# US 11,111,532 B2 · Method of characterizing a target ribonucleic acid (RNA) comprising forming a complementary polynucleotide which moves through a transmembrane pore > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,111,532 B2 - Document ID: US11111532B2 - Status: Granted - Kind: B2 - Application number: 15/028,637 - Filed: 2014-10-17 - Published: 2016-09-01 - Granted: 2021-09-07 - Earliest claimed: 2013-10-18 - Assignees: Oxford Nanopore Technologies PLC - Applicants: Oxford Nanopore Technologies Ltd. - Inventors: Clive Gavin Brown; Daniel John Turner; James White - Web page: https://patentgrep.com/patent/US11111532B2 ## CPC - C12Q 1/6869: Methods for sequencing - C12Q 1/6883: for diseases caused by alterations of genetic material - G01N 27/4166: {measuring a particular property of an electrolyte} - C12Q 2600/118: Prognosis of disease development - C12Q 2600/158: Expression markers - C12Q 2600/178: miRNA, siRNA or ncRNA - 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 - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The invention relates to a new method of characterising a target ribonucleic acid (RNA) involving forming a complementary polynucleotide. The method uses a transmembrane pore. ## Claims ### Claim 1 A method of characterising a target ribonucleic acid (RNA) comprising a poly A tail, the method comprising: (a) forming a complementary polynucleotide from the target RNA by hybridising a primer to the poly A tail of the target RNA and using the primer to reverse transcribe the target RNA to form the complementary polynucleotide, wherein the primer comprises a leader sequence and/or a region to which a polynucleotide binding protein is capable of binding; (b) contacting the complementary polynucleotide with a transmembrane pore such that the complementary polynucleotide moves through the pore; and (c) taking one or more measurements as the complementary polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the complementary polynucleotide and thereby characterising the target RNA, wherein the method does not comprise polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR), and wherein the complementary polynucleotide is contacted with the pore when the complementary polynucleotide is fully or partially hybridized to the targt RNA. ### Claim 2 (depends on 1) A method according to claim 1, wherein the target RNA is eukaryotic. ### Claim 3 (depends on 1) A method according to claim 1, wherein the primer is a polyT-VN primer, which comprises a polyT region and a VN anchor where V is dAMP, dCMP or dGMP and N is dAMP, dCMP, dGMP or dTMP. ### Claim 4 (depends on 1) A method according to claim 1, wherein the complementary polynucleotide is coupled to the membrane. ### Claim 5 (depends on 1) A method according to claim 1, wherein the one or more characteristics are selected from (i) the length of the target RNA, (ii) the identity of the target RNA, (iii) the sequence of the target RNA, and (iv) the amount of the target RNA. ### Claim 6 (depends on 1) A method according to claim 1, wherein the one or more characteristics of the complementary polynucleotide are measured by electrical measurement and/or optical measurement. ### Claim 7 (depends on 6) A method according to claim 6, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement. ### Claim 8 (depends on 1) A method according to claim 1, wherein step (b) comprises contacting the complementary polynucleotide with a polynucleotide binding protein such that the protein controls the movement of the complementary polynucleotide through the pore. ### Claim 9 (depends on 8) A method according to claim 8, wherein the polynucleotide binding protein is a polymerase, exonuclease, helicase or a topoisomerase. ### Claim 10 (depends on 1) A method according to claim 1, wherein the method comprises (b) contacting the complementary polynucleotide with a transmembrane pore and a polynucleotide binding protein such that the protein controls the movement of the complementary polynucleotide through the pore; and (c) measuring the current passing through the pore as the complementary polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the complementary polynucleotide and thereby characterising the template RNA. ### Claim 11 (depends on 1) A method according to claim 1, wherein the pore is a transmembrane protein pore or a solid state pore. ### Claim 12 (depends on 11) A method according to claim 11, wherein the transmembrane protein pore is derived from a hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA. ### Claim 13 (depends on 12) A method according to claim 12, wherein the transmembrane protein is: (a) formed of eight identical subunits as shown in SEQ ID NO: 2 or (b) a variant thereof in which one or more of the eight subunits has at least 50% homology to SEQ ID NO: 2 based on amino acid identity over the entire sequence and retains pore activity; or (c) formed of seven identical subunits as shown in SEQ ID NO: 4 or (d) a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 4 based on amino acid identity over the entire sequence and retains pore activity. ### Claim 14 (depends on 1) A method according to claim 1, wherein the target RNA