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 strand DNA useful in a variety of applications including protein expression.
1. An oligonucleotide array for RNA-mediated assembly of a target RNA molecule, comprising:
(a) a plurality of first surface-bound oligonucleotides each having a segment sequence corresponding to a portion of a target RNA and a complementary RNAP promoter sequence operably-linked to said segment sequence's 3′ termini, wherein two or more of the segment sequences correspond to different portions of the target RNA; and
(b) a plurality of second surface-bound oligonucleotides each having a splint sequence corresponding to a portion of the target RNA that complements and partially overlaps at least two of the two or more segment sequences of the first surface-bound oligonucleotides corresponding to different portions of the target RNA, said second surface-bound oligonucleotides including an RNAP promoter sequence operably-linked to their splint sequence's 3′ termini,
wherein said first and second surface-bound oligonucleotides are linked at their 3′ termini to a surface of the oligonucleotide array.
2. The oligonucleotide array according to claim 1 , wherein the 5′ end of each segment sequence and each splint sequence corresponds to a GG dinucleotide in the target RNA molecule.
3. The oligonucleotide array according to claim 1 , wherein the target RNA molecule is a full-length RNA transcript of a gene.
4. The oligonucleotide array according to claim 1 , wherein the oligonucleotide array's surface comprises a silanized glass or amorphous carbon deposited on a gold film.
5. The oligonucleotide array according to claim 1 , wherein said surface-bound oligonucleotides include a spacer, a T7 RNAP promoter sequence, a CC dinucleotide and either the segment sequence or the splint sequence.
6. The oligonucleotide array according to claim 1 , wherein oligonucleotide array further comprises a third oligonucleotide encoding an RNAP promoter sequence to the complementary RNAP promoter sequence of the first and second surface-bound oligonucleotides to yield double-stranded RNAP promoters.