Nucleic acid ligands that bind to and inhibit DNA polymerases

Details Nucleic acid ligands that bind to and inhibit DNA polymerases Nucleic acid ligands that bind to and inhibit DNA polymerases

1997-10-28

2000-02-01

Zitomer, Stephanie

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 435194, 435810, 536 221, 536 254, 536 243, 935 77, 935 78, C12P 168, C12Q 1934, C07H 2102, C07H 2104

Patent

active

060201304

DESCRIPTION:

BRIEF SUMMARY
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 selected from Taq polymerase, a thermostable polymerase isolated from Thermus aquaticus or Tth polymerase, a thermostable DNA polymerase and reverse transcriptase isolated from Thermus thermophilus. However, the method of this invention can be extended to the identification and preparation of any thermal stable DNA 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 and Tth polymerase. The invention includes high-affinity DNA ligands which bind to Taq polymerase and Tth polymerase, thereby inhibiting their ability to polymerase DNA synthesis at ambient temperatures. Further included within this invention are nucleic acid switches. The thermal 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.


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 deoxynucleotides (A, T, C and G). The solution is then heated to a temperature sufficient to separate the complementary strands of DNA (approximately 95.degree. 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.degree. 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 (Wernars 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. USA 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

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