# US 11,299,779 B2 · Nucleic acid modification and identification method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,779 B2 - Document ID: US11299779B2 - Status: Granted - Kind: B2 - Application number: 16/301,680 - Filed: 2018-04-13 - Published: 2019-06-13 - Granted: 2022-04-12 - Earliest claimed: 2017-04-13 - Assignees: Imba—Insiiiut FüR Molekulare Biotechnologie GmbH - Applicants: Imba—Institut FüR Molekulare Biotechnologie GmbH - Inventors: Stefan L. Ameres; Brian Reichholf; Veronika A. Herzog; Johannes Zuber; Matthias Muhar - Web page: https://patentgrep.com/patent/US11299779B2 ## CPC - C12Q 1/6869: Methods for sequencing - C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids - C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12N 2310/00: Structure or type of the nucleic acid - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A method of identifying a polynucleic acid (PNA) is presented, including the steps of providing a PNA; modifying one or more nucleobases of the PNA by addition or removal of a hydrogen bonding partner, thereby altering the base pairing capacity of the one or more nucleobases; base pairing a complementary nucleic acid to the PNA, including base pairing to at least one modified nucleobase; identifying the sequence of the complementary nucleic acid at least at the position that is complementary to at least one modified nucleobase. ## Claims ### Claim 1 A method of identifying a polynucleic acid (PNA) comprising the steps of: providing a PNA; modifying one or more nucleobases of the PNA by addition or removal of a hydrogen bonding partner, thereby altering the base pairing capacity of the one or more nucleobases; base pairing a complementary nucleic acid to the PNA, including base pairing to at least one modified nucleobase; identifying the sequence of the complementary nucleic acid at least at the position that is complementary to at least one modified nucleobase; wherein base pairing to at least one modified nucleobase leads to base paring with another nucleotide than base pairing with a nucleobase that has not been modified, with said nucleobases being otherwise the same; wherein one or more cells are cultured or grown in at least two culturing or growth phases, wherein one culturing or growth phase comprises incorporation of a modified nucleotide into biosynthesized PNA, which is modified by addition or removal of a hydrogen bonding partner, and another culturing or growth phase that lacks such incorporation of the modified nucleotide into biosynthesized PNA or wherein modified nucleotides are incorporated into biosynthesized PNA at a different concentration as in the other one culturing or growth phase; or wherein the method comprises incorporation of a modified nucleotide into biosynthesized PNA of at least two different cells or into at least two different groups of cells. ### Claim 2 (depends on 1) The method of claim 1, wherein the modification leads to an altered base pairing behaviour, thereby altering the preferential base pairing between A and T/U and between C and G as compared to the natural nucleobases selected from A, T/U, C and G. ### Claim 3 (depends on 1) The method according to claim 1, wherein the step of modification is by inclusion of a thiol modified nucleobase. ### Claim 4 (depends on 3) The method according to claim 3 further comprising alkylating said thiol nucleobase with an alkylating agent that comprises the hydrogen bonding partner. ### Claim 5 (depends on 3) The method according to claim 3 further comprising oxidizing said thiol nucleobase. ### Claim 6 (depends on 1) The method according to claim 1, wherein the modification comprises alkylating on position 4 of a uridine with an alkylating agent that comprises the hydrogen bonding partner. ### Claim 7 (depends on 1) The method according to claim 1, wherein the PNA comprises one or more 4-thiouridine or 6-thioguanosine. ### Claim 8 (depends on 1) The method according to claim 1, wherein the PNA is synthesized in a cell with a modification that alters said base pairing capacity. ### Claim 9 (depends on 1) The method according to claim 1, wherein a modified nucleobase is incorporated into the PNA through biosynthesis in a cell. ### Claim 10 (depends on 1) The method according to claim 1, wherein the PNA comprises RNA or DNA. ### Claim 11 (depends on 1) The method according to claim 1, wherein for each nucleotide type selected from A, G, C, U or T the modified PNA comprises more natural nucleotides than modified nucleotides. ### Claim 12 (depends on 1) The method according to claim 1, wherein the PNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more and up to 30 modified nucleotides. ### Claim 13 (depends on 1) The method of claim 1, wherein providing a PNA comprises expressing the PNA in cell; said method further comprises: isolating the PNA from the cell; modifying one or more nucleobases of the PNA in the cell and/or after isolation; wherein the modification(s) in the cell or after the isolation or both together add or remove a hydrogen bonding partner of one or more nucleobase, thereby altering the base pairing capacity of the one or more nucleobases. ### Claim 14 (depends on 1) The method according to claim 1, wherein the biosynthesized PNA of the two culturing or growth phases or of the of at least two different cells or at least two different groups of cells are collected from said cells and wherein base pairing a complementary nucleic acid to the PNA comprises generation of complementary polynucleic acid strands by transcription. ### Claim 15 (depends on 14) The method of claim 14, further comprising determining the sequence of the complementary polynucleic acid strands and comparing the strand sequences, wherein an altered complementary nucleic acid as a result of the modification by addition or removal of a hydrogen bonding partner can be identified by comparison with the complementary nucleic acid without modification. ### Claim 16 (depends on 1) The method of claim 1, comprising comparing identified sequences of the complementary nucleic acid at least at the position that is complementary to at least one modified nucleobase in at least two cells or in at least two different growth phases in a cell, wherein said at least two cells or growth phases have differential gene expression between said at least two cells or said growth phases. ### Claim 17 (depends on 1) A kit for performing the method of claim 1 comprising: a thiol modified nucleobase, an alkylating agent suitable for alkylating the thiol modified nucleobase at the thiol group, wherein the alkylating agent comprises a hydrogen boding donor or acceptor, and a reverse transcriptase. ### Claim 18 (depends on 17) The kit of claim 17 further comprising at least one of primers and nucleotides selected from A, G, C, and T. ### Claim 19 (depends on 1) The method of claim 1, wherein the incorporation of the two different cells or two different groups of cells is compared. ### Claim 20 (depends on 9) The method of claim 9, wherein the modified nucleobase is a thiol modified nucleobase. ### Claim 21 (depends on 9) The method of claim 9, wherein modifying one or more nucleobases comprises attaching or removing a hydrogen bonding partner to the modified nucleobase. ### Claim 22 (depends on 14) The method of claim 14, wherein said biosynthesized polynucleic acid of the two culturing or growth phases of or the at least two different cells or at least two different groups of cells are also mixed. ### Claim 23 (depends on 14) The method of claim 14, further including labelling the polynucleic acid according to the cell origin of the polynucleic acid. ### Claim 24 (depends on 14) The method of claim 14, wherein said strands being generated by said generation of strands are DNA strands. ### Claim 25 (depends on 14) The method of claim 14, wherein said transcription is reverse transcription. ### Claim 26 (depends on 14) The method of claim 14, further including labelling the PNA according to the cell origin of the PNA. ### Claim 27 (depends on 16) The method of claim 16, wherein differential gene expression is caused by inhibition or stimulation of at least one gene in a cell. ### Claim 28 (depends on 17) The kit of claim 17, wherein the alkylating agent is iodoacetamide.