US 10,208,312 B2Granted

Cis/trans riboregulators

AssigneeTrustees of Boston University

InventorsJames J. Collins, Farren J. Isaacs, Charles R. Cantor, Daniel J. Dwyer

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Abstract

The present invention provides nucleic acid molecules, DNA constructs, plasmids, and methods for post-transcriptional regulation of gene expression using RNA molecules to both repress and activate translation of an open reading frame. Repression of gene expression is achieved through the presence of a regulatory nucleic acid element (the cis-repressive RNA or crRNA) within the 5′ untranslated region (5′ UTR) of an mRNA molecule. The nucleic acid element forms a hairpin (stem/loop) structure through complementary base pairing. The hairpin blocks access to the mRNA transcript by the ribosome, thereby preventing translation. In particular, in embodiments of the invention designed to operate in prokaryotic cells, the stem of the hairpin secondary structure sequesters the ribosome binding site (RBS). In embodiments of the invention designed to operate in eukaryotic cells, the stem of the hairpin is positioned upstream of the start codon, anywhere within the 5′ UTR of an mRNA. A small RNA (trans-activating RNA, or taRNA), expressed in trans, interacts with the crRNA and alters the hairpin structure. This alteration allows the ribosome to gain access to the region of the transcript upstream of the start codon, thereby activating transcription from its previously repressed state.

A recombinant eukaryotic cell comprising, a first exogenous mRNA nucleic acid sequence and a second exogenous mRNA nucleic acid sequence encoding a trans-activating RNA, wherein:

a. the first exogenous mRNA nucleic acid sequence comprises a internal ribosome entry site (IRES) upstream of a start codon and nucleic acid sequence encoding a gene to be expressed, and a cis-repressive nucleic acid sequence located within a 5′ UTR upstream of the start codon,
wherein the cis-repressive nucleic acid sequence comprises at least 4 nucleic acids that are complementary to, or substantially complementary to at least 4 nucleic acids in the 5′UTR and can, upon complementary base pairing of the at least 4 nucleic acids of the cis-repressive nucleic acid sequence with the at least 4 nucleic acids in the 5′UTR form a duplex that is part of a repressive stem-loop structure, wherein the loop of the stem-loop structure is between 4-10 nucleotides;
and
b. the second exogenous nucleic acid sequence comprises an inducible promoter operatively linked to a nucleic acid sequence encoding the trans-activating RNA, wherein the trans-activating RNA comprises (i) a 5′ non-stem nucleic acid sequence, comprising at least 4 nucleotides that are complementary to, or substantially complementary to at least 4 nucleotides of the cis-responsive nucleic acid sequence, and (ii) a stem-loop structure comprising a RNA duplex and loop;
wherein:
when the trans-activating RNA is not present, the cis-repressive nucleic acid sequence and 5′UTR form the repressive stem-loop structure, wherein the repressive stem-loop structure blocks access of a ribosome to the IRES thereby repressing the translation of the gene, and
when the trans-activating RNA is present, the 5′ non-stem nucleic acid sequence of the trans-activating RNA forms a duplex with at least 4 nucleotides of the cis-repressive nucleic acid sequence, wherein the duplex disrupts the repressive stem-loop structure to expose the IRES, thereby allowing translation of the gene.

4 more elements

Facts

Application

15/364,659Filed 30 Nov 2016 · continuation

Priority

15 Nov 2002

Published

US 2018/0142247 A124 May 2018

Granted

19 Feb 2019

Term ends

Not estimatedThe source records don’t support an estimate.

Classification6 codes

C12N 15/67
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for enhancing…
C12N 15/11
Recombinant DNA-technology: dNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity
C12Q 1/6897
Involving nucleic acids: involving reporter genes operably linked to promoters
C12N 2310/53
Physical structure: partially self-complementary or closed

Family

Application60/426,891Us provisional application · filed 15 Nov 2002

PCT/US03/36506Parent of this continuation · filed 14 Nov 2003

US 2018/0142247 A1Pre-grant publication · published 24 May 2018

Application10/535,128Parent of this continuation

US 9,534,224 B2Parent of this continuation

US 2007/0136827 A1Published application

US 10,208,312 B2This patent

From the US filing history. Foreign family members aren’t listed.

Abstract

Claims26