Compositions and methods relating to synthetic RNA polynucleotides created from synthetic DNA oligonucleotides
Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides.
1. A method, the method comprising:
(a) selecting a nuclease for guiding to a nucleic acid target using a guide RNA;
(b) incubating a reaction mixture in a reaction chamber, the reaction mixture comprising:
a DNA polymerase,
an RNA polymerase,
a first synthetic single stranded (ss) DNA oligonucleotide,
a second synthetic single stranded (ss) DNA oligonucleotide,
wherein the first ss DNA oligonucleotide comprises a promoter sequence for the RNA polymerase and a sequence at the 3′ end that hybridizes to a complementary sequence in the second ss DNA oligonucleotide;
dNTPs, and
rNTPS,
to produce the guide RNA,
(c) associating the selected nuclease with the guide RNA from (b) to make an RNA guided nuclease,
wherein the RNA guided nuclease comprises the guide RNA and the guide RNA is at least partially transcribed from the second oligonucleotide.
2. The method according to claim 1 , wherein the RNA guided nuclease is a Cas nuclease.
3. The method according to claim 1 , wherein the RNA guided nuclease is a Cpf1 nuclease.
4. The method according to claim 1 , wherein the first ss DNA oligonucleotide comprises a variable region between the promoter sequence and the sequence at the 3′ end that hybridizes to the complementary sequence in the second ss DNA oligonucleotide.
5. The method according to claim 4 , wherein the variable region encodes a targeting region complementary to a target sequence.
6. The method according to claim 5 further comprising (d) contacting the RNA guided nuclease with a population of DNA molecules, wherein at least a portion of the DNA molecules comprise the target sequence.
7. The method according to claim 6 , wherein the RNA guided nuclease cleaves the DNA molecules comprising the target sequence.
8. The method according to claim 6 further comprising detecting cleavage of the DNA molecules comprising the target sequence.
9. The method according to claim 1 , wherein the DNA polymerase is a strand displacing polymerase.
10. The method according to claim 1 , wherein the 5′ end of the guide RNA comprises a sequence module containing a modified nucleotide.
11. The method according to claim 1 , wherein the second ss DNA oligonucleotide comprises a tracrRNA sequence.
12. The method according to claim 1 further comprising producing a plurality of said reaction mixes in a multiwell plate, wherein the sequence of the first ssDNA oligonucleotide differs in each reaction mix.
13. The method according to claim 12 , wherein the second ss DNA oligonucleotide is the same in each reaction.
14. The method according to claim 1 , further comprising combining in the reaction mixture a third synthetic ss DNA oligonucleotide and a fourth synthetic ss DNA oligonucleotide, wherein the 5′ end of the third synthetic ss DNA oligonucleotide hybridizes to the 5′ end of the second ss DNA oligonucleotide and the 3′ end of the third ss DNA oligonucleotide hybridizes to the 3′ end of the fourth ss DNA oligonucleotide for forming a single RNA polynucleotide comprising the sequences from first, second, third and fourth oligonucleotide excluding the RNA promoter sequence.
15. The method according to claim 14 , further comprising combining in the reaction mixture a DNA ligase.