Inhibition of DNA polymerases by uracil-DNA glycosylase-cleavable oligonucleotide ligands
Inventors
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Assignees
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CepheidCepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Cepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Publication Number
US-12168786-B2
Publication Date
2024-12-17
Expiration Date
Abstract
Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising modifying the aptamer by uracil-DNA glycosylase enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. In some aspects, the oligonucleotide aptamers are circular and comprise one or more deoxyuridine nucleotides providing for aptamer-specific recognition and modification of the circular aptamer by the uracil-DNA glycosylase enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing uracil-DNA glycosylase enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one uracil-DNA glycosylase enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific modification of the aptamer by the uracil-DNA glycosylase enzymatic activity.
Core Innovation
Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising modifying the aptamer by uracil-DNA glycosylase enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. In some aspects, the oligonucleotide aptamers are circular and comprise one or more deoxyuridine nucleotides providing for aptamer-specific recognition and modification of the circular aptamer by the uracil-DNA glycosylase enzymatic activity. Kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one uracil-DNA glycosylase enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific modification of the aptamer by the uracil-DNA glycosylase enzymatic activity are provided.
Aspects of the invention relate generally to improved methods of blocking DNA polymerase activity with oligonucleotide aptamers at low reaction temperatures, and restoring the enzyme activity upon raising the reaction temperature (e.g., hot-start methods). Ideally, an aptamer should completely block DNA polymerase at low temperatures and provide no blockage effect at the desired elevated reaction temperature, but the aptamer structure usually represents a compromise between these two key requirements; new methods are needed to improve control of aptamer activity in reaction mixtures containing DNA polymerases.
Particular aspects provide methods for activating an aptamer-inactivated DNA polymerase by providing a reaction mixture suitable for DNA synthesis comprising a DNA polymerase, a uracil-DNA glycosylase enzymatic activity, and a DNA polymerase-binding oligonucleotide aptamer that comprises a hairpin structure having a stem sequence portion and a loop sequence portion wherein the loop sequence portion comprises one or more deoxyuridine nucleotide(s) modifiable by the uracil-DNA glycosylase enzymatic activity, and modifying the aptamer by the uracil-DNA glycosylase enzymatic activity to form a modified aptamer having less or no inhibitory effect on the DNA polymerase, thereby activating or enhancing the DNA synthesis activity of the DNA polymerase to start and/or increase DNA synthesis in the reaction mixture.
Claims Coverage
Independent claim identified: claim 1. The claim contains two main inventive features.
Oligonucleotide aptamer capable of forming a hairpin structure
A DNA polymerase-binding nucleic acid sequence that is capable of forming a hairpin structure having a stem sequence portion and a loop sequence portion.
Loop sequence comprising SEQ ID NO:23 with deoxyuridine substitutions
The loop sequence portion comprises a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:23) wherein one or more thymidine nucleotides at positions 1, 2, 10, 11 and 12 of the SEQ ID NO:23 sequence are substituted by one or more deoxyuridine nucleotides modifiable by a uracil-DNA glycosylase enzymatic activity.
Claim 1 is directed to an oligonucleotide aptamer that binds DNA polymerase and forms a hairpin with a loop having the specified SEQ ID NO:23 sequence with one or more thymidine-to-deoxyuridine substitutions that are modifiable by a uracil-DNA glycosylase enzymatic activity.
Stated Advantages
Improved methods of blocking DNA polymerase activity at low reaction temperatures and restoring enzyme activity upon raising the reaction temperature (hot-start methods).
Aptamers can be quickly engineered by SELEX and readily and inexpensively manufactured by chemical synthesis.
The described approach provides a solution to the prior art difficulty of achieving complete block-and-release of DNA polymerase by aptamers by activating aptamer-inactivated polymerases via uracil-DNA glycosylase-induced aptamer modification.
Methods can facilitate DNA synthesis and DNA amplification (including PCR and isothermal amplification) and can be useful for fast PCR with short cycle times.
Documented Applications
DNA synthesis, including DNA amplification in the reaction mixture (e.g., isothermal amplification and PCR).
Detecting a presence of a target DNA in the reaction mixture and measuring an amount of a target DNA in the reaction mixture.
Kits for activating an aptamer-inactivated DNA polymerase comprising a uracil-DNA glycosylase enzymatic activity and a DNA polymerase-binding oligonucleotide aptamer with deoxyuridine in the loop sequence.
Reaction mixtures comprising a DNA polymerase and a DNA polymerase-binding oligonucleotide aptamer with deoxyuridine in the loop sequence, optionally further comprising a uracil-DNA glycosylase enzymatic activity and components provided in a dried state.
Oligonucleotide aptamers in combination with a DNA polymerase, including circular stem-loop aptamers and noncircular hairpin aptamers.
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