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US-12624385-B2 - Riboregulators and methods of use thereof

US12624385B2US 12624385 B2US12624385 B2US 12624385B2US-12624385-B2

Abstract

This disclosure provides riboregulators specific for particular viruses or for particular human transcription factors. The viral-specific riboregulators may be used to detect the presence of the particular virus, and this may enable diagnosis of an infection. The transcription factor specific riboregulators may be used to detect the presence and/or measure the level of the particular transcription factor, and this may enable diagnosis or prognosis of a particular condition such as cancer.

Inventors

  • Luis Ruben Soenksen Martinez
  • Nicolaas Angenent-Mari
  • Alexander S. Garruss
  • James J. Collins
  • George M. Church
  • Katherine Collins
  • Diogo M. CAMACHO

Assignees

  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE
  • MASSACHUSETTS INSTITUTE OF TECHNOLOGY

Dates

Publication Date
20260512
Application Date
20201211

Claims (20)

  1. 1 . A toehold riboregulator having: (a) a nucleic acid sequence comprising any one of SEQ ID NOs: 43841, 9602, 62866, 19367, 164989, 111698, and 236638, or (b) nucleotides 21-103 of any one of SEQ ID NOs: 43841, 9602, 62866, 19367, 164989, 111698, and 236638, or (c) nucleotides 21-100 of any one of SEQ ID NOs: 43841, 9602, 62866, 19367, 164989, 111698, and 236638, or (d) RNA versions of (a), (b) or (c).
  2. 2 . The toehold riboregulator of claim 1 , comprising any one of SEQ ID NOs: 43841, 9602, 62866, and 19367, or RNA versions thereof.
  3. 3 . The toehold riboregulator of claim 1 , having; (a) a nucleic acid sequence comprising SEQ ID NO: 9602, or (b) nucleotides 21-103 of SEQ ID NO: 9602, or (c) nucleotides 21-100 of SEQ ID NO: 9602, or (d) RNA versions of (a), (b) or (c).
  4. 4 . The toehold riboregulator of claim 1 , having: (a) a nucleic acid sequence comprising SEQ ID NO: 19367, or (b) nucleotides 21-103 of SEQ ID NO: 19367, or (c) nucleotides 21-100 of SEQ ID NO: 19367, or (d) RNA versions of (a), (b) or (c).
  5. 5 . The toehold riboregulator of claim 1 , having: (a) a nucleic acid sequence comprising SEQ ID NO: 164989, or (b) nucleotides 21-103 of SEQ ID NO: 164989, or (c) nucleotides 21-100 of SEQ ID NO: 164989, or (d) RNA versions of (a), (b) or (c).
  6. 6 . The toehold riboregulator of claim 1 , having: (a) a nucleic acid sequence comprising SEQ ID NO: 111698, or (b) nucleotides 21-103 of SEQ ID NO: 111698, or (c) nucleotides 21-100 of SEQ ID NO: 111698, or (d) RNA versions of (a), (b) or (c).
  7. 7 . The toehold riboregulator of claim 1 , having: (a) a nucleic acid sequence comprising SEQ ID NO: 236638, or (b) nucleotides 21-103 of SEQ ID NO: 236638, or (c) nucleotides 21-100 of SEQ ID NO: 236638, or (d) RNA versions of (a), (b) or (c).
  8. 8 . The toehold riboregulator of claim 1 covalently attached, at its 3′ end, to a nucleic acid encoding a reporter protein or reporter RNA.
  9. 9 . The toehold riboregulator of claim 1 , wherein the toehold riboregulator is specific for dengue virus, human rhino virus, or smallpox virus.
  10. 10 . The toehold riboregulator of claim 1 , wherein the toehold riboregulator is specific for a human mRNA encoding a transcription factor selected from E2F3, NCOR1, or ZNF175.
  11. 11 . A method comprising: contacting a sample with a toehold riboregulator of claim 1 , covalently attached, at its 3′ end, to a nucleic acid encoding a reporter protein or reporter RNA, under conditions sufficient to allow the toehold riboregulator to hybridize to its respective trigger nucleic acid, and detecting and optionally measuring expression of the reporter protein or reporter RNA, wherein the toehold riboregulator is an RNA.
  12. 12 . The method of claim 11 , wherein the sample is obtained from a human subject.
  13. 13 . The method of claim 12 , wherein the subject is suspected of having cancer.
  14. 14 . The method of claim 12 , wherein the subject is suspected of having a viral infection.
  15. 15 . The method of claim 11 , wherein the toehold riboregulator comprises an RNA version of any one of SEQ ID NOs: 43841, 9602, 62866, and 19367.
  16. 16 . The method of claim 11 , wherein the toehold riboregulator is a plurality of toehold riboregulators comprising RNA versions of SEQ ID NO: 43841 and SEQ ID NO: 62866.
  17. 17 . The method of claim 11 , wherein the subject is suspected of having a smallpox virus infection or having been exposed to smallpox virus.
  18. 18 . The method of claim 11 , wherein the subject is suspected of having dengue virus infection or having been exposed to dengue virus.
  19. 19 . The method of claim 11 , wherein the subject is suspected of having human rhino virus infection or having been exposed to human rhino virus.
  20. 20 . The method of claim 11 , wherein the sample has been treated to amplify RNA prior to contact with the toehold riboregulator.

