Chromatin activity precipitation method and system
Methods and systems for identifying binding sites in macromolecules using small molecule mimics of naturally occurring molecules is disclosed. A reactive probe is provided that mimics small molecule cofactors. A target macromolecule is irreversibly bound to the probe in vivo to selectively pull down or precipitate probe-bound macromolecules. The macromolecules may be, but are not limited to, DNA, RNA, and proteins.
1. A method of identifying binding sites in macromolecules using small molecule mimics of naturally occurring molecules comprising:
a. providing a reactive probe that mimics small molecule cofactors; and
b. irreversibly binding a target macromolecule to the probe to selectively pull down or precipitate probe-bound macromolecules that comprise target nucleic acids, wherein both captured and uncaptured macromolecule fragments are sequenced in parallel to detect which fragments are enriched or depleted.
2. The method of claim 1 wherein the macromolecules include at least one of the following: DNA, RNA.
3. The method of claim 1 wherein the probe includes a photo-crosslinker, and an alkyne or an azide.
4. The method of claim 3 wherein the photo-crosslinker is diazirine or benzophenone.
5. The method of claim 4 further comprising exposing the photo-crosslinker to UV light wherein the target macromolecule interacting with the probe is covalently attached to the probe, forming a probe-bound macromolecular complex.
6. The method of claim 5 wherein the cross-linking of the macromolecular complex to the target macromolecule is performed using aldehyde, formaldehyde, or paraformaldehyde.
7. The method of claim 6 further comprising enriching the probe-bound macromolecule complex by adding an enrichment group and a detection group to the alkyne or azide portion of the probe using copper (I) cycloaddition.
8. The method of claim 6 further comprising performing click chemistry on azide or alkyne-coated beads.
9. The method of claim 7 wherein the enrichment group is biotin or avidin, and the detection group is at least one of the following: fluorophores, nanoparticles, and quantum dots.
10. The method of claim 7 wherein the macromolecular complex includes at least one of the following: biotin, GST tags, and HA tags.
11. The method of claim 10 wherein the macromolecular complex is removed from any unbound macromolecules by affinity purification through binding to avidin or streptavidin.
12. The method of claim 11 wherein fractions from the avidin-bound, streptavidin-bound, and/or the unbound macromolecules are harvested for sequencing.
13. The method of claim 12 wherein, post-elution, the cross-linking in the avidin-bound or streptavidin-bound fractions will be reversed by heat treatment from which fractions of the macromolecules will be separated.
14. The method of claim 5 wherein the macromolecule irreversibly binding the probe is identified by LC-MS, DNA or RNA sequencing, or both, which yields a set of macromolecules binding the probe.