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 !!