Patents by Inventor Denis A. MALYSHEV
Denis A. MALYSHEV has filed for patents to protect the following inventions. This listing includes patent applications that are pending as well as patents that have already been granted by the United States Patent and Trademark Office (USPTO).
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Publication number: 20240124926Abstract: A method of characterizing a nucleic acid that includes steps of (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides to produce an extended primer hybrid and to form a stabilized ternary complex including the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template, wherein the mixture contains nucleotide cognates of four different base types, wherein the nucleotide cognate of the first base type has a reversible terminator, and wherein nucleotide cognates of the second, third and fourth base types are extendable; (b) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and (c) determining the presence of a base multiplet in the template nucleic acid, the base multiplet including the first base type followed by the next base.Type: ApplicationFiled: December 6, 2023Publication date: April 18, 2024Inventors: Arnold Oliphant, Denis Malyshev
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Publication number: 20240101590Abstract: Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker than can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.Type: ApplicationFiled: February 22, 2023Publication date: March 28, 2024Inventors: Floyd E. ROMESBERG, Denis A. MALYSHEV, Lingjun LI, Thomas LAVERGNE, Zhengtao LI
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Patent number: 11884971Abstract: A method of characterizing a nucleic acid that includes steps of (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides to produce an extended primer hybrid and to form a stabilized ternary complex including the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template, wherein the mixture contains nucleotide cognates of four different base types, wherein the nucleotide cognate of the first base type has a reversible terminator, and wherein nucleotide cognates of the second, third and fourth base types are extendable; (b) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and (c) determining the presence of a base multiplet in the template nucleic acid, the base multiplet including the first base type followed by the next base.Type: GrantFiled: August 2, 2021Date of Patent: January 30, 2024Assignee: Pacific Biosciences of California, Inc.Inventors: Denis Malyshev, Arnold Oliphant
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Publication number: 20230235339Abstract: A recombinantly expressed nucleotide triphosphate transporter efficiently imports the triphosphates of unnatural nucleotides into cells, and the endogenous cellular machinery incorporates those nucleotides into cellular nucleic acids. UBPs can therefore form within the cell's nucleic acids. Moreover, neither the presence of the unnatural triphosphates nor the replication of the UBP represents a significant growth burden. The UBP is not efficiently excised by nucleic acid repair pathways, and therefore can be retained as long as the unnatural triphosphates are available in the growth medium. Thus, the resulting cell is the first organism to stably propagate an expanded genetic alphabet.Type: ApplicationFiled: August 4, 2022Publication date: July 27, 2023Inventors: Floyd E. ROMESBERG, Denis A. MALYSHEV
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Publication number: 20230227900Abstract: A method for determining the presence of an allele, including (a) binding a polymerase to a double stranded nucleic acid that includes a primer hybridized to a template, the template including a first allele of a locus; (b) adding a nucleotide to the primer via catalytic activity of the polymerase, thereby producing an extended nucleic acid; (c) dissociating the polymerase from the extended nucleic acid; (d) detecting dissociation of the polymerase from the extended nucleic acid; and (e) comparing the dissociation of the polymerase from the extended nucleic acid to dissociation of the polymerase from a second double stranded nucleic acid, the second double stranded nucleic acid including a primer hybridized to the same position of the locus as the primer of the extended nucleic acid.Type: ApplicationFiled: September 27, 2022Publication date: July 20, 2023Applicant: Pacific Biosciences of California, Inc.Inventors: Denis MALYSHEV, Sean STROMBERG
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Publication number: 20230183798Abstract: Provided herein include a method for modifying polymerase-nucleic acid complexes, including (a) providing a plurality of surface-immobilized polymerase-nucleic acid complexes in a vessel, wherein the nucleic acid includes a primed-template nucleic acid, wherein at least a subset of the surface-immobilized polymerase-nucleic acid complexes include ternary complexes further including nucleotides; and (b) washing the surface with an aqueous solution including a diol, sulfoxide or polyol, thereby removing the nucleotides from the vessel and retaining the surface-immobilized polymerase-nucleic acid complexes in the vessel.Type: ApplicationFiled: April 30, 2021Publication date: June 15, 2023Inventors: Hari K.K. Subramanian, Aaron W. Feldman, Chih-Yuan Chen, Denis Malyshev, Greg Richmond
