1. Nucleic Acid Engineering and Its Applications Dan Luo, Ph.D. Assistant Professor Department of Biological and Environmental Engineering Cornell University Ithaca, New York (National Planning Workshop – Nanoscale Science and Engineering for Agriculture and Food Systems, Washington DC Nov. 18 th, 2002) Molecular BioEngineering Laboratory

2. Outline What is Nucleic Acid Engineering (what we are doing) What is Nucleic Acid Engineering (what we are doing) Introduction: “Bottom-up” Nanotechnology Introduction: “Bottom-up” Nanotechnology DNA as generic, rather than genetic materials DNA as generic, rather than genetic materials Nucleic Acid Delivery (what we are doing in agricultural research) Nucleic Acid Delivery (what we are doing in agricultural research) Introduction: non-viral DNA delivery systems Introduction: non-viral DNA delivery systems Deliver DNA to agriculturally important animals Deliver DNA to agriculturally important animals The Applications of Nucleic Acid Engineering (Where we are going from here?) The Applications of Nucleic Acid Engineering (Where we are going from here?) Nano-patterning Nano-patterning Nano-barcoding Nano-barcoding Nano-signal amplification Nano-signal amplification Nano-wiring Nano-wiring

3. Two Directions in Nanotechnology Etching, etc. Control the “building blocks” Controlled Assembly Control the “Knife” Novel materials and/or devices

4. DNA —Amazing Nanoscale Building Blocks DNA molecules posses amazing properties Mechanical Flexible rigid (when <persistence length 50nm) Physical small (width=2.0 nm, length=0.34 nm/bp) linear or circular (but no branching) Chemical stable non-toxic commercially available in large quantities and high purity DNA molecules are highly manipulable self-assembly (“Velcro”) enzymatic reactions

5. DNA molecules have been regarded almost exclusively as genetic information carriers and not polymeric molecules or construction materials, until Nadarin Seeman. DNA Nanotechnology http://www.nyu.edu/pages/chemistry/ http://seemanlab4.chem.nyu.edu/ homepage.html

6. G0=Y0G1=Y0+3Y1G2=G1+6Y2=Y0+3Y1+6Y2 Construct Basic Branching Building Block: Y-DNA DL-DNA No self-ligation Controlled growth High purity High yield Monodispersed

7. Lane 1 : Y 0a Lane 2 : Y 0b Lane 3 : Y 0c Lane 4 : Y 0a -Y 0b (step-wise) Lane 5 : Y 0a -Y 0c (step-wise) Lane 6 : Y 0b -Y 0c (step-wise) Lane 7 : Y 0a -Y 0b -Y 0c (step-wise) Lane 8 : Y 0a -Y 0c -Y 0b (step-wise) Lane 9 : Y 0b -Y 0c -Y 0a (step-wise) Lane 10: Y 0a Y 0b Y 0c (all-in-one) Synthesis of Y 0 -DNA Oligo Partial Y Y

8. Dendrimer-Like DNA Synthesis Strategy

9. High Generation DL-DNA: Schematic G0G0 Y0Y0 Y1Y1 G1G1 Y2Y2 G2G2 Y3Y3 G3G3 Y4Y4 G4G4

10. G1G1 G2G2 G3G3 G4G4 G5G5 High Generation DL-DNA

11. Blank Control Buffer Control

12. Importance of Non-viral DNA Delivery in basic research (basic agricultural research) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=PubMed ~ 1 paper/hour Papers published with the key word “transfection(s)” (total 78930 as of 11/16/2002) Currently about 1 paper published every hour, 24/7

13.  DNA-complex formation  Uptake  Endocytosis (endosome)  Escape from endosome  Degradation (endosome)  Intracellular release  Degradation (cytosol)  Nuclear targeting  Nuclear entry and expression Low uptake Slow release with less stability Lacking nuclear targeting DNA       ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕          Dan Luo and W. Mark Saltzman “Synthetic DNA delivery systems” Nature Biotechnology 18, 33-37, 2000 DNA Delivery Pathways and Barriers

14. Plug-and-play Multi-gene Delivery System D. Luo and W. M. Saltzman. Enhancement of transfection by physical concentration of DNA at the cell surface. Nature Biotechnology 18, 893-895, 2000

15. DNA Delivery to Agriculturally Important Animals DNA vs. Protein Delivery (cost; safety; simplicity; etc.) Bolus vs. Controlled Release Delivery DNA Encapsulation in Molded-Nanowells Multi-gene Delivery in Controlled Release Polymers DNA-polymer Hybrid Materials for Delivery *Zimmerman, D. in New Swine Growth Enhancers (Ames, Iowa, 1989).

16. DNA Nano-Patterning

17. DNA Nano-barcoding Coding Capacity with only 2 colors = 2 n, where n is the number of positions For 3 colors (above) and 2 positions: = 3 2 = 9 nanobarcodes

18. DNA nano-wiring: nanoscale electronics?

19. Conclusion “There’s Plenty of Room at the Bottom.” --- --- Richard Feynman (Dec. 1959) There’s plenty of room for the bottom-up approach, and there’s plenty of room for Agriculture and Food Systems