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