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Patent number: 11634451Abstract: Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker than can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.Type: GrantFiled: July 22, 2019Date of Patent: April 25, 2023Assignee: THE SCRIPPS RESEARCH INSTITUTEInventors: Floyd E. Romesberg, Denis A. Malyshev, Lingjun Li, Thomas Lavergne, Zhengtao Li
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Publication number: 20220372552Abstract: A method for identifying a nucleotide in a primed template nucleic acid, including the steps of (a) providing a vessel having a primed template nucleic acid, polymerase and a nucleotide cognate of a first base type; (b) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the first base type bound at a base position of the primed template nucleic acid; (c) delivering a nucleotide cognate of a second base type to the vessel, whereby the vessel retains the primed template nucleic acid and the polymerase from step (b); (d) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the second base type bound at the base position of the primed template nucleic acid; and (e) identifying the type of nucleotide at the base position of the primed template nucleic acid.Type: ApplicationFiled: June 21, 2022Publication date: November 24, 2022Applicant: Pacific Biosciences of California, Inc.Inventors: Brittany A. ROHRMAN, Denis MALYSHEV, Morassa Mohseni MIDDLETON, Arnold OLIPHANT
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Patent number: 11492666Abstract: A method for determining the presence of an allele, including (a) binding a polymerase to a double stranded nucleic acid that includes a primer hybridized to a template, the template including a first allele of a locus; (b) adding a nucleotide to the primer via catalytic activity of the polymerase, thereby producing an extended nucleic acid; (c) dissociating the polymerase from the extended nucleic acid; (d) detecting dissociation of the polymerase from the extended nucleic acid; and (e) comparing the dissociation of the polymerase from the extended nucleic acid to dissociation of the polymerase from a second double stranded nucleic acid, the second double stranded nucleic acid including a primer hybridized to the same position of the locus as the primer of the extended nucleic acid.Type: GrantFiled: February 14, 2018Date of Patent: November 8, 2022Assignee: PACIFIC BIOSCIENCES OF CALIFORNIA, INC.Inventors: Denis Malyshev, Sean Stromberg
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Patent number: 11466279Abstract: A recombinantly expressed nucleotide triphosphate transporter efficiently imports the triphosphates of unnatural nucleotides into cells, and the endogenous cellular machinery incorporates those nucleotides into cellular nucleic acids. UBPs can therefore form within the cell's nucleic acids. Moreover, neither the presence of the unnatural triphosphates nor the replication of the UBP represents a significant growth burden. The UBP is not efficiently excised by nucleic acid repair pathways, and therefore can be retained as long as the unnatural triphosphates are available in the growth medium. Thus, the resulting cell is the first organism to stably propagate an expanded genetic alphabet.Type: GrantFiled: October 2, 2019Date of Patent: October 11, 2022Assignee: THE SCRIPPS RESEARCH INSTITUTEInventors: Floyd E. Romesberg, Denis A. Malyshev
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Patent number: 11421262Abstract: A method for identifying a nucleotide in a primed template nucleic acid, including the steps of (a) providing a vessel having a primed template nucleic acid, polymerase and a nucleotide cognate of a first base type; (b) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the first base type bound at a base position of the primed template nucleic acid; (c) delivering a nucleotide cognate of a second base type to the vessel, whereby the vessel retains the primed template nucleic acid and the polymerase from step (b); (d) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the second base type bound at the base position of the primed template nucleic acid; and (e) identifying the type of nucleotide at the base position of the primed template nucleic acid.Type: GrantFiled: July 22, 2019Date of Patent: August 23, 2022Assignee: PACIFIC BIOSCIENCES OF CALIFORNIA, INC.Inventors: Brittany A. Rohrman, Denis Malyshev, Morassa Mohseni Middleton, Arnold Oliphant
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Patent number: 11339428Abstract: A method for identifying a nucleotide in a template nucleic acid by (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have an extendable 3? end; (b) contacting the plurality with: (i) blocked nucleotides to produce a first subset of the primer-template nucleic acid hybrids that include a blocked nucleotide at the 3? end, and (ii) a ternary complex inhibitor to produce a second subset of the primer-template nucleic acid hybrids that include a ternary complex inhibitor; (c) forming ternary complexes that each include a polymerase, a primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; and (d) detecting the ternary complexes, thereby identifying a nucleotide in the template nucleic acid.Type: GrantFiled: April 12, 2019Date of Patent: May 24, 2022Assignee: PACIFIC BIOSCIENCES OF CALIFORNIA, INC.Inventors: Chad Fleischer, Denis Malyshev
