Inhibition of nucleic acid polymerases by endonuclease V-cleavable circular oligonucleotide ligands
Inventors
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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-11970717-B2
Publication Date
2024-04-30
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. The oligonucleotide aptamers of the present invention are circular and comprise one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the circular 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
Aspects of the present 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). The patent states that DNA polymerase activity can be reduced when primers hybridize to non-complementary DNAs and that existing aptamer approaches struggle to both completely block polymerase at low temperatures and provide no blockage at elevated reaction temperatures, creating a need for new methods to improve control of aptamer activity in reaction mixtures containing DNA polymerases.
Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases by 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. The oligonucleotide aptamers of the invention are circular and comprise one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the circular aptamer by the endonuclease V enzymatic activity; the disclosure also provides exemplary oligonucleotide aptamers, mixtures, reaction mixtures, and kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity.
Claims Coverage
Overview: One independent claim is present with five main inventive features extracted from the claim language.
Circular oligonucleotide aptamer forming a hairpin structure
A circular nucleic acid sequence that forms a hairpin structure, having a stem and a loop portion, that binds to a DNA polymerase.
Loop comprises conserved nucleotide sequence SEQ ID NO:18
The loop portion comprises a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18) that may be substituted with deoxyinosine at one or more of positions 4, 5, and 7.
Incorporation of deoxyinosine nucleotides in stem and/or loop
The stem and/or the loop portion comprises one or more deoxyinosine nucleotides.
Endonuclease V cleavability (example Thermotoga maritima)
One or more deoxyinosine nucleotides renders the oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity, and the endonuclease V enzymatic activity may comprise Thermotoga maritima endonuclease V enzymatic activity.
Complexing with a DNA polymerase
The oligonucleotide aptamer can be complexed with a DNA polymerase.
The independent claim covers a circular hairpin oligonucleotide aptamer that binds to a DNA polymerase, specifies a conserved loop sequence (SEQ ID NO:18) with possible deoxyinosine substitutions at defined positions, requires incorporation of one or more deoxyinosine nucleotides in the stem and/or loop to render the aptamer cleavable by endonuclease V (including Thermotoga maritima endonuclease V), and contemplates the aptamer complexed with a DNA polymerase.
Stated Advantages
Improved control of aptamer activity for hot-start DNA synthesis by enabling activation of aptamer-inactivated DNA polymerases via endonuclease V-mediated cleavage.
Aptamers can be quickly engineered in a test tube using SELEX and readily and inexpensively manufactured by chemical synthesis.
Specific cleavage of deoxyinosine-containing circular aptamers by endonuclease V provides aptamer-specific recognition and cleavage to reduce or eliminate aptamer binding and thereby activate DNA polymerase.
Reaction components, including DNA polymerase, oligonucleotide aptamer, and endonuclease V enzymatic activity, may be provided in a dried state for convenience of storage and preparation of reaction mixtures.
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 a reaction mixture.
Kits for activating an aptamer-inactivated DNA polymerase comprising an endonuclease V enzymatic activity and a DNA polymerase-binding circular oligonucleotide aptamer.
Reaction mixtures and compositions comprising a DNA polymerase, an endonuclease V-cleavable circular oligonucleotide aptamer, and an endonuclease V enzymatic activity for use in DNA synthesis.
Compositions comprising a DNA polymerase complexed with a circular oligonucleotide aptamer.
Use in hot-start methods to block DNA polymerase activity at low temperatures and restore enzyme activity upon raising reaction temperature.
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