Synthetic Nanopore Force- Spectroscopy Andre Marziali Department of Physics and Astronomy University of British Columbia
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
DNA Sequence Detection Jonathan Nakane, Matthew Wiggin, Andre Marziali, Biophys J. 2004 Carolina Tropini, Andre Marziali, Biophys J. 2007
Single Nucleotide Resolution 1000 5 measurements per data point 3G12G 7C 100 7T 10C 10 PC rev PC seconds 0 1-10 -20-30 -40-50 -60-70 -80-90 -100 0.1 0.01 0.001 0.0001 mv Dissociation time for the match is 100 times the nearest same base mismatch.
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) 1 0.1 0.01 1E-3 PM+7C Data PM Data 7C Data PM+7C Fit 1E-4 1E-3 0.01 0.1 1 10 Time (s) % called 1.0 0.8 0.6 0.4 0.2 0.0 500 nmof target DNA rs7242-g rs7242-t rs7242-gt -- Haplotype % called for SNP_G % called for SNP_T
Synthetic-organic hybrid nanosensor array A25 : 75 115 mv
Label-free genotyping assay Sandwich Assay
Solid-State Nanopore Fabrication Individual Nanopores fabricated with single nanometre precision in thin SiNx membranes TEM 1.5 nm 3.5 nm 10 nm
Noise reduction 700 600 Current (pa) 500 400 300 200 100 0-100 0 1 2 3 4 5
Noise reduction 200 mv Applied Potential Vincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 1/f Noise S = a + a f + a f pa Hz 2 2 1 2 3 / Thermal Noise Dielectric Noise
λ-dna (48.5 kbp dsdna) at +150 mv in 1M KCl, ~ 7nm pore A25 : 75 115 mv
Synthetic Nanopore Force Spectroscopy 3 nm
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 = 0.003 0.01 1E-4 1E-3 0.01 0.1 1 10 Time (s) 0.01 0.1 1 10 100 Time (s) Perfect Match: 5 -GGTTTGGTTGGTGG-3 Mismatch: 5 -GGTTTGCTTGGTGG-3
Single Base Pair Resolution Comparison of PM& MM Analytes: Dissociation Timescales During Force Spectroscopy DNA Sequences used : Characteristics Timescale, τ α 10 1 0.1 (1) (2) (1) (2) (4) (4) (2) PM pore1 MM pore1 MM pore2 PM 5 -GGTTTGGTTGGTGG-3-37.7 k B T MM 5 -GGTTTGCTTGGTGG-3-27.4 k B T PM= Perfect Match, MM= MisMatch -225-200 -175-150 -125-100 -75-50 Applied Force (mv)
DNA-pore interactions Current (pa) 700 600 500 400 300 200 100 0-100 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0.5 Time (s) 100 80 60 40 20 0 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 0.3 0.2 0.1 Energy E b E b + E x 0.0 0.01 0.1 1 10 100 1000 Timescale (s)
Nanopore Force Spectroscopy DNA-DNA Interactions - Genotyping (SNP) Receptor-Ligand Interactions - Avidin-biotin (model system) - Protein detection Protein Structural Transition - β-sheet melting/annealing
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) 400 500 600 700 800 900 10 Survival Probability 0.1 400 mv 500 mv 600 mv 700 mv 800 mv 0.01 1E-3 0.01 0.1 1 10 Time (sec) <τ> 1 0.1 0.01 1E-3 40 50 60 70 80 90 100 110 Force (pn) Merkel et al. (1999) Nature. 397:50 53. & Izrailev, et al. (1997) Biophys. J. 72:1568 1581. & Pincet et al. (2005) Biophys. J. 89:4374 4381.
Nanopore Force Spectroscopy DNA-DNA Interactions - Genotyping (SNP) Receptor-Ligand Interactions - Avidin-biotin (model system) - Protein detection Protein Structural Transition - β-sheet melting/annealing
Prion Protein Structure Dynamics Progress on diagnostics & cures is limited by not knowing: 1. Structure of misfolded PrP 2. Mechanism of conversion
Nanopore Analysis of Prion Proteins Measured signal: ionic current in the pore Key Advantages: Resolution of ~Å Observe repeated structural transitions on 1 molecule
Evidence of Structural Transitions
Long-Term Observation of a Single Molecule dg ~10kcal/mol
Prion Protein Heterogeneity
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