Nucleic Acid Engineering and Its Applications
Molecular BioEngineering Laboratory
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. 18th, 2002)
Outline
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What is Nucleic Acid Engineering
(what we are doing)
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Nucleic Acid Delivery
(what we are doing in agricultural research)
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Introduction: “Bottom-up” Nanotechnology
DNA as generic, rather than genetic materials
Introduction: non-viral DNA delivery systems
Deliver DNA to agriculturally important animals
The Applications of Nucleic Acid Engineering
(Where we are going from here?)
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Nano-patterning
Nano-barcoding
Nano-signal amplification
Nano-wiring
Two Directions in Nanotechnology
Control the “building blocks”
Novel materials and/or devices
Controlled Assembly
Control the “Knife”
Etching, etc.
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
DNA Nanotechnology
DNA molecules have been regarded almost exclusively as genetic
information carriers and not polymeric molecules or construction materials,
until Nadarin Seeman.
http://www.nyu.edu/pages/chemistry/
http://seemanlab4.chem.nyu.edu/ homepage.html
Construct Basic Branching Building Block: Y-DNA
•No self-ligation
•Controlled growth
•High purity
•High yield
•Monodispersed
DL-DNA
G0=Y0
G1=Y0+3Y1
G2=G1+6Y2=Y0+3Y1+6Y2
Synthesis of Y0-DNA
Lane 1 : Y0a
Lane 2 : Y0b
Lane 3 : Y0c
Y
Partial Y
Oligo
Lane 4 : Y0a-Y0b (step-wise)
Lane 5 : Y0a-Y0c (step-wise)
Lane 6 : Y0b-Y0c (step-wise)
Lane 7 : Y0a-Y0b-Y0c (step-wise)
Lane 8 : Y0a-Y0c-Y0b (step-wise)
Lane 9 : Y0b-Y0c-Y0a (step-wise)
Lane 10: Y0aY0bY0c (all-in-one)
Dendrimer-Like DNA Synthesis Strategy
High Generation DL-DNA: Schematic
Y0
G0
G1
G2
G3
G4
Y1
Y2
Y3
Y4
High Generation DL-DNA
G5
G4
G3
G2
G1
Blank Control
Buffer Control
Importance of Non-viral DNA Delivery in
basic research (basic agricultural research)
9000
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Papers published with the key word “transfection(s)”
(total 78930 as of 11/16/2002)
Currently about 1 paper published every hour, 24/7
~ 1 paper/hour
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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=PubMed
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DNA Delivery Pathways and Barriers
Slow release with less stability
Low uptake
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DNA
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

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Lacking nuclear targeting
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 DNA-complex formation
 Uptake
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 Endocytosis (endosome)
 Escape from endosome
 Degradation (endosome)
 Intracellular release
 Degradation (cytosol)
Dan Luo and W. Mark Saltzman “Synthetic DNA delivery systems”
Nature Biotechnology 18, 33-37, 2000

 Nuclear targeting
 Nuclear entry and
expression
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
Fig. 14 Multi-gene delivery via a magnetic field
More particles are
Most particles float attracted to cell surface
No magnetic field
With magnetic field
DNA Delivery to Agriculturally Important Animals
Percent changes between pST-treated pigs and controls
Daily Gain
Feed/Gain
Backfat
+15.2%
-21.1%
-24.8%
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Loin Eye
+18.5%
Muscle
+9.9%
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).
DNA Nano-Patterning
DNA Nano-barcoding
Coding Capacity with only 2 colors = 2n, where n is the number of positions
For 3 colors (above) and 2 positions: = 32 = 9 nanobarcodes
DNA nano-wiring: nanoscale electronics?
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