Druk Functional Polymers for Targeted Delivery of Nucleic Acid Drugs 2009 Macromolecular Bioscience
Active Nanostructures for Nucleic Directed synthesis of Organic Functional Polymers
1
POSTER TEMPLATE BY: www.PosterPresentations.co m Active Nanostructures for Nucleic Directed synthesis of Active Nanostructures for Nucleic Directed synthesis of Organic Functional Polymers Organic Functional Polymers Nadrian Seeman a , William A. Goddard III b , James Canary a , Erik Winfree b a New York University b California Institute of Technology 1 5’-GCATAGT T T T T T GTCTAC 2 5’-GCATAGT T Un Uc T T GTCTAC 3 5’-GCATAGT T Un UccUn T GTCTAC 4 5’-GCATAGT Uc UnnUccUn T GTCTAC 5 5’-GCATAGT Uc UnnUccUnnUcGTCTAC 6 5’-GCATAGUcUnnUccUnnUccUnGTCTAC Thermal denaturing studies and circular dichroism measurements show stability of nylon-nucleic acid duplexes with DNA. DNA duplex of strands containing pendent groups prior to coupling are less stable than control. Stabilities of DNA duplex of strands after coupling are comparable to control. Circular dichroism spectra of DNA duplex with 6 shows typical signature for B-like secondary structure. Single strand 3 in B-form conformation with pendent groups in yellow. Intrastrand crosslinkages PEG UnUc Sequence Coupling Yield T1 5’-TTUn4TTTTTTTTUc4TTTT dsNNA/DNA: no coupling; dsNNA/RNA: T1>T2>T3; ssNNA: high yield. T2 5’-TTUn4TTTTTTTTTUc4TTT T3 5’-TTUn4TTTTTTTTTTUc4TT Hs1 5’-TGUn4ACGTGCGAUc4TTCG dsNNA/DNA: no coupling; dsNNA/RNA: no coupling; ssNNA: high yield. Hs2 5’-TGUn4ACGTGCGATUc4TCG Hs3 5’-TGUn4ACGTGCGATTUc4CG Hl1 5’-TGUn12ACGTGCGAUc4TTCG dsNNA/DNA: Hl1≈Hl2>Hl3. Hl2 5’-TGUn12ACGTGCGATUc4TCG Hl3 5’-TGUn12ACGTGCGATTUc4CG Uc1 5’-TGTACGTGCGAT Uc4TCG (16mer) No coupling observed (negative control). Uc2 5’-TGACGTGCGAT Uc4TCG (15mer) QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Uc 4 CO 2 H Un 12 DM T-M M H 2 N QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. U c 4 C Un 12 HN O O O O O OH O O O O O NH 2 R = NH O O N O S O P O O O O - R U c4 Un4 O O O O O O O O O O O NH 2 NH 2 OH O Uc Un U n12 NNA = nylon-nucleic acid Interstrand crosslinkages over distances Crosslink (Uc4-Un4) Yield Major groove Good Major groove Good Major groove Fair Minor groove Excellent M1 M2 M3 N1 Coupling occurs more efficiently between Uc4 and Un4 across the minor groove than across the major groove. minor groove crosslink major groove crosslink Minor groove crosslink increases Tm by 17°C QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. NH 2 QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. U n4 CO 2 H U n4 NH U n4 C U n4 O DM T-M M QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. NH U n4 C U n4 O Coupling of double strand was successful with Un12 + Uc4 but not with Un4 + Uc4. Dispersal complexes are used to link SWNTs to DNA hooks. DNA hooks bind to a 5 nt toehold and displace the protection strand by branch migration. Increasing the length of the toehold to 7 nt and switching the dispersal buffer from Na + to Mg 2+ results in extensive formation of SWNT-DNA “ladders”. SWNTs labeled with “red” and “blue” sequences attach to respective hook positions. Orientation occurs through cooperative action of many hooks. In a one pot reaction, blue SWNTs will only attach at blue sites while reds will only attach at red sites. Assembled FETs are deposited on SiO2 and contacted with Au covered Pd electrodes using standard ebeam lithography techniques. Device shown below has clear switching and exhibits signal gain. DNA origami NA hooks with “red” and “blue” sequences on opposite faces. Additional DX tiles forming a DNA ribbon are added as structural reinforcement. Entire scaffold is ligated for additional strength. Process Overview Synthesize and characterize functional nanomachines and devices using: General strategies for synthesizing DNA structures that self organize to provide a