Detection of local protein structures along DNA using solid-state nanopores nanopore Stefan Kowalczyk Adam Hall Cees Dekker RecA-DNA filament (Nano Letters cover September 2009) Bremen 29-06-2009
Main topics: Solid-state nanopores for proteins on DNA Nuclear pore complex (NPC) dsdna RecA
Main topics: Solid-state nanopores for proteins on DNA Nuclear pore complex (NPC) http://sspatel.googlepages.com/nuclearporecomplex
Biopolymer translocation through nanopores Stefan Kowalczyk Michiel van den Hout Gary Skinner Adam Hall Ralph Smeets Meng-Yue Wu Ulrike Ziese Serge Lemay Nynke Dekker Cees Dekker
Outline Brief motivation Fabrication of solid-state nanopores Translocation of dsdna through solid-state pores Translocation of RNA Translocation of protein-coated DNA Very first data on artificial nuclear pores
Nanopores in biology: ion channels nuclear pore complexes viral infection protein secretion bacterial gene transfer etc etc http://www.chemsoc.org/exemplarchem/entries/2002/tim_smith/transp http://www.scripps.edu/~stoffler/proj/npc/npc.html
Dreams of nanopore-based DNA sequencing
Nanopores for biotechnology and biophysics: ssdna/rna through α-hemolysin biopores (Kasianowicz, Branton, Akeson, Deamer, Meller, ) Blockades of 100 bp poly(da) through an α-hemolysin membrane protein from Meller et al. PNAS 97, 1079 (2000)
Solid-state nanopores present advantages over biological pores within a lipid membrane: Flexibility in pore diameter and pore length High stability (temperature, ph, salt,..) Adjustable surface properties of the pore Allows integration into devices and arrays
SiO 2 or SiN nanopores from silicon processing
Fine tuning by glass making with a TEM beam True nanometer control of the nanopore size 5 nm High-intensity TEM slowly closes the pore, with live imaging Stop imaging to freeze the geometry for nm-sized pore A. Storm et al, Nature Mater. 2, 537 (2003)
Measuring the ionic current through a nanopore
dsdna translocation through a 10 nm nanopore A. Storm et al, Phys. Rev. E71, 051903 (2005)
Rich variety of experimental results for dsdna Folding phenomena Power-law length dependence of translocation time τ ~ L α Sign reversal of current signal at low salt Noise studies, evidence for nanobubbles 1M 0.1M
Nanopore technique basically applicable to any charged molecule -polynucleotides -proteins - protein-dna complexes - polyelectrolytes -nanotubes -nanowires - quantum dots etc Three examples of recent research 1. double and single strand RNA 2. proteins on DNA 3. artificial nuclear pore complexes
Comparison of different polynucleotides G. Skinner, M. van den Hout et al, Nano Letters (2009)
ds RNA ss RNA G. Skinner, M. van den Hout et al, Nano Letters (2009)
Current blockade amplitudes differ at high fields dg Conductance change dg (ns)
Translocation of proteins and protein-coated DNA Screening of local structures along DNA, e.g., proteins, transcription factors, nucleosomes, etc
Our model system: RecA protein on DNA DNA RecA
AFM imaging of RecA-coated DNA dsdna RecA-coated dsdna R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
Translocation of RecA-coated double-strand DNA Blockades are 15 times bigger than for dsdna Consistent with large cross section dg => d=8.5 nm R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
Translocation of RecA-coated double-strand DNA RecA + DNA RecA only
Some lessons learned: 1) Dwell times of bare DNA and fully RecA-coated DNA are equal within error (surprisingly?) 2) Poissonian process ( no memory ) 3) Two different regimes (constant and exponential event rate vs. voltage) R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
Next step: Read out DNA along its length
Local patches of RecA protein along DNA Height scale [0-2 nm] S. Kowalczyk, et al, submitted
Local patches of RecA protein along DNA translocation data conductance histogram (dg = di/v) S. Kowalczyk, et al, submitted
S. Kowalczyk, et al, submitted
What is the best resolution we can achieve? baseline DNA RecA
Total translocation time is inversely proportional with voltage S. Kowalczyk, et al, submitted
Resolution of protein along DNA: ~ 60 bp (probably even better in a very recent data set, ~ 30 bp) S. Kowalczyk, et al, submitted
The Optical Tweezer-Nanopore System Same trick with optical tweezers? V A. Hall, et al
The Optical Tweezer-Nanopore System 20 100 10 50 0 0-10 -50 A. Hall, et al
Applying RecA to the Hybrid System 1M KCl A. Hall, et al
First data on captured partly-reca coated DNA Under investigation A. Hall, et al
Salt dependence of RecA-DNA translocations (preliminary data) dsdna dsdna RecA-DNA RecA-DNA Crossover ~0.35M KCl; in agreement with R. Smeets, et al, Nano Letters, 2006
Salt dependence of RecA-DNA translocations (preliminary data) 0.2M KCl Current increases from DNA Current decreases from RecA-DNA
Main topics: Solid-state nanopores for proteins on DNA Nuclear pore complex (NPC) http://sspatel.googlepages.com/nuclearporecomplex
The cell as a collection of protein machines The only way to get from the nucleus to the cytoplasm is through a nuclear pore complex
Use solid-state nanopores as a chassis to build biomimetic artificial nuclear pore complexes in collaboration with Roderick Lim and Ueli Aebi (Basel) http://sspatel.googlepages.com/nuclearporecomplex
Small Molecules can diffuse freely through the Nuclear Pore, Larger molecules require active transport, Cartoon Biology:
F. Alber, et al, Nature 450, 695-701, 2007
F. Alber, et al, Nature 450, 695-701, 2007
Selective gating / virtual gating Roderick Lim, et al, Science (2007)
Recent simultations from Klaus Schulten s group (Urbana) MD evidence that FG-nups form brushes L. Miao and K. Schulten, Structure, 17, (2009)
Building a minimalistic NPC starts with protein purification Thanks to Roderick Lim and Larisa Kapinos-Schneider! (Basel University)
Next task: chemistry thinking How to attach those FG-proteins to the SiN? Our hero cross-linker: NH2- -SH
Transport of Importin-Beta through bare and modified nanopores FG-nups
TEM images of the same 40 nm nanopore Before (a) and after (b,c,d) attachment of FG-nups
Power spectral analysis before and after attachment:
Example traces of Importin-Beta translocations through a bare and modified nanopore ImpB through a bare pore ImpB through a modified pore
Translocation time histograms for ImpB translocations through bare and modified nanopore <T dwell > = 0.12 ± 0.02 ms <T dwell > = 2.7 ± 0.5 ms
Some indications/confirmations that the FG-nups bind to our solid-state nanopore, and that we can do transport measurements: Ellipsometry data indicates extra layers of expected thickness TEM images show some stuff in the pore that s very sensitive to the electron beam Power spectra before and after are clearly different Open pore current decreases systematically upon binding of nups (dependence of pore size and type of nups) Translocation data show 20-fold (!) increase in translocation time for Importin-Beta for bare vs. FGnanopore, with equal event amplitudes More experiments on the way..
Summing up: We have used solid-state nanopores for : - variety of experiments on dsdna - experiments on ssrna and dsrna - experiments on RecA proteins along DNA - biomimetic nuclear pore complexes
15 faculty openings at Delft as well!! One-line summary: Solid state nanopores are versatile new probes for biophysics postdoc openings!!