US 12,486,533 B2
Direct RNA nanopore sequencing with help of a stem-loop reverse polynucleotide
Yiwei Huang, Erlangen (DE)
Assigned to Siemens Healthineers AG, Erlangen (DE)
Filed by Siemens Healthcare GmbH, Erlangen (DE)
Filed on Oct. 17, 2022, as Appl. No. 18/047,063.
Application 18/047,063 is a division of application No. 16/677,451, filed on Nov. 7, 2019, granted, now 11,505,824.
Claims priority of application No. 18205148 (EP), filed on Nov. 8, 2018.
Prior Publication US 2023/0295713 A1, Sep. 21, 2023
Int. Cl. C12Q 1/6869 (2018.01); C12N 15/10 (2006.01); C12Q 1/6806 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12N 15/102 (2013.01); C12Q 1/6806 (2013.01); C12N 2310/531 (2013.01)] 15 Claims
 
1. A method of characterizing a target RNA polynucleotide comprising:
(i) providing an RNA polynucleotide;
(ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form, wherein the stem-loop form comprises a stem portion and a loop portion, wherein the stem portion comprises one or more 5-methyl-isoC-isoG base-pairs and a restriction enzyme recognition site;
(iii) optionally performing a reverse transcription of the RNA polynucleotide;
(iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang;
(v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv);
(vi) contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore and the cholesterol tether anchors the RNA polynucleotide in the vicinity of the transmembrane pore; and
(vii) taking one or more measurements during the movement of the RNA polynucleotide through the transmembrane pore, wherein the measurements are indicative of one or more characteristics of the RNA polynucleotide.