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. AB C Design options

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