Molecular sensor selection
View Patent ↗Provided herein, among other things, is an automatable procedure that employs in vitro directed evolution to create DNA sequences that encode a ligand-responsive ribozyme and which, when transcribed, can control expression of genes they are coupled to. The method also allows creation of functional RNA sequences that bind target molecules, without requiring any modification or immobilization of the target.
1. A method for selecting a ligand-responsive ribozyme, comprising:
(a) in vitro transcribing a DNA library to produce RNA;
(b) cleaving the RNA to yield cleaved and uncleaved RNA;
(c) reverse transcribing the cleaved and uncleaved RNA to cDNA without isolating the cleaved or uncleaved RNA;
(d) selectively amplifying the cDNA such that cDNA molecules corresponding to RNA molecules that have been cleaved are amplified by a different amount relative to cDNA molecules corresponding to uncleaved RNA;
(e) preparing the amplified cDNA of (d) for in vitro transcription;
(f) repeating steps (a)-(e) one or more times; and,
(g) determining the sequence of one or more of the molecules in the product of step (f).
2. The method of claim 1 , wherein the selective amplifying step (d) is done by polymerase chain reaction (PCR).
3. The method of claim 1 , wherein the selective amplifying of step (d) comprises ligating a prefix oligonucleotide to either the cDNA molecules corresponding to RNA molecules that have been cleaved or the cDNA molecules that correspond to uncleaved RNA.
4. The method of claim 3 , wherein the ligating is done using a splint oligonucleotide that splints the joint between the prefix oligonucleotide and the cDNA.
5. The method of claim 1 , wherein said selective amplifying of step (d) selectively amplifies the cDNA molecules that correspond to cleaved RNA relative to cDNAs that correspond to uncleaved RNA.
6. The method of claim 1 , wherein the in vitro transcribing step (a) is performed in either the presence of or absence of a target ligand molecule.
7. The method of claim 6 , wherein the target ligand is a small molecule.
8. The method of claim 1 , wherein said in vitro transcribing step (a) is performed in the presence of a mixture of target molecules, and wherein the target molecules are different during each repeat of steps (a)-(e).
9. The method of claim 8 , wherein said mixture used in each repeat of steps (a)-(e) is one of two compositions, and the selective amplifying step (d) amplifies cDNA corresponding to cleaved RNA in repeats in which one composition is used, and the selective amplifying step (d) amplifies cDNA corresponding to uncleaved RNA in repeats in which the other composition is used.
10. The method of claim 1 , wherein the steps are implemented on a liquid-handling robot.
11. The method of claim 1 , wherein the determining step (g) is done by high-throughput sequencing.
12. A method to isolate an RNA aptamer comprising the steps of:
(a) in vitro transcribing a DNA library to produce RNA;
(b) cleaving the RNA to yield cleaved and uncleaved RNA;
(c) reverse transcribing the cleaved and uncleaved RNA to cDNA without isolating the cleaved or uncleaved RNA;
(d) selectively amplifying the cDNA such that cDNA molecules corresponding to RNA molecules that have been cleaved are amplified by a different amount relative to cDNA molecules corresponding to uncleaved RNA;
(e) preparing the amplified cDNA of (d) for in vitro transcription;
(f) repeating steps (a)-(e) one or more times; and,
(g) determining the sequence of one or more of the molecules in the product of step (f).
13. The method of claim 1 , the reverse transcription is done using a reverse transcription primer that comprises a 3′ sequence that hybridizes to the RNA and a 5′ tail that does not hybridize to the RNA.
14. The method of claim 1 , wherein the selective amplifying of step (d) uses DNA polymerase to add additional nucleotides.
15. The method of claim 1 , wherein the selective amplifying of step (d) uses the 5′ end of the reverse transcription primer as a template or splint.
16. The method of claim 1 , wherein the selective amplifying step (d) adds a promoter sequence.