Inhibition of nucleic acid polymerases by endonuclease V-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-11840728-B2
Publication Date
2023-12-12
Expiration Date
Abstract
Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V 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 aptamer comprises one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.
Core Innovation
Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V 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 aptamer comprises one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the aptamer by the endonuclease V enzymatic activity.
The background identifies the problem that existing aptamer-based 'hot start' methods cannot simultaneously completely block DNA polymerase at low temperatures and provide no blockage at the desired elevated reaction temperature, and that new methods are needed to improve control of aptamer activity in reaction mixtures containing DNA polymerases. Aspects of the present invention provide a solution by activating an aptamer-inactivated DNA polymerase in a reaction mixture comprising a DNA polymerase, an endonuclease V-cleavable oligonucleotide aptamer present in an amount effective to inhibit DNA synthesis activity, and an endonuclease V enzymatic activity that cleaves the aptamer to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating the DNA synthesis activity of the DNA polymerase.
In particular aspects, the oligonucleotide aptamer may have a stem-loop (hairpin) structure with one or more deoxyinosine nucleotides located in the stem and/or loop, and the loop portion may comprise nucleotide sequences including 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:21) or loop sequences selected from SEQ ID NOs:22-28. Kits and reaction mixtures are provided comprising a DNA polymerase-binding oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity and an endonuclease V enzymatic activity.
Claims Coverage
One independent claim is present, covering a hairpin-forming oligonucleotide aptamer with deoxyinosine substitutions in the stem and/or loop including a specified loop sequence (SEQ ID NO:21) with optional deoxyinosine substitutions at positions 4, 5, and 7.
Hairpin aptamer comprising deoxyinosine in stem and/or loop
A nucleic acid sequence that forms a hairpin structure, having a stem and a loop portion, that binds to a DNA polymerase, wherein the stem and/or the loop portion comprises one or more deoxyinosine nucleotides, and wherein the loop portion comprises a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:21) that may be substituted with deoxyinosine at one or more of positions 4, 5, and 7.
The independent claim covers an oligonucleotide aptamer that binds a DNA polymerase and is defined by a hairpin (stem-loop) structure containing one or more deoxyinosine nucleotides with a loop sequence of SEQ ID NO:21 optionally substituted with deoxyinosine at positions 4, 5, and/or 7.
Stated Advantages
Activation of aptamer-inactivated DNA polymerases by endonuclease V cleavage of the aptamer to increase DNA synthesis in the reaction mixture.
Specific recognition and cleavage of aptamers enabled by incorporation of one or more deoxyinosine nucleotides.
Provides an improved solution to the hot-start problem by enabling substantial or complete inhibition of DNA polymerase at assembly temperatures and subsequent reactivation at reaction temperatures, providing an advantage over other 'hot start' technologies.
Documented Applications
DNA synthesis in reaction mixtures.
DNA amplification, including PCR.
Isothermal amplification reactions.
Detecting the presence of a target DNA and measuring an amount of a target DNA in the reaction mixture.
Kits comprising a DNA polymerase-binding oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity and an endonuclease V enzymatic activity for DNA synthesis, amplification, and detection.
Reaction mixtures comprising a DNA polymerase, an endonuclease V-cleavable oligonucleotide aptamer, and an endonuclease V enzymatic activity, optionally provided in a dried state for subsequent dissolution.
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