Synthetic Nanopore Force- Spectroscopy
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1 Synthetic Nanopore Force- Spectroscopy Andre Marziali Department of Physics and Astronomy University of British Columbia
2 Nanopore force spectroscopy DNA-DNA Interactions - Genotyping (SNP) Receptor-Ligand Interactions - Avidin-biotin (model system) - Protein detection τ =τd e Eb f xbarrier k BT Protein Structural Transition - β-sheet melting/annealing
3 DNA Sequence Detection Jonathan Nakane, Matthew Wiggin, Andre Marziali, Biophys J Carolina Tropini, Andre Marziali, Biophys J. 2007
4 Single Nucleotide Resolution measurements per data point 3G12G 7C 100 7T 10C 10 PC rev PC seconds mv Dissociation time for the match is 100 times the nearest same base mismatch.
5 Nanopore-based genotyping Demonstration on organic pores: SNP from gene SERPINE1 (rs7242) is associated with increase risk in SEPSIS patient P survival Comparison of Force Spectroscopy Results for Mixed vs. Pure Analytes (-50mV) E-3 PM+7C Data PM Data 7C Data PM+7C Fit 1E-4 1E Time (s) % called nmof target DNA rs7242-g rs7242-t rs7242-gt -- Haplotype % called for SNP_G % called for SNP_T
6 Synthetic-organic hybrid nanosensor array A25 : mv
7 Label-free genotyping assay Sandwich Assay
8 Solid-State Nanopore Fabrication Individual Nanopores fabricated with single nanometre precision in thin SiNx membranes TEM 1.5 nm 3.5 nm 10 nm
9 Noise reduction Current (pa)
10 Noise reduction 200 mv Applied Potential Vincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, /f Noise S = a + a f + a f pa Hz / Thermal Noise Dielectric Noise
11 λ-dna (48.5 kbp dsdna) at +150 mv in 1M KCl, ~ 7nm pore A25 : mv
12 Synthetic Nanopore Force Spectroscopy 3 nm
13 Single Base Pair Resolution DNA duplex dissociation under force α-hl data SiN x data Force applied -50mV Force applied -150mV 1 1 PM PM Survival Probability 0.1 MM τ α PM = 0.77 Survival Probability 0.1 MM τ α MM = 0.24 τ α PM = 6.74 τ α MM = E-4 1E Time (s) Time (s) Perfect Match: 5 -GGTTTGGTTGGTGG-3 Mismatch: 5 -GGTTTGCTTGGTGG-3
14 Single Base Pair Resolution Comparison of PM& MM Analytes: Dissociation Timescales During Force Spectroscopy DNA Sequences used : Characteristics Timescale, τ α (1) (2) (1) (2) (4) (4) (2) PM pore1 MM pore1 MM pore2 PM 5 -GGTTTGGTTGGTGG k B T MM 5 -GGTTTGCTTGGTGG k B T PM= Perfect Match, MM= MisMatch Applied Force (mv)
15 DNA-pore interactions Current (pa) Time (s) Applied Potential (mv) Fit: 0.4 E = k T ln( τ / τ ) 2 3k T B i j B t / i Ae τ i i Coefficient Energy E b E b + E x Timescale (s)
16 Nanopore Force Spectroscopy DNA-DNA Interactions - Genotyping (SNP) Receptor-Ligand Interactions - Avidin-biotin (model system) - Protein detection Protein Structural Transition - β-sheet melting/annealing
17 Strength of Molecular Bonds biotin (Neutr)Avidin Effective charge z ~ 0.3 x b Kramer s rate theory: = b τ τ e B D E f x k T barrier 1 Applied Voltage (mv) Survival Probability mv 500 mv 600 mv 700 mv 800 mv E Time (sec) <τ> E Force (pn) Merkel et al. (1999) Nature. 397: & Izrailev, et al. (1997) Biophys. J. 72: & Pincet et al. (2005) Biophys. J. 89:
18 Nanopore Force Spectroscopy DNA-DNA Interactions - Genotyping (SNP) Receptor-Ligand Interactions - Avidin-biotin (model system) - Protein detection Protein Structural Transition - β-sheet melting/annealing
19 Prion Protein Structure Dynamics Progress on diagnostics & cures is limited by not knowing: 1. Structure of misfolded PrP 2. Mechanism of conversion
20 Nanopore Analysis of Prion Proteins Measured signal: ionic current in the pore Key Advantages: Resolution of ~Å Observe repeated structural transitions on 1 molecule
21 Evidence of Structural Transitions
22 Long-Term Observation of a Single Molecule dg ~10kcal/mol
23 Prion Protein Heterogeneity
24 Thanks Vincent Tabard-Cossa Jason Dwyer Matthew Wiggin Nahid Jetha Dhruti Trivedi Carolina Tropini Chris Feehan St. Paul s Hospital icapture center Scott Tebbutt Keith Walley Prion Collaboration Dr. Neil Cashman (UBC) Will Guest Dr. David Wishart (U of A) Bow Suriyamongkol
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