DNA/RNA, RNA, or RNA/RNA; it can be formed from two single-strands or from a single nucleic acid (oligonucleotide) but should be capable of forming at least one double-strand region and a 5' single-strand overhang. It may optionally contain a plurality of single-strand regions and a plurality of double-strand regions. If the synthetic nucleic acid is an oligonucleotide, it should be capable of folding in such a way as to contain at least one double-strand region at a temperature lower than the reaction temperature as described above. The oligonucleotide may have a length in the range of 8-200

nucleotides. Any double-strand region in the inhibitor preferably has a length of 4-35 nucleotides.

The 5' single-strand overhang should be at least 4 nucleotides and preferably less than 100 nucleotides in length, for example 4-40 nucleotides, for example 6-10 nucleotides, and should contain one or more non-standard nucleotides such as U or I positioned between the second and tenth position of the overhang counted from the double- strand region, for example in the fourth position where the one or more non-standard nucleotides may be 1 to 5 uracils or 1 to 5 inosines. For example, the sequences shown in Table 1 were all found to be effective as reversible binding oligonucleotides.

In addition, a synthetic nucleic acid may optionally have a 3' end that is resistant to exonuclease activity and/or non-extendable by a polymerase. The 3' end of the oligonucleotide can be blocked from extension by modification, such as dideoxynucleotides, spacer

molecules, inverted bases or amino-modified nucleotides. The 3' end can be made resistant to exonuclease degradation by the addition of phosphorothioate linkages between one or more bases at or near the 3' end or the use of inverted bases at the 3' end. The oligonucleotide can be made non-amplifiable by adding non-replicable bases in the internal sequence, such as carbon spacers, l',2'-Dideoxyribose, abasic site, or thymine dimers.

Table 1 provides examples of synthetic nucleic acid molecules capable of forming hairpins and that were found to be effective in the assays described herein. The exemplified synthetic nucleic acid molecules have spacers of T _n or X _n where T ₍₄ -g) or X(i- ₄ ) , a 5' end containing a modified base, U ₍ i- ₅₎ , or I ₍ i- ₃₎ and has a U or an I at position 4 counted from the double-stranded region. The 5' end varies as shown.

Table 1

Oligonucleotides tested and effective in Hot Start PCR

0 iqo Sequence containinq uracil (U) or Inosine (I) ' = phosphorothioate bonds

Le ;nqth

28 TUUUUUCTATCCTTA I I I I I AAGGA*T*A*G (SEQ ID NO : 3)

24 TUUUUUAGCTAGGTTTTCCTA*G*C*T (SEQ ID NO :4)

24 TUUUUUGCAGCGA I I I I I CGC*T*G*C (SEQ ID NO: 5)

30 TU U U U UG AG ACTCG RCTTTTG ACG AGT* C*T* C (SEQ ID NO : 6)

34 TUUUUUCTATCCTTAACGTTTTCGTTAAGGA*T*A*G (SEQ ID NO : 7)

30 TUUUUUACAC ^" n ^" CCGG ^" n ^~ TTCCGGAAG*T*G*T (SEQ ID NO : 8)

31 I TUUUUUCTATCCTTAACGXCGTTAAGGA*T*A*G (SEQ ID NO :9) Oliqo Seguence containing uracil (U) or Inosine (I) ^* = phosphorothioate bonds

Length

34 TUUUUUCTATCCTTAACGXXXXCGTTAAGGA*T*A*G (SEQ ID NO: 10)

36 IUUUUUGIAIGGI IAAGGI M i l l GG 11 AAGGA* 1 *A*G (SbQ ID NO: 11)

40 IUUUUUGIAIGGI IAAGGI 111111111 GG 11 AAGGA* 1 *A*G (bbO. ID NU:1

34 IUUUUUGIAIGGI IAAG1I 111 GG 11 AAGGA* 1 *A*G (bbO. ID NO: 13)

34 IUUUUUGIAIGGI IAAG1I 111 GG 11 AAGG*A* 1 *A*G (bbO. ID NO: 14)

34 IUUUUUAIGIGGI IAAG1I 111 GG 11 AAGGAGAmvd / (SbQ ID NO: l )

34 IUUUUUGI1IGGI I11GGI 11 IGGI 1 AAGGA* 1 *A*G (bbQ ID NO: 16)

34 1 AUGGAG 1 A 1 GO 11 AAGG 1111 GG 11 AAGGA* 1 *A*G (bbQ ID NO : 1 /)

34 IUUUGAGIAIGGI 1 AAGG 1111 GG 11 AAGGA* 1 *A*G (bbQ ID NO: 18)

34 11111 IGIAIGGI 1 AAGG 1111 GG 1 IAAGGA*I*A*G (bbQ ID NO: 19)

