Nucleic acid ligand inhibitors to DNA polymerases

Details Nucleic acid ligand inhibitors to DNA polymerases Nucleic acid ligand inhibitors to DNA polymerases

1999-03-01

2001-02-06

Zitomer, Stephanie W. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S194000, C536S023100, C536S025400

Reexamination Certificate

active

06183967

ABSTRACT:

FIELD OF THE INVENTION
Described herein are methods for identifying and preparing high-affinity nucleic acid ligands to DNA polymerases, specifically thermostable DNA polymerases. In a preferred embodiment the DNA polymerase is Taq polymerase, a thermostable polymerase isolated from
Thermus aquaticus;
Tth polymerase, a thermostable DNA polymerase isolated from
Thermus thermophilus;
or TZ05 polymerase, isolated from another Thermus species. However, the method of this invention can be extended to the identification and preparation of any thermally stable DNA polymerase. Some of these thermostable DNA polymerases also have the ability to reverse transcribe RNA to copy DNA. Examples of DNA polymerases with reverse transcription ability include Tth and TZ05 polymerase. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential Enrichment. Also described herein is an improved method for performing the Polymerase Chain Reaction using the nucleic acid ligands of this invention. Specifically disclosed herein are high-affinity nucleic acid ligands to Taq polymerase, Tth polymerase, and TZ05 polymerase. The invention includes high-affinity DNA ligands which bind to Taq polymerase, Tth polymerase and TZ05 polymerase, thereby inhibiting their polymerase activity at a predetermined range of temperatures. Further included within this invention are nucleic acid switches. The temperature dependent binding of the nucleic acid ligands to DNA polymerases of this invention are examples of ligands whose desirable properties can be switched on or off based on any number of reaction conditions, such as pH and salt concentration.
BACKGROUND OF THE INVENTION
The Polymerase Chain Reaction (PCR), is a recently developed technique which has had a significant impact in many areas of science. PCR is a rapid and simple method for specifically amplifying a target DNA sequence in an exponential manner. (Saiki et al. (1985) Science 230:1350; Mullis and Faloona (1987) Methods Enzymol. 155:335). Briefly, the method consists of synthesizing a set of primers that have nucleotide sequences complementary to the DNA that flanks the target sequence. The primers are then mixed with a solution of the target DNA, a thermostable DNA polymerase and all four deoxynucleotide triphosphates (dATP, dTTP, dCTP and dGTP). The solution is then heated to a temperature sufficient to separate the complementary strands of DNA (approximately 95° C.) and then cooled to a temperature sufficient to allow the primers to bind to the flanking sequences. The reaction mixture is then heated again (to approximately 72° C.) to allow the DNA synthesis to proceed. After a short period of time, the temperature of the reaction mixture is once again raised to a temperature sufficient to separate the newly formed double-stranded DNA, thus completing the first cycle of PCR. The reaction mixture is then cooled and the cycle is repeated. Thus, PCR consists of repetitive cycles of DNA melting, annealing and synthesis. Twenty replication cycles can yield up to a million fold amplification of the target DNA sequence. The ability to amplify a single DNA molecule by PCR has applications in environmental and food microbiology (Wemars et al. (1991) Appl. Env. Microbiol. 57:1914-1919; Hill and Keasler (1991) Int. J. Food Microbiol. 12:67-75), clinical microbiology (Wages et al. (1991) J. Med. Virol. 33:58-63; Sacramento et al. (1991) Mol. Cell Probes 5:229-240; Laure et al. (1988) Lancet 2:538), oncology (Kumar and Barbacid (1988) Oncogene 3:647-651; McCormick (1989) Cancer Cells 1:56-61; Crescenzi et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:4869), genetic disease prognosis (Handyside et al (1990) Nature 344:768-770), blood banking (Jackson (1990) Transfusion 30:51-57) and forensics (Higuchi et al. (1988) Nature (London) 332:543).
The availability of thermostable DNA polymerases such as Taq DNA polymerase has both simplified and improved PCR. Originally only heat-sensitive polymerases, such as
E. coli
DNA polymerase were available for use in PCR. Heat-sensitive polymerases, however, are destroyed at the temperatures required to melt double-stranded DNA and additional polymerase has to be added after each PCR cycle. Taq DNA polymerase, isolated from the thermophilic bacterium
Thermus aquaticus,
is stable up to 95° C. and its use in PCR has eliminated the necessity of repetitive addition of temperature sensitive polymerases after each thermal cycle. Additionally, because Taq polymerase can be used at higher temperatures it has improved the specificity and sensitivity of PCR. The reason for the improved specificity is that at higher temperatures the binding of primers to sites other that the desired ones (referred to as mispriming) is significantly reduced.
Since its discovery, the Polymerase Chain Reaction has been modified for various applications, such as in situ PCR, in which the detection limit of traditional in situ hybridization has been pushed to the single copy level (Haase et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:4971-4975), and reverse transcriptase PCR (RT-PCR), wherein an RNA sequence is converted to its copy DNA (cDNA) by reverse transcriptase (RT) before being amplified by PCR, making RNA a substrate for PCR (Kawasaki (1991) Amplification of RNA in
PCR Protocols, A Guide to Methods and Applications,
Innis et al., Eds. Academic Press Inc., San Diego, Calif., 21-27). Mesophilic viral reverse transcriptases, however, are often unable to synthesize full-length cDNA molecules because they cannot “read through” stable secondary structures of RNA molecules. This limitation has recently been overcome by use of a polymerase isolated from
Thermus thermophilus
(Tth polymerase). Tth polymerase is a thermostable polymerase that can function as both reverse transcriptase and DNA polymerase (Myers and Gelfand (1991) Biochemistry 30:7661-7666). The reverse transcription performed at an elevated temperature using Tth polymerase eliminates secondary structures of template RNA, making it possible for the synthesis of full-length cDNA.
Although significant progress has been made in PCR technology, the amplification of nontarget oligonucleotides due to side-reactions, such as mispriming of background DNA and/or primer oligomerization still presents a significant problem. This is especially true in diagnostic applications in which PCR is carried out in a milieu containing background DNA while the target DNA may be present in a single copy (Chou et al. (1992) Nucleic Acid Res. 20:1717-1723). The generation of nonspecifically amplified products has been attributed to polymerase activity at ambient temperature that extends nonspecifically annealed primers. (Chou et al. (1992) Nucleic Acid Res. 20:1717-1723, Li et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:4580). Accordingly, the inhibition of polymerase activity at ambient temperature is important to control the generation of non-specific products.
Two methods have been reported which minimize these side reactions. In the first method, termed “manual hot start” PCR, a component critical to polymerase activity (e.g. divalent ions and/or the polymerase itself) is not added to the reaction mixture until the temperature of the mixture is high enough to prevent nonspecific primer annealing. (Chou et al. (1992) Nucleic Acid Res. 20:1717-1723; D'Aquila et al. (1991) Nucleic Acid Res. 19:3749). Thus, all of the reagents are heated to 72° C. before adding the final reagent, usually the polymerase. In wax-mediated “hot start” PCR, a component(s) crucial to polymerase activity is physically separated from the rest of the reaction mixture at low temperature by a wax layer which melts upon heating in the first cycle. (Chou et al. (1992) Nucleic Acids Res. 20:1717; Horton et al. (1994) BioTechniques 16:42). “Hot start” PCR has certain drawbacks; the requirement of reopening of tubes before initiating thermocycling increases crossover contamination and repetitive pipetting makes it tedious in handling a large number of sampl

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