Methods and compositions relating to super-resolution imaging and modification
This disclosure provides methods for generating super-resolution patterns of molecules on substrates.
1. A method for obtaining partial sequence information from a target protein, comprising
denaturing and elongating a protein,
attaching docking strands to particular amino acids in the protein,
capturing the protein on a substrate,
repeatedly contacting the captured protein with fluorescently-labeled imager strands that transiently bind to the docking strand, and
imaging the substrate, thereby locating the particular amino acids in the protein.
2. The method of claim 1 , wherein the protein is modified with a click chemistry reacting group prior to or at the same time as docking strands are attached.
3. The method of claim 2 , wherein the click chemistry reacting group is trans-cyclooctene (TCO).
4. The method of claim 3 , wherein the substrate comprises a plurality of counter-acting click chemistry groups.
5. The method of claim 4 , wherein the counter-acting click chemistry groups are 1,2,4,5-tetrazine (TZ).
6. The method of claim 1 , wherein the particular amino acids are amine-containing amino acids, optionally wherein the docking strands are attached to the amine-containing through an NHS-ester or an imidoester.
7. The method of claim 1 , wherein the particular amino acids are lysines, optionally wherein the docking strands are attached to the lysines through an NHS-ester or an imidoester.
8. The method of claim 1 , wherein the particular amino acids are sulfhydryl-containing amino acids, optionally wherein the docking strands are attached to the sulfhydryl-containing amino acids through a maleimide group, a haloacetyl group, or a pyridyldisulfide.
9. The method of claim 1 , wherein the particular amino acids are cysteines, optionally wherein the docking strands are attached to the cysteines through a maleimide group, a haloacetyl group, or a pyridyldisulfide.
10. The method of claim 1 , wherein the particular amino acids are carboxyl-containing amino acids, optionally wherein the docking strands are attached to the carboxyl-containing amino acids through a carbodiimide.
11. The method of claim 1 , wherein the particular amino acids are aspartic acid, optionally wherein the docking strands are attached to the aspartic acid through a carbodiimide.
12. The method of claim 1 , wherein the particular amino acids are glutamic acid, optionally wherein the docking strands are attached to the glutamic acid through a carbodiimide.
13. The method of claim 1 , wherein the captured target protein is denatured, stretched, and immobilized on the substrate.
14. The method of claim 13 , wherein the target protein is denatured using a denaturant selected from the group consisting of SDS, detergent, organic solvents, acids and bases.
15. The method of claim 1 , wherein the protein is denatured and/or elongated using an amphiphilic agent.
16. The method of claim 15 , wherein the protein is denatured and/or elongated using SDS.
17. The method of claim 1 , wherein the protein is captured on the substrate in an electric field.
18. The method of claim 1 , wherein imager strands for a single amino acid are contacted to the protein and imaged consecutively.
19. The method of claim 1 , wherein imager strands for different amino acids are contacted to the protein consecutively, optionally wherein different amino acids are imaged consecutively or simultaneously.
20. The method of claim 1 , wherein the location of the particular amino acids is determined with a 5 nm resolution.
21. The method of claim 1 , wherein the docking strands comprise a photo-cleavable linker, and optionally the docking strands are removed by ultraviolet irradiation once imaging based on the particular amino acid is complete.