Active Nanostructures for Nucleic Directed synthesis of Organic Functional Polymers

1. 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) Active Nanostructures for Nucleic Directed synthesis of Organic Functional Polymers Nadrian Seemana, William A. Goddard IIIb, James Canary a, Erik Winfreeb aNew York University bCalifornia Institute of Technology Massively parallel self assembly of single wall carbon nanotube FETs using self assembled DNA crystal scaffolds 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. 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. Process Overview 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”. Nylon-Nucleic Acids Form Stable Duplexes with DNA 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. Interstrand crosslinkages over distances Intrastrandcrosslinkages Crosslink (Uc4-Un4) Yield M1 M2 M3 N1 Major groove Good Major groove Good Major groove Fair Minor groove Excellent 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. NNA = nylon-nucleic acid 15’-GCATAGT T T T T T GTCTAC 25’-GCATAGT T Un Uc T T GTCTAC 35’-GCATAGT T Un UccUn T GTCTAC 45’-GCATAGT Uc UnnUccUn T GTCTAC 55’-GCATAGT Uc UnnUccUnnUcGTCTAC 65’-GCATAGUcUnnUccUnnUccUnGTCTAC minor groove crosslink major groove crosslink Minor groove crosslink increases Tm by 17°C Single strand 3 in B-form conformation with pendent groups in yellow. Thermal denaturing studies and circular dichroism measurements show stability of nylon-nucleic acid duplexes with DNA. Coupling of double strand was successful with Un12 + Uc4 but not with Un4 + Uc4. Coupling occurs more efficiently between Uc4 and Un4 across the minor groove than across the major groove. 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. Meso-scale Molecular Dynamics Simulation of DNA Origami The Mesoscale DNA Forcefield • 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 Meso vs Atomistic B-DNA • Meso helical parameters within acceptable range for BDNA • Over 100x speedup