scaffold designed to produce polymers with the diversity and precision that ribosomes exhibit in building proteins: developed by the Seeman lab Methods to crosslink DNA strands across the minor groove and offer the potential to develop polymers whose topology can be directed by the single-stranded DNA topology of unusual motifs: developed by the Canary lab Self assembled an all-single wall carbon nanotube (SWNT) field effect transistor (FET) on DNA origami using solution phase molecular linker mediated attachment: developed by the Winfree/Goddard labs A Mesoscale model of DNA to investigate the structural and thermodynamics properties of these systems: developed by the Goddard lab. Small piece of experimental origami 1856 DNA base-pairs 2.5nm x 17.7nm x 60.5nm 2-d structure not known Atomistic system >360,000 atoms: 60,000 DNA atoms, 1836 Na+ ions, 300,000 waters Estimated 2 weeks for 15ns of simulation on 100 processors (4 years for 1.5 microseconds) Coarse Grain system ~100,000 atoms 3 weeks for 1.5 microseconds (75x speedup) Backbone-base structure Bead for Phosphate/Ribose/Nucleoside Bead for water molecule Bead for Ions quasi-Bead for Hydrogens ▪ Bonds, angles not calculated during dynamics ▪ Move as rigid body with parent nucleotide Statistics obtained from explicit water atomistic simulations All beads are neutral, Morse potential for nonbonds (VDW + Coulomb) Bond stretch: Harmonic Angle bend: Cosine Harmonic Torsion: Harmonic Hydrogen Bonds: Dreiding van der Waals: Morse Potential 2 2 1 ) ( eq Bonds r r V 2 2 1 ) cos (cos eq Angles V d n V Torsions cos 1 2 1 4 10 12 0 cos 6 5 0 0 r r r r HBond D V 1 5 . 0 2 1 5 . 0 0 0 0 2 R R R R R ij ij ij e e D V CRMS Helical Parameters Simulation Details Meso Crysta l Atoms Rise Twist Length CPU Time Meso - 5.583 2.727 3.62 (1.28) 29.22 (8.06) 2.5 micro- sec 12.5 days Atoms 2.727 3.023 - 3.5 (0.32) 34.69 (2.47) 25 nano- sec 14 days Meso helical parameters within acceptable range for BDNA Over 100x speedup
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PEG UnUc. Sequence. Coupling Yield. T1. 5’-TT Un4 TTTTTTTT Uc4 TTTT. dsNNA/DNA: no coupling; dsNNA/RNA: T1>T2>T3; ssNNA: high yield. T2. 5’-TT Un4 TTTTTTTTT Uc4 TTT. T3. 5’-TT Un4 TTTTTTTTTT Uc4 TT. Hs1. 5’-TG Un4 ACGTGCGA Uc4 TTCG. dsNNA/DNA: no coupling; dsNNA/RNA: no coupling; - PowerPoint PPT Presentation
Transcript of Active Nanostructures for Nucleic Directed synthesis of Organic Functional Polymers
POSTER TEMPLATE BY:
www.PosterPresentations.com
Active Nanostructures for Nucleic Directed synthesis of Organic Functional Polymers Active Nanostructures for Nucleic Directed synthesis of Organic Functional Polymers Nadrian Seemana, William A. Goddard IIIb, James Canary a, Erik Winfreeb
aNew York UniversitybCalifornia Institute of Technology
1 5’-GCATAGT T T T T T GTCTAC
2 5’-GCATAGT T Un Uc T T GTCTAC
3 5’-GCATAGT T Un UccUn T GTCTAC
4 5’-GCATAGT Uc UnnUccUn T GTCTAC
5 5’-GCATAGT Uc UnnUccUnnUcGTCTAC
6 5’-GCATAGUcUnnUccUnnUccUnGTCTAC
Thermal denaturing studies and circular dichroism measurements show stability of nylon-nucleic acid duplexes with DNA.Thermal denaturing studies and circular dichroism measurements show stability of nylon-nucleic acid duplexes with DNA.
DNA duplex of strands containing pendent groups prior to coupling are less stable than control.
Stabilities of DNA duplex of strands after coupling are comparable to control.
Circular dichroism spectra of DNA duplex with 6 shows typical signature for B-like secondary structure.
Single strand 3 in B-form conformation with pendent groups in yellow.
Intrastrand crosslinkages
PEG UnUc Sequence Coupling Yield
T1 5’-TTUn4TTTTTTTTUc4TTTTdsNNA/DNA: no coupling; dsNNA/RNA: T1>T2>T3;ssNNA: high yield.
T2 5’-TTUn4TTTTTTTTTUc4TTT
T3 5’-TTUn4TTTTTTTTTTUc4TT
Hs1 5’-TGUn4ACGTGCGAUc4TTCGdsNNA/DNA: no coupling;dsNNA/RNA: no coupling;ssNNA: high yield.
