nucleic acid engineering and its applications dan luo, ph.d. assistant professor department of...
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Nucleic Acid Engineering and Its ApplicationsNucleic Acid Engineering and Its Applications
Dan Luo, Ph.D.Dan Luo, Ph.D.Assistant ProfessorAssistant Professor
Department of Biological and Environmental EngineeringDepartment of Biological and Environmental EngineeringCornell UniversityCornell UniversityIthaca, New YorkIthaca, New York
(National Planning Workshop – Nanoscale Science and Engineering for Agriculture and Food Systems, (National Planning Workshop – Nanoscale Science and Engineering for Agriculture and Food Systems, Washington DC Nov. 18Washington DC Nov. 18thth, 2002), 2002)
Molecular BioEngineering LaboratoryMolecular BioEngineering Laboratory
Outline
What is Nucleic Acid Engineering What is Nucleic Acid Engineering (what we are doing)(what we are doing) Introduction: “Bottom-up” NanotechnologyIntroduction: “Bottom-up” Nanotechnology DNA as generic, rather than genetic materialsDNA as generic, rather than genetic materials
Nucleic Acid Delivery Nucleic Acid Delivery (what we are doing in agricultural research)(what we are doing in agricultural research) Introduction: non-viral DNA delivery systemsIntroduction: non-viral DNA delivery systems Deliver DNA to agriculturally important animalsDeliver DNA to agriculturally important animals
The Applications of Nucleic Acid EngineeringThe Applications of Nucleic Acid Engineering(Where we are going from here?)(Where we are going from here?) Nano-patterningNano-patterning Nano-barcodingNano-barcoding Nano-signal amplificationNano-signal amplification Nano-wiringNano-wiring
Two Directions in Nanotechnology
Etching, etc.C
on
tro
l th
e “b
uild
ing
blo
cks”
Controlled Assembly
Co
ntro
l the “K
nife”
Novel materials and/or devicesNovel materials and/or devices
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 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
G0=Y0G0=Y0
G1=Y0+3Y1G1=Y0+3Y1
G2=G1+6Y2=Y0+3Y1+6Y2G2=G1+6Y2=Y0+3Y1+6Y2
Construct Basic Branching Building Block: Y-DNA
DL-DNA
•No self-ligation•Controlled growth•High purity•High yield•Monodispersed
Lane 1 : Y0a
Lane 2 : Y0b
Lane 3 : Y0c
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)
Synthesis of Y0-DNA
Oligo
Partial Y
Y
Dendrimer-Like DNA Synthesis Strategy
High Generation DL-DNA: Schematic
G0
Y0
Y1 G1
Y2
G2
Y3
G3
Y4
G4
G1
G2
G3
G4
G5
High Generation DL-DNA
Blank Control Buffer Control
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
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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
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
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Dan Luo and W. Mark Saltzman “Synthetic DNA delivery systems” Nature Biotechnology 18, 33-37, 2000
DNA Delivery Pathways and Barriers
Plug-and-play Multi-gene Delivery System
Modules: 1 BFP, 2 RFP, 0 YFP and 1 Au-MB
Core (X0)
B2Y0-(X0)-R4M2
Fig. 14 Multi-gene delivery via a magnetic field
No magnetic field With magnetic field
More particles are attracted to cell surface Most particles float
D. Luo and W. M. Saltzman. Enhancement of transfection by physical concentration of DNA at the cell surface. Nature Biotechnology 18, 893-895, 2000
DNA Delivery to Agriculturally Important Animals
Percent changes between pST-treated pigs and controls Daily Gain Feed/Gain Backfat Loin Eye Muscle
+15.2% -21.1% -24.8% +18.5% +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 positionsFor 3 colors (above) and 2 positions: = 32 = 9 nanobarcodes
DNA nano-wiring: nanoscale electronics?
Conclusion
““There’s Plenty of Room at the Bottom.”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 the bottom-up approach, and there’s plenty of room for Agriculture and Food Systemsthere’s plenty of room for Agriculture and Food Systems