Oligonucleotide trapping
Novel methods and compositions for identifying one or more factors associated with a nucleic acid sequence (e.g., DNA and/or RNA) of interest are provided.
1. A method for identifying one or more factors bound to one or more targeting moieties of a plurality of oligonucleotide paints in a biological sample by in situ hybridization, comprising the steps of:
hybridizing the plurality of oligonucleotide paints to different chromosomal regions of a chromosome in the biological sample by in situ hybridization, thereby forming hybridized oligonucleotide paints, wherein each of the oligonucleotide paints comprises a 5′ primer sequence, a nucleotide sequence capable of hybridizing to a specific region of the chromosome, 3′ primer sequence, and one or more targeting moieties, and the nucleotide sequence capable of hybridizing to the specific region of the chromosome is located between the 5′ primer sequence and the 3′ primer sequence and is different in each of the oligonucleotide paints and the 5′ primer sequence and the 3′ primer sequence comprises either nucleotide sequences of a universal primer or nucleotide sequences fully complementary to the universal primer;
removing the oligonucleotide paints unbound to the different chromosomal regions of the chromosome from the hybridized oligonucleotide paints;
covalently attaching the targeting moieties of the hybridized oligonucleotide paints to the one or more factors in the biological sample; and
identifying the one or more factors bound to the one or more targeting moieties of the plurality of oligonucleotide paints in the biological sample by identifying the factors attached to the targeting moieties of the hybridized oligonucleotide paints.
2. The method of claim 1 , wherein the one or more factors are selected from the group consisting of a protein, a peptide, a carbohydrate, a lipid, a chemical moiety and any combination thereof.
3. The method of claim 1 , wherein the oligonucleotide paints are between about 20 base pairs and about 32 base pairs in length.
4. The method of claim 1 , wherein the nucleotide sequence capable of hybridizing to the specific region of the chromosome is between about 32 base pairs and about 40 base pairs in length.
5. The method of claim 1 , further comprising the step of retrieving the hybridized oligonucleotide paints after the attaching step, wherein the oligonucleotide paints further include a retrievable moiety.
6. The method of claim 5 , wherein the retrievable moiety is one component of a binding pair.
7. The method of claim 5 , wherein the retrievable moiety is biotin.
8. The method of claim 5 , wherein the hybridized oligonucleotide paints are retrieved by binding the retrievable moiety.
9. The method of claim 1 , further comprising activation of the targeting moieties of the hybridized oligonucleotide paints before the attaching step.
10. The method of claim 9 , wherein the activation of the targeting moieties of the hybridized oligonucleotide paints is performed by heat activation of the targeting moieties, light activation of the targeting moieties, or chemical activation of the targeting moieties or any combination thereof.
11. The method of claim 1 , wherein the one or more targeting moieties are covalently attached to a protein, a peptide, a carbohydrate, a lipid, or a chemical moiety or any combination thereof.
12. The method of claim 11 , wherein the chemical moiety is a methyl group or an acetyl group.
13. The method of claim 1 , wherein the one or more factors are selected from the group consisting of histone proteins, scaffold proteins, transcription factors, DNA binding proteins, DNA repair factors, and nucleases or any combination thereof.
14. The method of claim 1 , wherein the chromosome is within a cell from the biological sample.
15. The method of claim 1 , wherein the oligonucleotide paints are ligated oligonucleotide paints.
16. The method of claim 1 , wherein the oligonucleotide paints are hapten-conjugated oligonucleotide paints.
17. The method of claim 1 , wherein the oligonucleotide paints unbound to the different chromosomal regions of the chromosome are removed by washing a support having the biological sample immobilized on the support.
18. The method of claim 17 , wherein the different chromosomal regions are present in single or low copy numbers.
19. The method of claim 1 wherein one of the one or more targeting moieties is a protein capture moiety, a lipid capture moiety, a sugar capture moiety or a nucleic acid capture moiety.
20. The method of claim 1 wherein one of the one or more targeting moieties is
21. The method of claim 1 wherein the plurality of oligonucleotide paints comprises a set of oligonucleotide paints bound to a plurality of sub-chromosomal regions.
22. The method of claim 21 wherein the plurality of sub-chromosomal regions comprises at least 50 regions.
23. A method for identifying one or more factors bound to one or more targeting moieties of a plurality of oligonucleotide paints in a biological sample by in situ hybridization, comprising the steps of:
hybridizing the plurality of oligonucleotide paints to different regions of genomic DNA or mitochondrial DNA in the biological sample by in situ hybridization, thereby forming hybridized oligonucleotide paints, wherein each of the oligonucleotide paints comprises a 5′ primer sequence, a nucleotide sequence capable of hybridizing to a specific region of the genomic DNA or the mitochondrial DNA, 3′ primer sequence, and one or more targeting moieties, and the nucleotide sequence capable of hybridizing to the specific region of the genomic DNA or the mitochondrial DNA is located between the 5′ primer sequence and the 3′ primer sequence and is different in each of the oligonucleotide paints and the 5′ primer sequence and the 3′ primer sequence comprises either nucleotide sequences of a universal primer or nucleotide sequences fully complementary to the universal primer;
removing the oligonucleotide paints unbound to the different regions of the genomic DNA or the mitochondrial DNA from the hybridized oligonucleotide paints;
covalently attaching the targeting moieties of the hybridized oligonucleotide paints to the one or more factors in the biological sample; and
identifying the one or more factors bound to the one or more targeting moieties of the plurality of oligonucleotide paints in the biological sample by identifying the factors attached to the targeting moieties of the hybridized oligonucleotide paints.
24. The method of claim 23 , wherein the one or more factors are selected from the group consisting of a protein, a peptide, a carbohydrate, a lipid, and a chemical moiety or any combination thereof.
25. A method for identifying one or more factors bound to one or more targeting moieties of a plurality of oligonucleotide paints in a biological sample by in situ hybridization, comprising the steps of:
hybridizing the plurality of oligonucleotide paints to different regions of genomic DNA or mitochondrial DNA in the biological sample by in situ hybridization, thereby forming hybridized oligonucleotide paints, wherein the plurality of oligonucleotide paints has retrievable moieties, and wherein each of the oligonucleotide paints comprises a 5′ primer sequence, a nucleotide sequence capable of hybridizing to a specific region of the genomic DNA or the mitochondrial DNA, 3′ primer sequence, and one or more targeting moieties, and the nucleotide sequence capable of hybridizing to the specific region of the genomic DNA or the mitochondrial DNA is located between the 5′ primer sequence and the 3′ primer sequence and is different in each of the oligonucleotide paints and the 5′ primer sequence and the 3′ primer sequence comprises either nucleotide sequences of a universal primer or nucleotide sequences fully complementary to the universal primer;
removing the oligonucleotide paints unbound to the different regions of the genomic DNA or mitochondrial DNA from the hybridized oligonucleotide paints;
covalently attaching the targeting moieties of the hybridized oligonucleotide paints to the one or more factors in the biological sample; and
identifying the one or more factors bound to the one or more targeting moieties of the plurality of oligonucleotide paints in the biological sample by identifying the factors attached to the targeting moieties of the hybridized oligonucleotide paints.
26. The method of claim 25 , wherein the one or more factors are selected from the group consisting of a protein, a peptide, a carbohydrate, a lipid, and a chemical moiety or any combination thereof.