detection of local protein structures along dna …...present advantages over biological pores...
TRANSCRIPT
Detection of local protein structures along DNA using solid-state nanopores
Stefan Kowalczyk Adam Hall
Cees Dekker
Bremen 29-06-2009
nanopore
RecA-DNA filament
(Nano Letters cover September 2009)
Main topics:
RecA
dsDNA
• Solid-state nanopores for proteins on DNA
• Nuclear pore complex (NPC)
http://sspatel.googlepages.com/nuclearporecomplex
Main topics:
• Solid-state nanopores for proteins on DNA
• Nuclear pore complex (NPC)
Stefan KowalczykMichiel van den HoutGary SkinnerAdam HallRalph SmeetsMeng-Yue WuUlrike ZieseSerge LemayNynke DekkerCees Dekker
Biopolymer translocation through nanopores
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
http://www.chemsoc.org/exemplarchem/entries/2002/Tim_Smith/transp http://www.scripps.edu/~stoffler/proj/NPC/npc.html
Nanopores in biology:• ion channels
• nuclear pore complexes
• viral infection
• protein secretion
• bacterial gene transfer
• etc etc
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 lengthHigh stability (temperature, pH, salt, ..)Adjustable surface properties of the pore Allows integration into devices and arrays
SiO2
or SiN nanopores from silicon processing
Fine tuning by ‘glass making’
with a TEM beam True nanometer control of the nanopore size
High-intensity TEM slowly closes the pore, with live imagingStop imaging to ‘freeze’ the geometry for nm-sized pore
5 nm
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
Sign reversal of current signal at low salt
τ
~ Lα
1M 0.1M
Power-law length dependence of translocation time
Noise studies, evidence for nanobubbles
Nanopore technique basically applicable to any charged molecule
- polynucleotides
- proteins -
protein-DNA complexes
-
polyelectrolytes
- nanotubes - nanowires -
quantum dots
etc
Three examples of recent research1. double and single strand RNA2. proteins on DNA3. 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
dGC
ondu
ctan
ce c
hang
e 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
RecADNA
dsDNA RecA-coated dsDNA
R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
AFM imaging of RecA-coated DNA
Blockades are 15 times bigger than for dsDNA
Consistent with large cross sectiondG => d=8.5 nm
R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
Translocation of RecA-coated double-strand DNA
Translocation of RecA-coated double-strand DNA
RecA + DNA RecA only
3) Two different regimes (constant and exponential event rate vs. voltage)
1) Dwell times of bare DNAand fully RecA-coated DNAare equal within error(surprisingly?)
2) Poissonian
process (“no memory”)
R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
Some lessons learned:
Next step: “Read out”
DNA along its length
Local patches of RecA protein along DNA
S. Kowalczyk, et al, submitted
Height scale [0-2 nm]
translocation data conductance histogram
(dG = dI/V)
Local patches of RecA protein along DNA
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
VV
The Optical Tweezer-Nanopore System
Same trick with optical tweezers?
A. Hall, et al
20
10
0
-10
100
50
0
-50
The Optical Tweezer-Nanopore System
A. Hall, et al
Applying RecA to the Hybrid System
1M KCl
A. Hall, et al
A. Hall, et al
First data on captured partly-RecA
coated DNA
Under investigation…
Salt dependence of RecA-DNA translocations (preliminary data)
“Crossover”
~0.35M KCl; in agreement with R. Smeets, et al, Nano
Letters, 2006
dsDNA
RecA-DNA
dsDNA
RecA-DNA
Salt dependence of RecA-DNA translocations (preliminary data)
0.2M KCl
• Current increases from DNA• Current decreases from RecA-DNA
http://sspatel.googlepages.com/nuclearporecomplex
Main topics:
• Solid-state nanopores for proteins on DNA
• Nuclear pore complex (NPC)
The cell as a collection of
protein machines
The only way to get from the nucleusto the cytoplasm is through a nuclear pore complex
Use solid-state nanopores as a chassis to build biomimetic artificial nuclear pore complexes
http://sspatel.googlepages.com/nuclearporecomplex
in collaboration with Roderick Lim and Ueli Aebi (Basel)
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
Roderick Lim, et al, Science (2007)
“Selective gating”
/ “virtual gating”
Recent simultations
from Klaus Schulten’s
group (Urbana)
L. Miao and K. Schulten, Structure, 17, (2009)
MD evidence thatFG-nups
form
brushes
Building a “minimalistic NPC”…
Thanks to Roderick Lim and Larisa Kapinos-Schneider! (Basel University)
…
starts withprotein purification
Next task: chemistry thinking…How to attach those FG-proteins to the SiN…?
Our hero cross-linker:
NH2- -SH
Transport of Importin-Beta throughbare and modified nanopores
FG-nups
TEM images of the same 40 nm nanoporeBefore (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
<Tdwell
> = 0.12 ±
0.02 ms <Tdwell > = 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. FG-
nanopore, 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
One-line summary:
Solid state nanopores are versatile new probes for biophysics
postdoc openings!!
15 faculty openings at Delft as well !!