Poly(UG) polymerase, constructs, and methods of making and using the same
Methods, kits, and compositions of matter relating to poly(UG) polymerases are disclosed. In one embodiment, a method includes: contacting an RNA substrate with a poly(UG) polymerase; and allowing the poly(UG) polymerase to add a poly(UG) sequence to the end of the RNA substrate by retaining contact between the RNA substrate and the poly(UG) polymerase for a period of time from about 1 second to about 28 days. The poly(UG) polymerase can be Caenorhabditis elegans RDE-3.
1. A method for adding a poly(UG) sequence to an end of an RNA substrate, the method comprising:
(a) expressing a poly(UG) polymerase from a construct comprising a poly(UG) polymerase activity sequence linked to a heterologous promoter;
(b) contacting the RNA substrate with the poly(UG) polymerase; and
(c) allowing the poly(UG) polymerase to add a poly(UG) sequence to the end of the RNA substrate by retaining contact between the RNA substrate and the poly(UG) polymerase for a period of time from about 1 second to about 28 days.
2. The method of claim 1 , wherein the poly(UG) polymerase is C. elegans RDE-3.
3. The method of claim 1 , wherein the poly(UG) polymerase comprises at least a portion of NCBI Reference Sequence NP_491834.1.
4. The method of claim 1 , wherein the method is performed in vitro.
5. The method of claim 1 , wherein the method additionally comprises
(d) priming cDNA synthesis with a poly(CA) primer.
6. The method of claim 1 , wherein the period of time of step (c) is a first length of time and is sufficient to introduce a poly(UG) tail having a length sufficient to form a hairpin between a 5′ portion of the poly(UG) tail with a 3′ portion of the poly(UG) tail, and wherein the method additionally comprises;
(d) waiting a second length of time sufficient to allow formation of the hairpin; and
(e) priming cDNA or cRNA synthesis with the poly(UG) tail as a primer.
7. The method of claim 5 , wherein the poly(UG) polymerase is C. elegans RDE-3.
8. The method of claim 6 , wherein the poly(UG) polymerase is C. elegans RDE-3.
9. The method of claim 1 , wherein the sequence of the RNA substrate is unknown.
10. The method of claim 1 , wherein the poly(UG) polymerase is a fusion protein comprising a poly(UG) polymerase activity domain and an RNA-interaction domain.
11. The method of claim 10 , wherein the poly(UG) polymerase activity domain includes at least a portion of C. elegans RDE-3.
12. The method of claim 10 , wherein the RNA-interaction domain is MS2 coat protein or another RNA-binding protein.