IGEM:Harvard/2006/DNA nanostructures

Roadblocks and Solutions

1. Big Concept
   • Is this really better than currently available antithrombins?
     • Not in efficacy, necessarily. But it's more generalizable, engineerable, and whether or not the triggerability and the bioavailability features work in our favor is something drug companies spend years testing. In essence, the thrombin problem is just an example application of the greater idea of a triggerable drug box - it is our proof of concept.

1. Nitty-Gritty Details
   • How is this going to stay in the body?
     • The "lock," or the box structure, will be covered in a non-immunologic polymer, such as PEG (REFERENCE) or PLGA (REFERENCE), allowing it to traverse the bloodstream, or perhaps eventually enter the cell (REFERENCE) and drop cargo.
       Part of the functionality of the "key," or the oligo clasp-opening strand, is that it will be quickly cleared if it does not bind the lock within the time that naked DNA is cleared, approximately 5-10 minutes. This allows a quick pulse-controlled attack against thrombin, though the possibility of a constant stream of key time-released or released by IV is not impossible.
       And the opened structures will be cleared, thrombin attached, thus permanently and immediately lowering the levels of that protein from the blood.

1. • How big is this stuff going to be anyway? And how much thrombin will it bind?
     • The thrombin-aptamer is approximately 3nm long, 2nm wide. If the open-faced tetrahedron is used, it could be designed to be big enough to just fit one aptamer within, or four aptamers, one on each side. The limiting factor here is the need to keep the aptamer within the confines of the structure. However, my personal preference is for a closed-face structure because, w/ an open-faced one which needs to be constrained based on geometry, the odds of difficulty folding are higher.

1. • Is there a chance that the aptamer sequence will mistakenly bind the scaffold or oligos?
     • ClustalW says chances are not that great.

Presentation Outline

• Specific goal(s) of the project

• • Proof of concept. The idea of a generalizable, injectable, triggerable, clearable, simple-to-engineer protein manipulation system is a goal well worth working towards, as is building a useful DNA nanostructure.

• • state an existing problem and the impact if we solve it

• • • Anti-thrombotics are needed for patients who have a tendency towards thrombosis, embolisms, and stroke - a highly-molar-controlled, triggerable form could be extremely helpful for patients that demand fast action and close regulation.

• Initial ideas for how to solve the problem
  • Unique/interesting features of our approach

• • • Generalizability: Because DNA aptamer designed to bind a protein in the bloodstream can be easily engineered into the structure, the design can be generalized simply.
    • Molar-Triggerability: Because the strand-replacement-clasp system functions as a "lock and key" on a 1:1 molar level, tight control of thrombin inactivation and pulse-inactivation (due to the quick 5-10 min clearance of "key" strand) are obtainable.
    • Coolness Factor: Because it's iGEM, and it'll look awesome.
    • Novelness: Even if we fail on further levels, simply building a box is novel

• • what pieces of the project do you already have a good idea that they can & will work? what is novel?

• • • Jack-able:
      • Tetrahedron concept, and possibly sequence, if we go w/ the open-surface model (a la http://www.sciencemag.org.ezp1.harvard.edu/cgi/content/full/310/5754/1661/FIG1)
      • Thrombin-aptamer sequence
      • Endo-incorporation of aptamer into oligo sequences (a la http://www3.interscience.wiley.com.ezp1.harvard.edu/cgi-bin/fulltext/110526995/PDFSTART)
      • DNA tweezer strand-replacement sequence - though we can easily create one ourselves to hold a vertex of the tetrahedron together

• • • Novel:
      • Design and sequence, if not going with the open-tetrahedron

• Logistics

• • outline of project milestones and suggestions for division of labor

1) Design a box with strand-displaced clasp (group) - we should design at least two different kinds of boxes. 2) Write code to design box with oligo staples without aptamers, for testing, and with aptamers; (4 people at least, 2 per box) 3) Test closed box w/o aptamers to see if it actually opens (gel studies should be good enough to show this). Simultaneously, test closed box w/ aptamers to see if it still opens (2 people at least). 4) Test closed box w/o aptamers to see if it opens in presence of thrombin. (2 people at least) Simultaneously, test open box to see if aptamers on inner box surface actually bind thrombin (again, gel studies - or perhaps Western blot) (1 person). 5) Test closed boxes w/ aptamers in in vitro system with thrombin, adding strand-displacement oligo after. See if it sequesters (1 person) 6) PEG-enclosure?

• • costs ($$ & time)

• Potential iGEM problems

• • articulating how this fits into iGEM

• • BioBricks

• Brief Summary

• • we are trying to solve problem X with approach Y

• • if we are successful, what will we be able to deliver in November
    • Openable, triggerable box that binds thrombin and has a chance of being biostable
  • if we are unsuccessful, what will we be able to deliver in November
    • Box designs, possibly a non-opening box

Bibliography

Thrombin

1. Velan T and Chandler WL. Effects of surgical trauma and cardiopulmonary bypass on active thrombin concentrations and the rate of thrombin inhibition in vivo. Pathophysiol Haemost Thromb. 2003 May-Jun;33(3):144-56. DOI:10.1159/000077823 |
2. Jackson CM and Nemerson Y. Blood coagulation. Annu Rev Biochem. 1980;49:765-811. DOI:10.1146/annurev.bi.49.070180.004001 |

Thrombin-Aptamer

3. Schultze P, Macaya RF, and Feigon J. Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG). J Mol Biol. 1994 Feb 4;235(5):1532-47. DOI:10.1006/jmbi.1994.1105 |
4. Liu Y, Lin C, Li H, and Yan H. Aptamer-directed self-assembly of protein arrays on a DNA nanostructure. Angew Chem Int Ed Engl. 2005 Jul 11;44(28):4333-8. DOI:10.1002/anie.200501089 |

DNA Bioavailability

5. Patil SD, Rhodes DG, and Burgess DJ. DNA-based therapeutics and DNA delivery systems: a comprehensive review. AAPS J. 2005 Apr 8;7(1):E61-77. DOI:10.1208/aapsj070109 |
6. Houk BE, Martin R, Hochhaus G, and Hughes JA. Pharmacokinetics of plasmid DNA in the rat. Pharm Res. 2001 Jan;18(1):67-74. DOI:10.1023/a:1011078711008 |
7. Kawabata K, Takakura Y, and Hashida M. The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharm Res. 1995 Jun;12(6):825-30. DOI:10.1023/a:1016248701505 |

PEG Covering

8. Lee M and Kim SW. Polyethylene glycol-conjugated copolymers for plasmid DNA delivery. Pharm Res. 2005 Jan;22(1):1-10. DOI:10.1007/s11095-004-9003-5 |