is messenger RNA (mRNA) or microRNA (miRNA). ### Claim 15 (depends on 14) A method according to claim 14, wherein the mRNA or miRNA can be used to diagnose or prognose a disease or condition. ### Claim 16 (depends on 1) A method of determining whether or not a patient has or is at risk of developing a disease or condition associated with an altered amount and/or alternate splicing of messenger RNA (mRNA), comprising determining the amount and/or identity of the mRNA in a sample from the patient using a method according to claim 1 and thereby determining whether or not the patient has or is at risk of developing the disease or condition. ### Claim 17 A method of characterising a target ribonucleic acid (RNA) comprising a polyA tail, comprising: (a) forming a complementary polynucleotide from the target RNA by hybridising a primer to the polyA tail of the target RNA and using the primer to reverse transcribe the target RNA to form the complementary polynucleotide; (b) contacting the complementary polynucleotide with a transmembrane pore such that the complementary polynucleotide moves through the pore; and (c) taking one or more measurements as the complementary polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the complementary polynucleotide and thereby characterising the target RNA; wherein the complementary polynucleotide is contacted with the pore when the complementary polynucleotide is fully or partially hybridized to the target RNA. ### Claim 18 (depends on 17) A method according to claim 17, wherein the method does not comprise polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR). ### Claim 19 (depends on 17) A method according to claim 17, wherein the target RNA is eukaryotic. ### Claim 20 (depends on 17) A method according to claim 17, wherein the primer is a polyT-VN primer, which comprises a polyT region and a VN anchor where V is dAMP, dCMP or dGMP and N is dAMP, dCMP, dGMP or dTMP. ### Claim 21 (depends on 17) A method according to claim 17, wherein the complementary polynucleotide is coupled to the membrane. ### Claim 22 (depends on 17) A method according to claim 17 wherein the one or more characteristics are selected from (i) the length of the target RNA, (ii) the identity of the target RNA, (iii) the sequence of the target RNA, and (iv) the amount of the target RNA. ### Claim 23 (depends on 17) A method according to claim 17, wherein the one or more characteristics of the complementary polynucleotide are measured by electrical measurement and/or optical measurement. ### Claim 24 (depends on 23) A method according to claim 23, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement. ### Claim 25 (depends on 17) A method according to claim 17, wherein step (b) comprises contacting the complementary polynucleotide with a polynucleotide binding protein such that the protein controls the movement of the complementary polynucleotide through the pore. ### Claim 26 (depends on 25) A method according to claim 25, wherein the polynucleotide binding protein is a polymerase, exonuclease, helicase or a topoisomerase. ### Claim 27 (depends on 17) A method according to claim 17, wherein the method comprises (b) contacting the complementary polynucleotide with a transmembrane pore and a polynucleotide binding protein such that the protein controls the movement of the complementary polynucleotide through the pore; and (c) measuring the current passing through the pore as the complementary polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the complementary polynucleotide and thereby characterising the template RNA. ### Claim 28 (depends on 17) A method according to claim 17, wherein the pore is a transmembrane protein pore or a solid state pore. ### Claim 29 (depends on 28) A method according to claim 28, wherein the transmembrane protein pore is derived from a hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA. ### Claim 30 (depends on 29) A method according to claim 29, wherein the transmembrane protein is: (a) formed of eight identical subunits as shown in SEQ ID NO: 2 or (b) a variant thereof in which one or more of the eight subunits has at least 50% homology to SEQ ID NO: 2 based on amino acid identity over the entire sequence and retains pore activity; or (c) formed of seven identical subunits as shown in SEQ ID NO: 4 or (d) a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 4 based on amino acid identity over the entire sequence and retains pore activity. ### Claim 31 (depends on 17) A method according to claim 17, wherein the target RNA is messenger RNA (mRNA) or microRNA (miRNA). ### Claim 32 (depends on 31) A method according to claim 31, wherein the mRNA or miRNA can be used to diagnose or prognose a disease or condition. ### Claim 33 (depends on 17) A method of determining whether or not a patient has or is at risk of developing a disease or condition associated with an altered amount and/or alternate splicing of messenger RNA (mRNA), comprising determining the amount and/or identity of the mRNA in a sample from the patient using a method according to claim 17 and thereby determining whether or not the patient has or is at risk of developing the disease or condition.