Description

RELATED APPLICATIONS This application is a national stage filing under 35 U.S.C. § 371 of International Patent Application Serial No. PCT/US2020/064695, filed Dec. 11, 2020, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/948,175, filed Dec. 13, 2019, entitled “RIBOREGULATORS AND METHODS OF USE THEREOF”, the entire contents of each of which are incorporated by reference herein. FEDERALLY SPONSORED RESEARCH This invention was made with U.S. Government support under DE-FG02-02ER63445 awarded by the U.S. Department of Energy and HDTRA1-14-1-0006 awarded by the Department of Defense/Defense Advanced Research Projects Agency. The Government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 10, 2022, is named H049870709US01-SEQ-MAT, and is 69,080,020 bytes in size. BACKGROUND OF INVENTION Engineered ribonucleic acid (RNA) molecules with targeted biological functions play an important role in synthetic biology (1), particularly as programmable response elements for small molecules, proteins, and nucleic acids. Examples include riboswitches, riboregulators, and ribozymes, many of which hold great promise for a variety of in vitro and in vivo applications (1, 2). Toehold riboregulators are a class of versatile prokaryotic riboregulators inducible by the presence of a fully programmable trans-RNA trigger sequence (2-6, 15, 16). These RNA synthetic biology modules have displayed impressive dynamic range and orthogonality when used both in vivo as genetic circuit components (2, 5, 6), and in vitro as nucleic acid diagnostic tools using cell-free protein synthesis (CFPS) systems (3, 4, 15, 16). SUMMARY OF INVENTION This disclosure provides novel toehold riboregulators and uses thereof. The toehold riboregulators are specific for a particular viral nucleic acid or a particular human transcription factor nucleic acid. The toehold riboregulators may be used to detect the presence of and/or measure the level of such nucleic acids. The presence and/or level of such nucleic acids may be associated with a viral infection or another condition such as a cancer. Thus, in one aspect, this disclosure provides a toehold riboregulator comprising (a) a nucleic acid sequence comprising any one of SEQ ID NOs: 1-244,000, or(b) nucleotides 21-103 of any one of SEQ ID NOs: 1-244,000, or(c) nucleotides 21-100 of any one of SEQ ID NOs: 1-244,000, or(d) RNA versions of (a), (b) or (c). In a related aspect, this disclosure provides a toehold riboregulator comprising (a) a nucleic acid sequence comprising any one of SEQ ID Nos: 164989, 43841, 9602, 40182, 62866, 111698, 236638, and 19367, or(b) nucleotides 21-103 of any one of SEQ ID NOs: 164989, 43841, 9602, 40182, 62866, 111698, 236638, and 19367, or(c) nucleotides 21-100 of any one of SEQ ID NOs: 164989, 43841, 9602, 40182, 62866, 111698, 236638, and 19367, or(d) RNA versions of (a), (b) or (c). In another related aspect, this disclosure provides a toehold riboregulator comprising (a) a nucleic acid sequence comprising any one of SEQ ID Nos: 43841, 9602, 62866, and 19367, or(b) nucleotides 21-103 of any one of SEQ ID NOs: 43841, 9602, 62866, and 19367, or(c) nucleotides 21-100 of any one of SEQ ID NOs: 43841, 9602, 62866, and 19367, or(d) RNA versions of (a), (b) or (c). In another related aspect, this disclosure provides a toehold riboregulator comprising (a) a nucleic acid sequence comprising any one of SEQ ID Nos: 43841 and 62866, or(b) nucleotides 21-103 of any one of SEQ ID NOs: 43841 and 62866, or(c) nucleotides 21-100 of any one of SEQ ID NOs: 43841 and 62866, or(d) RNA versions of (a), (b) or (c). Any one of these toehold riboregulators may be covalently attached (or conjugated or operably linked), at its 3′ end, to a nucleic acid encoding a reporter protein or reporter RNA. In some embodiments, as defined herein, the riboregulator is specific for a virus selected from the group consisting of astrovirus, cardiovirus, chikungunya virus, cosavirus, coxsackie virus, dengue virus, ebola virus, hantavirus, human immunodeficiency virus, human parvo virus, human rhino virus, influenza virus: h1n1, influenza virus: h3n2, lassa virus, leishmanial virus, Marburg virus, papilloma virus, poliovirus, rabies virus, smallpox virus, west nile virus, yellow fever virus, an zika virus. In some embodiments, as defined herein, the riboregulator is specific for a virus selected from the group consisting of dengue virus, human rhino virus, or smallpox virus. In some embodiments, as defined herein, the riboregulator is specific for dengue virus. In some embodiments, as defined herein, the riboregulator is specific for human rhino virus. In some embodiments, as defined herein, the riboregulator is specific for small