Hs2 5’-TGUn4ACGTGCGATUc4TCG
Hs3 5’-TGUn4ACGTGCGATTUc4CG
Hl1 5’-TGUn12ACGTGCGAUc4TTCG
dsNNA/DNA: Hl1≈Hl2>Hl3.Hl2 5’-TGUn12ACGTGCGATUc4TCG
Hl3 5’-TGUn12ACGTGCGATTUc4CG
Uc1 5’-TGTACGTGCGAT Uc4TCG (16mer)No coupling observed (negative control).Uc2 5’-TGACGTGCGAT Uc4TCG (15mer)
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Uc4 CO2H
Un12
DMT-MMH2N
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
U c 4 C
Un12
HN
O
OO
OO
OH
O
OO
OO
NH2
R =
NH
O
ON
O
SO
PO
O
O
O- RUc4
Un4
OOO OO
OO OOOO NH2
NH2
OH
OUc
Un
Un12
NNA = nylon-nucleic acid
Interstrand crosslinkages over distances
Crosslink (Uc4-Un4) YieldMajor groove Good
Major groove Good
Major groove Fair
Minor groove Excellent
M1
M2
M3
N1
Coupling occurs more efficiently between Uc4 and Un4 across the minor groove than across the major groove.Coupling occurs more efficiently between Uc4 and Un4 across the minor groove than across the major groove.
minor groove crosslink
major groove crosslink
Minor groove crosslink increases Tm by 17°C
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
NH2
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Un4 CO2H
Un4 NH
Un4 C
Un4
O
DMT-MM
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
NH
Un4C
Un4
O
Coupling of double strand was successful with Un12 + Uc4 but not with Un4 + Uc4. Coupling of double strand was successful with Un12 + Uc4 but not with Un4 + Uc4.
Dispersal complexes are used to link SWNTs to DNA hooks. DNA hooks bind to a 5 nt toehold and displace the protection strand by branch migration. Increasing the length of the toehold to 7 nt and switching the dispersal buffer from Na+ to Mg 2+
results in extensive formation of SWNT-DNA “ladders”.
SWNTs labeled with “red” and “blue” sequences attach to respective hook positions. Orientation occurs through cooperative action of many hooks. In a one pot reaction, blue SWNTs will only attach at blue sites while reds will only attach at red sites.
Assembled FETs are deposited on SiO2 and contacted with Au covered Pd electrodes using standard ebeam lithography techniques. Device shown below has clear switching and exhibits signal gain.
DNA origami NA hooks with “red” and “blue” sequences on opposite faces. Additional DX tiles forming a DNA ribbon are added as structural reinforcement. Entire scaffold is ligated for additional strength.
Process Overview
Synthesize and characterize functional nanomachines and devices using: General strategies for synthesizing DNA structures that self organize to provide a scaffold designed to produce polymers with the diversity and precision that ribosomes exhibit in building proteins: developed by the Seeman lab Methods to crosslink DNA strands across the minor groove and offer the potential to develop polymers whose topology can be directed by the single-stranded DNA topology of unusual motifs: developed by the Canary lab Self assembled an all-single wall carbon nanotube (SWNT) field effect transistor (FET) on DNA origami using solution phase molecular linker mediated attachment: developed by the Winfree/Goddard labs A Mesoscale model of DNA to investigate the structural and thermodynamics properties of these systems: developed by the Goddard lab.
Small piece of experimental origami 1856 DNA base-pairs 2.5nm x 17.7nm x 60.5nm 2-d structure not known
Atomistic system >360,000 atoms: 60,000 DNA atoms, 1836
Na+ ions, 300,000 waters Estimated 2 weeks for 15ns of simulation on
100 processors (4 years for 1.5 microseconds) Coarse Grain system
~100,000 atoms 3 weeks for 1.5 microseconds (75x speedup)
Backbone-base structure Bead for Phosphate/Ribose/Nucleoside Bead for water molecule Bead for Ions quasi-Bead for Hydrogens
▪ Bonds, angles not calculated during dynamics▪ Move as rigid body with parent nucleotide
Statistics obtained from explicit water atomistic simulations
All beads are neutral, Morse potential for nonbonds (VDW + Coulomb)
Bond stretch: Harmonic
Angle bend: Cosine Harmonic
Torsion: Harmonic
Hydrogen Bonds: Dreiding
van der Waals: Morse Potential
221 )( eq
Bonds rrV
221 )cos(cos eq
AnglesV
dnVTorsions cos121
41012
0 cos65 00
rr
rr
HBond DV
15.0215.00
00 2 RRRRR
ijij
ijeeDV
CRMS Helical Parameters Simulation Details
Meso Crystal Atoms Rise Twist LengthCPU Time
Meso - 5.583 2.727 3.62 (1.28) 29.22 (8.06) 2.5 micro-sec 12.5 days
Atoms 2.727 3.023 - 3.5 (0.32) 34.69 (2.47) 25 nano-sec 14 days
Meso helical parameters within acceptable range for BDNA Over 100x speedup
