US 11,834,659 B2
Trans-activated functional RNA by strand displacement and uses thereof
Ron Weiss, Newton, MA (US); and Giulio Alighieri, Cambridge, MA (US)
Assigned to Massachusetts Institute of Technology, Cambridge, MA (US)
Filed by Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Sep. 25, 2020, as Appl. No. 17/032,233.
Claims priority of provisional application 62/906,248, filed on Sep. 26, 2019.
Prior Publication US 2021/0095286 A1, Apr. 1, 2021
Prior Publication US 2021/0395732 A9, Dec. 23, 2021
Int. Cl. C12N 15/11 (2006.01); C12N 15/113 (2010.01); A61K 31/7105 (2006.01); C12N 9/22 (2006.01)
CPC C12N 15/113 (2013.01) [A61K 31/7105 (2013.01); C12N 9/22 (2013.01); C12N 2310/122 (2013.01); C12N 2310/141 (2013.01); C12N 2310/20 (2017.05)] 12 Claims
 
1. An engineered RNA comprising,
(i) an effector portion; and
(ii) a responder sequence,
wherein the effector portion comprises a coding sequence for a pre-microRNA (pre-miRNA),
wherein, in the absence of an input signal, the engineered RNA forms a first secondary structure in which the engineered RNA is not capable of being recognized by an actuator;
wherein, in the presence of the input signal, the responder sequence is capable of responding to the input signal such that the engineered RNA forms a second secondary structure, not at its lowest energy state, in which the engineered RNA is capable of being recognized by the actuator; and wherein the actuator is Drosha;
(a) wherein the engineered RNA comprises parts T-d-f-e-b-S-a-c,
wherein the coding sequence for a pre-miRNA comprises parts b-S-a,
wherein the responder sequence comprises parts T-d-f-e,
wherein, in the absence of an input signal which comprises an input RNA that is completely or partially complementary to parts T-d-f, the engineered RNA forms a first secondary structure in which part d completely or partially hybridizes to part b, part e completely or partially hybridizes to part f, part a partially hybridizes to part c, and parts a and b are incapable of hybridizing with each other such that the engineered RNA is not capable of being recognized by Drosha, and
wherein, in the presence of the input RNA that is completely or partially complementary to parts T-d-f, the engineered RNA forms a secondary structure in which parts T-d-f form a double strand with the input RNA, thereby releasing part e from part f and part b from part d, and in which part a partially hybridizes to part b to form a Drosha recognizable cleavage site not at its lowest energy state; or
(b) wherein the engineered RNA comprises parts T-f-d-c-a-S-b-e,
wherein the coding sequence for a pre-miRNA comprises parts b-S-a,
wherein the responder sequence comprises parts T-f-d and e,
wherein, in the absence of an input signal which comprises an input RNA that is completely or partially complementary to parts T-f-d, the engineered RNA forms a first secondary structure in which part d completely or partially hybridizes to part b, part e completely or partially hybridizes to part f, part a partially hybridizes to part c, and parts a and b are incapable of hybridizing with each other such that the engineered RNA is not capable of being recognized by Drosha, and
wherein, in the presence of the input RNA that is completely or partially complementary to parts T-f-d, the engineered RNA forms a secondary structure in which parts T-f-d form a double strand with the input RNA, thereby releasing part e from part f and part b from part d, and in which part a partially hybridizes to part b to form a Drosha recognizable cleavage site not at its lowest energy state; or
(c) wherein the engineered RNA comprises parts 5′ hairpin-toehold-antisense-ribozyme-stem-seed-sense-3′ hairpin,
wherein the coding sequence for a pre-miRNA comprises parts stem-seed-sense,
wherein the responder sequence comprises parts toehold-antisense-ribozyme,
wherein, in the absence of an input signal which comprises an input RNA that is completely or partially complementary to parts toehold-antisense-ribozyme, the engineered RNA forms a first secondary structure in which part 5′ hairpin completely or partially hybridizes to itself, part antisense completely or partially hybridizes to part sense, part ribozyme completely or partially hybridizes to itself, part stem completely or partially hybridizes to itself, part seed completely or partially hybridizes to itself, part 3′ hairpin completely or partially hybridizes to itself, and part stem is incapable of hybridizing to part sense, such that the engineered RNA is not capable of being recognized by Drosha,
wherein, in the presence of the input RNA that is completely or partially complementary to parts toehold-sense-ribozyme, the engineered RNA forms a secondary structure in which parts toehold-antisense-ribozyme hybridize with the input RNA, resulting in ribozyme-mediated cleavage that releases an RNA waste product comprising the input RNA hybridized to parts 5′ hairpin-toehold-sense and a portion of part ribozyme of the engineered RNA, and wherein, following the release of the RNA waste product, the remaining portion of the engineered RNA forms a secondary structure in which part stem partially or completely hybridizes to part sense to form a Drosha recognizable cleavage site not at its lowest energy state.