(DNA-based) cell targeting

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(DNA-based) cell targeting
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Overview

• Goals
• 3 designs
• Possible Implementation
• Future directions

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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.

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Design options
C
A
B
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A Aptamer-based

• Good
• Its simple.
• Its been done before with success.
• Bad
• Requires design of different aptamer sequences
  that can bind to cell-surface protein.

2 associations
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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
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C DNA-base pairing

• Good
• Control of relative amounts of protein that bind
  cell

3 associations
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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.

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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
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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
  wouldnt finish.
• Part 2 Conjugate a protein to biotinylated DNA
  (can do in parallel)
• Part 3 Testing
• More fluorescently labeled DNA

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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

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Implementation of C

• Part 0 Figure out if its 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.

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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

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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?

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Future directions

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