Thermostable polymerase inhibitor compositions and methods
Inventors
Lokhov, Sergey G. • Gall, Alexander A. • Baraznenok, Vera • Viazovkina, Ekaterina V. • Shishkina, Irina • Persing, David H.
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Assignees
Member
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-12385045-B2
Publication Date
2025-08-12
Expiration Date
Abstract
The present disclosure relates to aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.
Core Innovation
The present disclosure relates to aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided. The nucleic acid inhibitors can adapt a secondary structure to reversibly inhibit thermostable polymerases, wherein the inhibitory activity is temperature dependent.
In vitro synthesis of nucleic acid target sequences often relies in part on the use of thermostable DNA polymerases and at least one oligonucleotide primer designed to specifically bind to a target nucleic acid substrate. If the assay requires a period of time at a lower or ambient temperature, the target-specific primers may hybridize to non-target sequences or may form primer dimers, resulting in mispriming and the subsequent generation of non-specific products that can mask the product of interest and prematurely deplete the reaction mixture of necessary reagents.
Described are aptamers which can form a secondary structure and which can reduce generation of non-specific polymerase products in various assays which rely on thermostable polymerase activity but which can dissociate from the polymerase to facilitate target product generation and detection. The disclosure includes aptamers having stem-and-loop, dumbbell, and hairpin predicted secondary structures, compositions comprising thermostable polymerase and aptamer, methods for extending a primer in the presence of the aptamer, and kits comprising the aptamer and a thermostable polymerase.
Claims Coverage
The claims include three independent inventive features relating to aptamers and compositions: a dumbbell aptamer with a single folded oligonucleotide and defined split stem and loop structure; a ligated aptamer consisting of a specific sequence; and an aptamer comprising a specific sequence with defined substitution limits.
Dumbbell aptamer comprising a single folded oligonucleotide
A dumbbell aptamer comprising a single oligonucleotide in a folded configuration, the single oligonucleotide comprising nucleotides of SEQ ID NO:1, or a variant thereof, covalently linked to nucleotides of SEQ ID NO:3, or a variant thereof, covalently linked to nucleotides of SEQ ID NO:2, or a variant thereof, covalently linked to nucleotides of SEQ ID NO:3, wherein in the folded configuration the single oligonucleotide comprises a nucleotide stem and two nucleotide loops, the nucleotide stem comprises the nucleotides of SEQ ID NO:1, or the variant thereof, hybridized to the nucleotides of SEQ ID NO:2, or the variant thereof, the nucleotides of SEQ ID NO:1 or SEQ ID NO:2 are split into a 5′ portion and a 3′ portion due to a lack of a covalent bond between adjacent nucleotides defining a 5′ terminus and a 3′ terminus positioned adjacent to one another within the nucleotide stem, the variant of SEQ ID NO:1 has only 1 to 4 nucleotide substitutions, the variant of SEQ ID NO:2 has only 1 to 4 nucleotide substitutions, the variants of SEQ ID NO:1 and SEQ ID NO:2 are equal in length and 100% complementary along their entire length, and each of the two nucleotide loops individually comprise SEQ ID NO:3 or variants thereof with only 1 or 2 nucleotide substitutions.
Ligated aptamer consisting of SEQ ID NO:110
An aptamer consisting of SEQ ID NO:110 wherein the 5′ and 3′ ends are ligated.
Aptamer comprising SEQ ID NO:106 with defined substitution limits
An aptamer comprising SEQ ID NO:106 or a variant thereof, the aptamer further comprising a secondary structure comprising two nucleotide loops and a nucleotide stem, wherein the variant of SEQ ID NO:106 comprises fewer than 3 nucleotide substitutions in each of the two nucleotide loops, fewer than 9 nucleotide substitutions in the nucleotide stem, and fewer than 13 nucleotide substitutions in total.
The independent claims cover (1) a folded single-oligonucleotide dumbbell aptamer with a split stem and defined sequence variant constraints and loop composition, (2) a ligated aptamer consisting of a specific SEQ ID, and (3) an aptamer comprising a specified SEQ ID with explicit limits on allowable substitutions in loops and stem.
Stated Advantages
Improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis.
Reduce generation of non-specific polymerase products and decrease nonspecific product generation in PCR reactions.
Increase quantities of target amplicons in reactions containing aptamer and thermostable polymerase.
Provide reversible inhibition of thermostable polymerases that dissociates upon increasing reaction temperature (hot start functionality).
Documented Applications
Standard PCR.
Reverse transcriptase PCR.
In-situ PCR.
Quantitative PCR.
Minisequencing and other reactions involving primer extension in which a thermostable polymerase is present and a hot-start method is advantageous.
Amplification and analysis of DNA and RNA in medical genetics research and diagnosis, pathogen detection, forensic analysis, and animal and plant genetics applications.
Use with automated analysis platforms including the GeneXpert® system (sample extraction, amplification, and detection in a self-contained cartridge).
Kits for detecting a nucleic acid target sequence comprising a thermostable polymerase and an aptamer that can reversibly inhibit activity of the polymerase.
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