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Publication number: 20220144875Abstract: Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3?-O-oxime moiety such as with a reagent having an —ONH2 moiety to produce a nucleotide having a 3?-O—NH2 moiety such as wherein the reagent having the —ONH2 moiety further comprises alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, or (heteroalicyclyl)alkyl.Type: ApplicationFiled: September 17, 2021Publication date: May 12, 2022Inventors: Denis Malyshev, Aaron W. Feldman
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Publication number: 20220136045Abstract: A method of characterizing a nucleic acid that includes steps of (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides to produce an extended primer hybrid and to form a stabilized ternary complex including the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template, wherein the mixture contains nucleotide cognates of four different base types, wherein the nucleotide cognate of the first base type has a reversible terminator, and wherein nucleotide cognates of the second, third and fourth base types are extendable; (b) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and (c) determining the presence of a base multiplet in the template nucleic acid, the base multiplet including the first base type followed by the next base.Type: ApplicationFiled: August 2, 2021Publication date: May 5, 2022Inventors: Denis Malyshev, Arnold Oliphant
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Publication number: 20220106628Abstract: A method for identifying a nucleic acid template that include (a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality includes a first template hybridized to a first primer, and wherein a second hybrid of the plurality includes a second template hybridized to a second primer, the second primer having a ternary complex inhibitor moiety at the 3? end; (b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and (c) detecting the stabilized ternary complex to identify the first template.Type: ApplicationFiled: October 15, 2021Publication date: April 7, 2022Applicant: Omniome, Inc.Inventors: Hari K.K. SUBRAMANIAN, Chad FLEISCHER, Denis MALYSHEV
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Patent number: 11180520Abstract: Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3?-O-oxime moiety such as with a reagent having an —ONH2 moiety to produce a nucleotide having a 3?-O—NH2 moiety such as wherein the reagent having the —ONH2 moiety further comprises alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, or (heteroalicyclyl)alkyl.Type: GrantFiled: September 10, 2020Date of Patent: November 23, 2021Assignee: OMNIOME, INC.Inventors: Denis Malyshev, Aaron W. Feldman
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Patent number: 11180794Abstract: A method for identifying a nucleic acid template that includes (a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality includes a first template hybridized to a first primer, and wherein a second hybrid of the plurality includes a second template hybridized to a second primer, the second primer having a ternary complex inhibitor moiety at the 3? end; (b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and (c) detecting the stabilized ternary complex to identify the first template.Type: GrantFiled: October 18, 2019Date of Patent: November 23, 2021Assignee: OMNIOME, INC.Inventors: Hari K. K. Subramanian, Chad Fleischer, Denis Malyshev
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Publication number: 20210070795Abstract: Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3?-O-oxime moiety with a reagent having the structure R2-ONH2 to produce a nucleotide having a 3?-O—NH2 moiety, wherein R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted variant thereof.Type: ApplicationFiled: September 10, 2020Publication date: March 11, 2021Inventors: Denis Malyshev, Aaron W. Feldman
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Patent number: 10626138Abstract: Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker than can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.Type: GrantFiled: August 8, 2014Date of Patent: April 21, 2020Assignees: THE SCRIPPS RESEARCH INSTITUTE NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT NIH DIVISION OF EXTRAMURAL INVENTIONS AND TECHNOLOGY RESOURCES (DEITR)Inventors: Floyd E. Romesberg, Denis A. Malyshev, Lingjun Li, Thomas Lavergne, Zhengtao Li
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Publication number: 20200095591Abstract: A recombinantly expressed nucleotide triphosphate transporter efficiently imports the triphosphates of unnatural nucleotides into cells, and the endogenous cellular machinery incorporates those nucleotides into cellular nucleic acids. UBPs can therefore form within the cell's nucleic acids. Moreover, neither the presence of the unnatural triphosphates nor the replication of the UBP represents a significant growth burden. The UBP is not efficiently excised by nucleic acid repair pathways, and therefore can be retained as long as the unnatural triphosphates are available in the growth medium. Thus, the resulting cell is the first organism to stably propagate an expanded genetic alphabet.Type: ApplicationFiled: October 2, 2019Publication date: March 26, 2020Inventors: Floyd E. ROMESBERG, Denis A. MALYSHEV