US 12,448,636 B2
High-efficiency reconstitution of RNA molecules
Lukas Christoph Bachmann, San Diego, CA (US); and Samuel Lawrence Pfaff, San Diego, CA (US)
Assigned to Salk Institute for Biological Studies, La Jolla, CA (US)
Filed by Salk Institute for Biological Studies, La Jolla, CA (US)
Filed on Sep. 27, 2021, as Appl. No. 17/486,488.
Application 17/486,488 is a continuation of application No. PCT/US2020/025430, filed on Mar. 27, 2020.
Claims priority of provisional application 62/933,714, filed on Nov. 11, 2019.
Claims priority of provisional application 62/888,855, filed on Aug. 19, 2019.
Claims priority of provisional application 62/834,305, filed on Apr. 15, 2019.
Claims priority of provisional application 62/826,854, filed on Mar. 29, 2019.
Prior Publication US 2022/0145347 A1, May 12, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/34 (2006.01); C12N 15/86 (2006.01); C12Q 1/6813 (2018.01)
CPC C12P 19/34 (2013.01) [C12N 15/86 (2013.01); C12Q 1/6813 (2013.01); C12N 2750/14111 (2013.01)] 27 Claims
 
1. A system for expressing a target protein, comprising:
(a) a first synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
an RNA molecule encoding an N-terminal portion of the target protein, comprising a splice junction at a 3′-end of the RNA molecule encoding the N-terminal portion of the target protein;
a splice donor; and
a first dimerization domain comprising a first RNA hairpin, wherein the first RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a first stem and the region of the non-complementary RNA sequence forms a first loop; and
(b) a second synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
a second dimerization domain comprising a second RNA hairpin, wherein the second RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a second stem and the region of the non-complementary RNA sequence of the second RNA hairpin forms a second loop;
a branch point sequence;
a polypyrimidine tract;
a splice acceptor; and
an RNA molecule encoding a C-terminal portion of the target protein, comprising a splice junction at a 5′-end of the RNA molecule encoding the C-terminal portion of the target protein,
wherein the first loop hybridizes with the second loop, and
wherein the first and second loops avoid intramolecular annealing.