RNA-mediated gene assembly from DNA oligonucleotides
The present invention is directed to methods and materials for RNA-mediated gene assembly from oligonucleotide sequences. In some embodiments, the oligonucleotides used for gene assembly are provided in an array format. An RNA polymerase promoter is appended to surface-bound oligonucleotides and a plurality of RNA copies of each oligonucleotide are then produced with an RNA polymerase. These RNA molecules self-assemble into a desired full-length RNA transcript by hybridization and ligation. The resulting RNA transcript may then be converted into double stand DNA useful in a variety of applications including protein expression.
1. An RNA-mediated assembly method for providing a target RNA molecule, comprising:
(a) providing:
(i) a plurality of first oligonucleotides each having a segment sequence corresponding to a portion of a target RNA and an RNA polymerase (RNAP) promoter sequence operably-linked to the segment sequence's 3′ termini; and
(ii) a plurality of second oligonucleotides each having a splint sequence corresponding to a portion of the target RNA that complements and partially overlaps the segment sequence of the first oligonucleotides, the second oligonucleotides including an RNAP promoter sequence operably-linked to their splint sequence's 3′ termini;
(b) hybridizing a third oligonucleotide comprising a sequence complementary to the RNAP promoter sequence of the first and second pluralities of oligonucleotides to yield double-stranded RNAP promoters;
(c) transcribing with RNA polymerase, in the presence of rNTPs, the segment sequences of the plurality of first oligonucleotides to yield an RNA segment and the splint sequences of the plurality of second oligonucleotides to yield an RNA splint;
(d) assembling the RNA segments and RNA splints by hybridization to form RNA:RNA hybrids; and
(e) sealing nicks between the RNA segments in the RNA:RNA hybrid to provide a target RNA molecule.
2. The method according to claim 1 , wherein the plurality of first oligonucleotides or the plurality of second oligonucleotides is provided as a surface-bound oligonucleotide array.
3. The method according to claim 1 , wherein in step (c), transcription is carried out in the presence of a mixture of rNTPs and rNMPs.
4. The method according to claim 1 , further comprising removing terminal pyrophosphate groups from the RNA segments prior to step (e).
5. The method according to claim 4 , wherein removal of the terminal pyrophosphate groups is performed by treatment with 5′ pyrophosphohydrolase or RNA pyrophosphatase.
6. The method according to claim 1 , wherein the splint sequences are transcribed in the presence of 2′-O-Methyl ribonucleotides and sealing the nicks in step (e) is carried out with T4 DNA ligase or a truncated version thereof.
7. The method according to claim 1 , wherein the target RNA molecule is a full-length RNA transcript of a gene.
8. The method according to claim 2 , wherein the oligonucleotide array's surface comprises a silanized glass or amorphous carbon deposited on a gold film.
9. The method according to claim 2 , wherein the oligonucleotide array is provided by in situ photolithographic oligonucleotide array synthesis.
10. The method according to claim 2 , wherein the surface-bound oligonucleotides include a spacer, a T7 RNAP promoter sequence, a CC dinucleotide and either the segment sequence or the splint sequence.
11. The method according to claim 1 , wherein the third oligonucleotide includes a T7 RNAP promoter sequence and a dinucleotide GG.
12. The method according to claim 1 , wherein step (c) results in the synthesis of multiple copies of RNA segments and RNA splints from each of the segment sequences and splint sequences.