(DNA-based) cell targeting

1. (DNA-based) cell targeting

2. Overview • Goals • 3 designs • Possible Implementation • Future directions

3. Goal (what we want) • Control of substrate delivery to a cell. • What substrates? Proteins, sugars, DNA [nanostructures]. • What kind of control? Amount that gets to the cell surface, amount relative to other proteins targeted to cell. • Potentially important application in drug delivery, nanostructures.

4. Design options C A B

5. A: Aptamer-based • Good: • It’s simple. • It’s been done before with success. • Bad: • Requires design of different aptamer sequences that can bind to cell-surface protein. 2 associations

6. B: Biotin/Streptavidin • Good: • Depends on association b/w biotin and streptavidin. • Able to swap in and out target proteins. • Bad: • Depends on association b/w biotin-streptavidin interaction • Ugly: • Complexity • Expressing streptavidin on cell surface using OmpX • Nevertheless, proof of principle! 4 associations

7. C: DNA-base pairing • Good: • Control of relative amounts of protein that bind cell 3 associations

8. Control of relative amounts? • DNA bound cell surface: • ATCATC • Sequence conjugated to substrate A: TAGTAG • Sequence conjugated to substrate B: TAG • Substrate A binds less often but for longer periods of time. • Precise control of kinetics based on types of base-pairs used; repeated patterns.

9. C: DNA-base pairing • Good: • Control of relative amounts of protein that bind cell. • Bad: • If we can control amount of aptamer bound to cell surface, probably can get as much/ better control by delivering protein bound directly to the aptamer. 3 associations

10. Implementation of B • Part 1: Making E. coli express streptavidin on cell surface (which would be interesting by itself). See Rice, et al. • Challenges: Is this even possible? • Assay: Test for fluorescently labeled biotin-DNA binding to cell surface. • Might attempt to evolve proteins that can do this. • Time consuming!! Might start knowing that we wouldn’t finish. • Part 2: Conjugate a protein to biotinylated DNA (can do in parallel) • Part 3: Testing • More fluorescently labeled DNA

11. Implementation of B • Part 1: Infinite time for 3 people working on streptavidin fusion protein • Part 2 [in parallel]: 4 weeks for 2 people to work on substrate synthesis

12. Implementation of C • Part 0: Figure out if it’s worth it. • Part 1: Find and test aptamer that binds cell-surface protein • Literature search • Assay: use fluorescently labeled aptamer • Part 2: Conjugate a protein to single stranded DNA. • Part 3: Testing • Fluorescently labeled DNA again! • Part 4: Determining DNA annealing properties and whether proteins can simply be swapped with predictable behavior.

13. Implementation of C • Part 1: One week for literature search, one week for testing, 2 people. • Part 2: [In parallel] Four weeks for two people to conjugate a protein to single stranded DNA. • Part 3: One week for 2 people. • Part 4: Potentially a week based on faculty answers; testing is Parts 1-3 multiplied

14. Major Questions • What cell-surface protein(s) to target? What substrate do we want to use? • Eventual application? • Practicality? • Can we really hope to express Streptavidin on a cell surface in a summer? • How does this fit into iGEM? • Where are we going with this?

15. Future directions • Use aptamers to trigger signaling cascades; finer control of gene expression. • Create a dock for a DNA nanostructure