Modular kinetically-controlled functional rna constructs and related compositions, systems, and methods
AssigneeUniversity of Washington
InventorsJames M. Carothers, Jesse Zalatan, David Sparkman-Yager, Jason Fontana
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Abstract
Disclosed are kinetically-controlled RNA biosensor constructs and related, nucleic acids, vectors, cells, systems and methods useful for detecting ligands of interest. Also disclosed are computer implemented methods for designing kinetically-controlled biosensors, guide RNA molecules, and/or target promoter sequences, and constructs produced thereby. Exemplary embodiments include scRNAs and expression cassettes incorporating synthetic promoters for implementation of CRISPRa.
Claims28
A kinetically-controlled RNA biosensor construct, comprising from 5′ to 3′:
- a sensor domain that specifically binds to a ligand of interest;
- and
- an output domain configured to modulate a detectable output signal when folded into an active conformation;
- wherein the output domain, when transcribed, folds into the active conformation when the sensor domain is bound to the ligand of interest, and wherein the output domain, when transcribed, folds into an inactive conformation when the sensor domain is not bound to the ligand of interest;
- wherein the sensor domain comprises:
- an aptamer domain, wherein the aptamer domain comprises, 5′ to 3′, a stem sequence, an aptamer subsequence, and a stem target sequence;
- and
- an overhang sequence 5′ of the aptamer domain and/or a linker sequence 3′ of the aptamer domain;
- wherein if the sensor domain comprises a linker sequence, the aptamer domain comprises a linker target sequence between the stem sequence and the aptamer subsequence;
- and
- wherein the output domain comprises, from 5′ to 3′:
- a stem target sequence, and
- an output subsequence,
- wherein if the sensor domain comprises an overhang sequence, the output domain comprises an overhang target sequence between the stem target sequence and the output subsequence.
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