34 11111 IGIAIGGI 1 AAGG 1111 GGkkAAGG*A* 1 *A*G (bbQ ID NU: U

34 11111 lAIGIGGI 1 AAGG 1111 GGkkAAGGAGAinvd / (bbQ ID NU: 1

34 11111 IGIAIGGI 1 AAGG 111 IGGI 1 AAGGA* 1 *A*G (bbQ ID NU:

34 11111 IGIAIGGI 1 AAGG 111 IGGI 1 AAGG*A* 1 *A*G (bbQ ID NU: J

34 11111 lAIGIGGI 1 AAGG 111 IGGI 1 AAGGAGAmvd / (bbQ ID NU: 4

In an embodiment of the invention, one or more polymerases are added to the synthetic nucleic acid. The polymerases may be thermostable polymerases such as wild type or recombinant Archaeal DNA polymerases or bacterial DNA polymerases or variants (mutants) thereof including fusion proteins where the polymerase or variants thereof may be fused to a DNA binding domain such as Sso7d (for example, US Patent No.7,666,645). A variant of a bacterial

polymerase is exemplified at least 90%, 91%, 92% 93%, 95%, or 98% amino acid sequence homology or identity with SEQ ID NO: 25 prior to fusion to a DNA binding domain if such is present. Regardless of the presence of an additional DNA binding domain, the variant preferably includes one or more mutations at positions corresponding to 52 (not R), 278, 307, 402, and/or 578 (not R) in SEQ ID NO:25, for example, one or more of the following mutations: H278Q, H307R, H402Q. Additional mutations may be optionally introduced into the polymerase by routine methods of random or directed mutagenesis. Amplification procedures referred to herein include standard thermocycling or isothermal amplification reactions such as PCR amplification or LAMP (Gill, et al., Nucleos. Nucleot. Nucleic Acids, 27: 224-43 (2008); Kim, et al, Bioanalysis, 3 : 227-39 (2011);

Nagamine, et al., Mol. Cel. Probes, 16: 223-9 (2002); Notomi, et al., Nucleic Acids Res., 28: E63 (2000); and Nagamine, et al., Clin. Chem. , 47: 1742-3 (2001)), helicase displacement amplification (HDA), recombinase polymerase amplification (RPA), nicking enzyme

amplification reaction (NEAR) and/or strand displacement amplification (SDA). Variant polymerases described herein may be used in

amplification or sequencing reactions with or without the use of synthetic nucleic acids described herein.

Amino acid sequence for a wild type Bst polymerase

AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGR

FFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLL

AAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVR

KAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQ

MGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTS

ADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALT

QTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIA

DDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDY

GLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYL

PDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQ

VHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK (SEQ

ID NO: 25)

All references cited herein are incorporated by reference.

EXAMPLE

Assay to measure inhibition of polymerase activity prior to PCR cycling Inhibition of polymerase activity was measured at a temperature below that used in the PCR assay which followed. The assay was performed as follows:

Primers were made for PCR to produce a 2kb Lambda DNA amplicon . Additionally, the 3' end of the reverse primer contained 8 nucleotides that could anneal to Lambda DNA creating a false priming site producing a non-specific 737 bp amplicon.

The PCR assay was done in the presence of high levels of human genomic DNA and the reaction mixture was incubated with the thermostable polymerase at 25°C for 15 minutes prior to PCR cycling . These conditions created many opportunities to form non-specific products. The presence of a nucleic acid composition to inhibit polymerase activity prior to amplification was required to yield a 2kb amplicon, with minimal or no non-specific products. The reaction mix was set up on ice and contained the following reagents: Thermopol

Buffer, 0.4 pg/μΙ Lambda DNA, 2.0 ng/μΙ Jurkat genomic DNA, 0.2 mM dNTP and 0.2 μΜ primers.

Forward primer, L30350F: 5'CCTGCTCTGCCGCTTCACGC3' (SEQ ID No: l)

Reverse primer, L2kbalt4rv: 5'GGGCCGTGGCAGTCGCATCCC3' (SEQ ID No: 2)

0.25 μΙ to 0.50 μΙ of 2.0 units/μΙ Vent® DNA Polymerase (NEB, Ipswich, MA) with or without the nucleic acid composition (see Figure 2) was added to 25 μΙ or 50 μΙ of the reaction mix, and transferred to a PCR machine and cycled at 25°C for 15-30 minutes, then cycled 35 times at 98°C for 10 seconds, 45°C for 20 seconds, 72°C for 60 seconds, 72°C for 4 minutes. DNA products generated by PCR cycling were analyzed by agarose gel electrophoresis.

In the absence of a reversibly inhibiting synthetic nucleic acid, the polymerase failed to yield the expected 2kb Lamda amplicon.

Non-specific products including the 737 bp amplicon were observed. In the presence of oligonucleotide inhibitors, a robust yield of the expected 2kb Lambda amplicon was produced with minimal or no nonspecific products.