Single-pot methods for producing circular rnas
AssigneePeking University
InventorsWensheng WEI, Liang QU, Zongyi YI, Yong SHEN
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Abstract
The present application provides methods for producing circular RNAs (circRNAs) from a DNA construct encoding a linear RNA precursor, wherein the linear RNA precursor comprises from the 5′-end to the 3′ end: a 3′ catalytic Group I intron fragment, a 3′ exon sequence, an effector RNA sequence, a 5′ exon sequence, and a 5′ catalytic Group I intron fragment, wherein the method comprises an in vitro single-pot reaction. In some embodiments, the single-pot reaction does not comprise supplementing the reagent composition with GTP, a divalent metal ion such as Mg2+, or DNase I prior to circularization of a linear RNA precursor.
Claims25
A method of producing a circular RNA from a DNA construct encoding a linear RNA precursor, wherein the linear RNA precursor comprises from the 5′-end to the 3′ end:
- a 3′ catalytic Group I intron fragment, a 3′ exon sequence, an effector RNA sequence, a 5′ exon sequence, and a 5′ catalytic Group I intron fragment, wherein the method comprises an in vitro single-pot reaction step comprising contacting the DNA construct with a reagent composition comprising an RNA polymerase, adenosine 5′-triphosphate (ATP), uridine 5′-triphosphate (UTP), guanosine 5′-triphosphate (GTP) and cytosine 5′-triphosphate (CTP) under conditions that allow transcription of the DNA construct into the linear RNA precursor and circularization of the linear RNA precursor, wherein the circularization comprises activation of the 3′ catalytic Group I intron fragment and the 5′ catalytic Group I intron fragment to splice the 3′ exon sequence and the 5′ exon sequence from the linear RNA precursor, thereby forming the circular RNA comprising the effector RNA.
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