Methods for improving or altering promoter/enhancer properties
The present invention provides methods of reassembling polynucleotides and selecting polynucleotides with altered transcriptional regulatory activity.
1 . A method of reassembling polynucleotides involved in transcription, the method comprising,
providing a plurality of random polynucleotide segments from one or more transcriptional regulatory progenitor polynucleotides;
assembling the plurality of segments in a random fashion, thereby forming a plurality of reassembled polynucleotide; and
selecting a reassembled polynucleotide with a different transcriptional regulatory activity than the progenitor polynucleotides.
2 . The method of claim 1 , wherein the segments are from 5 bp to 5,000 bp long.
3 . The method of claim 1 , wherein the segments are less than 50 base pairs.
4 . The method of claim 1 , wherein the segments are greater than 49 base pairs.
5 . The method of claim 1 , wherein the assembling step comprises ligating the segments.
6 . The method of claim 5 , wherein the ligating step is performed by with a DNA ligase or a topoisomerase.
7 . The method of claim 1 , wherein the plurality of random segments comprises segments from at least two distinct promoter or enhancer polynucleotides.
8 . The method of claim 1 , wherein the plurality of random polynucleotide segments are obtained by random cleavage of one or more transcriptional regulatory progenitor polynucleotides.
9 . The method of claim 1 , wherein the plurality of random polynucleotide segments are obtained by random amplification of one or more part of one or more transcriptional regulatory progenitor polynucleotides.
10 . The method of claim 1 , wherein the reassembled polynucleotide comprises a promoter.
11 . The method of claim 1 , wherein the reassembled polynucleotide comprises an enhancer.
12 . The method of claim 1 , wherein the selection step comprises selecting a reassembled polynucleotide with increased transcriptional activity relative to the transcriptional activity of a progenitor polynucleotide.
13 . The method of claim 1 , wherein the selection step comprises selecting a reassembled polynucleotide with decreased transcriptional activity relative to the transcriptional activity of a progenitor polynucleotide.
14 . The method of claim 1 , wherein the selection step comprises selecting a reassembled polynucleotide with significant transcriptional activity in at least one cell or tissue type where the progenitor polynucleotide lacks activity.
15 . The method of claim 1 , wherein the selection step comprises selecting a reassembled polynucleotide without significant transcriptional activity in at least one cell or tissue type where the progenitor polynucleotide has activity.
16 . The method of claim 1 , wherein the selection step comprises selecting a reassembled polynucleotide with transcriptional activity that is activated in response to biotic or abiotic stimuli.
17 . The method of claim 1 , where the segments are formed by nicking and subsequent end-repair of DNA that is altered by radiation, oxidation, or a chemical agent.
18 . The method of claim 1 , wherein the selection step comprises selecting a reassembled polynucleotide with transcriptional activity at a different developmental stage of an organism relative to the transcriptional activity of a progenitor polynucleotide.
19 . The method of claim 1 , wherein the segments are formed by cleaving one or more progenitor polynucleotides with a restriction endonuclease.
20 . The method of claim 1 , wherein the segments are formed by cleaving one or more progenitor polynucleotides with DNaseI.
21 . The method of claim 1 , wherein the segments are formed by cleaving one or more progenitor polynucleotides mechanically.
22 . The method of claim 1 , wherein the segments are formed in a thermocyclic amplification reaction.
23 . The method of claim 22 , wherein the thermocyclic reaction is a polymerase chain reaction.
24 . The method of claim 23 , wherein the polymerase chain reaction is a mutagenic polymerase chain reaction.
25 . The method of claim 1 , wherein the selection step is performed by ligating the reassembled polynucleotide to a reporter gene and measuring reporter gene activity.
26 . The method of claim 1 , wherein the plurality of segments further comprises oligonucleotides.
27 . The method of claim 26 , wherein the oligonucleotide sequence corresponds to a transcription factor binding site.
28 . The method of claim 26 , wherein the nucleotide sequence of the oligonucleotides are not from a transcriptional regulatory polynucleotide.
29 . The method of claim 1 , wherein the reassembled polynucleotide is shorter than the progenitor polynucleotide.
30 . The method of claim 1 , wherein the reassembled polynucleotide is longer than the progenitor polynucleotide.
31 . The method of claim 1 , wherein the progenitor polynucleotides comprise allelic variants of a transcriptional regulator polynucleotide.
32 . The method of claim 1 , wherein the progenitor polynucleotides comprise plant transcriptional regulatory polynucleotides.
33 . The method of claim 1 , wherein the progenitor polynucleotides comprise yeast transcriptional regulatory polynucleotides.
34 . The method of claim 1 , wherein the progenitor polynucleotides comprise fungal transcriptional regulatory polynucleotides.
35 . The method of claim 1 , wherein the progenitor polynucleotides comprise mammalian transcriptional regulatory polynucleotides.
36 . The method of claim 1 , wherein the progenitor polynucleotides comprise viral transcriptional regulatory polynucleotides.
37 . The method of claim 1 , wherein the progenitor polynucleotides comprise bacterial transcriptional regulatory polynucleotides.
38 . The method of claim 1 , wherein the progenitor polynucleotides consist of one transcriptional regulatory polynucleotide.
39 . The method of claim 1 , wherein the transcriptional regulatory progenitor polynucleotides comprise more than one transcriptional regulatory polynucleotide.
40 . The method of claim 1 , wherein the transcriptional regulatory progenitor polynucleotides are less than 70% identical.
41 . The method of claim 1 , wherein the progenitor polynucleotides are less than 50% identical.
42 . The method of claim 1 , wherein the progenitor polynucleotides do not hybridize to each other following at least one wash in 0.2×SSC at 55° C. for 20 minutes.
43 . The method of claim 1 , wherein the polynucleotide segments are single stranded.
44 . The method of claim 1 , wherein the polynucleotide segments are double-stranded.
45 . The method of claim 44 , wherein the double-stranded segments have at least one overhanging single-stranded end.
46 . The method of claim 45 , wherein the overhanging single-stranded end comprises fewer than 10 base pairs.
47 . The method of claim 1 , wherein the assembling step does not comprise a polymerase.
48 . A reassembled polynucleotide of claim 1.