US-12624359-B2 - L-RNA aptamers and methods of identifying the same

Abstract

Disclosed herein is a method of identifying an L-RNA aptamer specific for a target nucleic acid. According to some embodiments of the present disclosure, the method comprises, in vitro transcribing the DNAs of the DNA library into D-RNAs, followed by identifying target-specific D-RNAs via negative and positive selections, and then producing the L-RNA aptamer based on the identified D-RNA. Also disclosed herein are two aptamers identified by the present method. According to some embodiments of the present disclosure, the two aptamers respectively comprise the nucleotide sequences of SEQ ID NOs: 1 and 2.

Inventors

• Chun Kit Kwok
• Danyang JI

Assignees

• CITY UNIVERSITY OF HONG KONG

Dates

Publication Date

20260512

Application Date

20221104

Claims (3)

1. 1 . An L-form ribonucleic acid (L-RNA) aptamer specific for the 3′-untranslated region (3′-UTR) of amyloid precursor protein (APP) messenger RNA (mRNA), comprising the nucleotide sequence of SEQ ID NO: 1.
2. 2 . An L-form ribonucleic acid (L-RNA) aptamer specific for a target nucleic acid having a parallel G-quadruplex structure, comprising the nucleotide sequence of SEQ ID NO: 2.
3. 3 . The L-RNA aptamer of claim 2 , wherein the target nucleic acid is the promoter of c-kit gene.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application relates to and claims the benefits of U.S. Provisional Application No. 63/281,503 filed Nov. 19, 2021, and U.S. Provisional Application No. 63/394,847 filed Aug. 3, 2022: the contents of these applications are incorporated herein by reference in their entirety. INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE This application incorporates by reference the Sequence Listing in the following XML file being submitted concurrently with: File name: HP0228US_US_seqlisting; created on Nov. 2, 2022; and having a size of 4.47 KB. The information in the Sequence Listing is incorporated herein in its entirety for all purposes. BACKGROUND OF THE INVENTION 1. Field of the Invention The present disclosure in general relates to the field of L-form ribonucleic acid (L-RNA) aptamers. More particularly, the present disclosure relates to a method for identifying L-RNA aptamers, which recognize and bind to target nucleic acids having G-quadruplex (G4) structures. 2. Description of Related Art Guanine (G)-rich sequences of single-stranded DNA and RNA can fold into stable, intra- or intermolecular secondary structures called G-quadruplexes (dG4s and rG4s). Four guanines interact with each other by Hoogsteen-hydrogen bonds to form a planar structure, G-quartet. Stacking of two or more G-quartets, connected by loop nucleotides forms a G4 structure, and it is further stabilized by monovalent cations (K+>Na+>Li+). Over the years, rG4s have been reported to have key roles in gene regulation and cellular processes, such as transcription, RNA splicing, translation, RNA stability, RNA localization and others. In addition, new studies have associated rG4s with diseases and cancers, making them one of the promising therapeutic targets for drug development. G4 targeting is a topic of emerging interest, and since the first report of G4-specific chemical in 1997, more than hundreds of G4 ligands have been developed. Currently, major approaches employed for G4 targeting include the development of G4-specific chemicals, peptides and antibodies. The generation and application of these G4 tools have greatly promoted the understanding of G4 structure and biology, and the elucidation of the three-dimensional (3D) high resolution structure of the binding complexes provided fundamental insights on the future enhancement of these G4 tools. Despite the significant progresses made, the selective targeting of G4 of interest is still challenging due to the structural similarity of G4s, with only limited success so far. In view of the foregoing, there is a continuing interest in developing a novel method for developing G4-targeting tools. SUMMARY The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later. As embodied and broadly described herein, one aspect of the disclosure is directed to a method of identifying an L-RNA aptamer specific for a target nucleic acid structure. The selection process starts from a deoxyribonucleic acid (DNA) library, in which the DNA library comprises a first plurality of DNAs respectively having randomized sequences. According to the embodiments of the present disclosure, the method comprises, (a) producing a first plurality of D-form RNAs (D-RNAs) respectively corresponding to the first plurality of DNAs via in vitro transcription;(b) mixing the first plurality of D-RNAs of step (a) with a first plurality of streptavidin-coated magnetic beads, which are pre-treated with transfer RNAs (tRNAs);(c) collecting and mixing the supernatant of the product of step (b) with a biotinylated target nucleic acid;(d) mixing the product of step (c) with a second plurality of streptavidin-coated magnetic beads, which are pre-treated with the tRNAs;(e) eluting the precipitate of step (d) with an elution buffer so as to produce a second plurality of D-RNAs that can bind to the target nucleic acid;(f) producing a plurality of complementary DNAS (cDNAs) respectively corresponding to the second plurality of D-RNAs of step (e) via reverse transcription;(g) producing a second plurality of DNAs respectively corresponding to the plurality of cDNAs of step (f) via polymerase chain reaction (PCR);(h) repeating steps (a) to (g) at least 4 times, in which the in vitro transcription was performed by using the product of step (g), so as to identify a D-RNA aptamer specific for the target nucleic acid; and(i) producing the L-RNA aptamer corresponding to the D-RNA aptamer of step (h). In general, the target nucleic acid may be DNA or RNA. According to some embodiments of the present disclosure, the target n