Towards DNA sequencing by force

Josep Maria Huguet, Núria Forns, Fèlix Ritort

Small Biosystems Lab, Facultat de Física, UBhttp://www.ffn.ub.es/ritort/

Steve B. Smith, Carlos BustamanteBustamante Lab

UPoN 2008, Lyon, June 2-6 2008

Outline

Introduction

Optical tweezers

Experiments

Model

Results

Conclusions

Outline

Introduction

Optical tweezers

Experiments

Model

Results

Conclusions

Structure of DNA

4 different types of complementary bases(A) Adenine(C) Cytosine A - T(G) Guanine C - G(T) Thymine

... ATGCTGCGAAACTTTGGCTGA

... ATG CTG CGA AAC TTT GGC TGA

... Met Leu Arg Asn Phe Gly Stop

4 bases

64 codons

20 aminoacids

Protein Structure, function

Cell needs access to genetic information-Transcription & translation (syntesis of proteins)-Replication (duplication of DNA)

Both strands of DNA must be separated to get to the bases

Strands

Bases

DNA double helix

UNZIPPING

Genetic information

Watson J. & Crick F. (1953). Nature 171 (4356)

Motivation

Can we infer the DNA sequence by force?

F F

UNZIPPING

We need a suitable experimental setup to

perform DNA unzipping

Outline

Introduction

Optical tweezers

Experiments

Model

Results

Conclusions

Optical tweezers

A focused laser beam produces an optical trapLight is deflected when a force is applied

We can apply and measure forces and distances

0-100 pN 0.1 pN resolution0-10 m 5 nm resolution

boi ppp

1 m

Ashkin, A. "Phys. Rev. Lett. 24, 156-159", (1970)

IEF 2

Outline

Introduction

Optical tweezers

Experiments

Model

Results

Conclusions

Experimental setup

UNZIPPING: Pulling apart both strands of dsDNA from the same end

Molecule Experimental setup

1m

Bockelmann et. al., Biophys. J.:82:1537-1553 (2002)

Force vs. Distance Curves (FDC)

- Pulling cycles. Folding-unfolding curves - Force vs. Total distances curves (FDC)- Slow pulling rate (5-25 nm/s). Close-to-equilibrium FDC

Raw data

Averaged data (Running Average)

Sawtooth-like shape

12

3

1

2

3

Outline

Introduction

Optical tweezers

Experiments

Model

Results

Conclusions

Description of the model

Potential energy of the bead in the trap

Elastic energy of the handles

Elastic energy the released ssDNA

Nearest-neighbour (NN) energy of the DNA molecule

Y

x

bx

Tk

Tk

bxFx B

B

1coth)(

L

x

Lxl

TkxF

p

B

4

1

/14

1)( 2

2

2

1)( kxxE

ssDNA – Freely Jointed Chain

dsDNA – Worm Like Chain

Bead in the trap – harmonic potential

S. Cocco et. al., PNAS:98:8608-8613 (2002)

NN model and FDC

--A-A--

--T-T--

--A-C--

--T-G--

--A-G--

--T-C--

--A-T--

--T-A--

--C-A--

--G-T--

--C-C--

--G-G--

--C-G--

--G-C--

--C-T--

--G-A--

--G-A--

--C-T--

--G-C--

--C-G--

--G-G--

--C-C--

--G-T--

--C-A--

--T-A--

--A-T--

--T-C--

--A-G--

--T-G--

--A-C--

--T-T--

--A-A--

DNA parameters

tottot

toteq

ntottot

xZdx

dxF

nxExZ

ln1

)(

),(exp)(

shbtot

DNAsshhbbtot

xxxx

nEnxExExEnxE

,,

The energy of DNA determined by the

sequence

0

n

iiDNA nE

where i is the energy to open the i th base

pair

Total energy of the system (energy landscape) Equilibrium FDC

Outline

Introduction

Optical tweezers

Experiments

Model

Results

Conclusions

Comparison with experiments

M-fold is a web server that provides the stacking energies of DNAhttp://frontend.bioinfo.rpi.edu/zukerm/dna/credit.html

J. Santalucia, Jr., PNAS:95:1460-1465 (1998)

--A-A--

--T-T--

--A-C--

--T-G--

--A-G--

--T-C--

--A-T--

--T-A--

--C-A--

--G-T--

--C-C--

--G-G--

--C-G--

--G-C--

--C-T--

--G-A--

--G-A--

--C-T--

--G-C--

--C-G--

--G-G--

--C-C--

--G-T--

--C-A--

--T-A--

--A-T--

--T-C--

--A-G--

--T-G--

--A-C--

--T-T--

--A-A--

DNA parameters

Correcting the energies

-Correction of the 10 DNA stacking energies-Similar values, but corrected up to 10%-Corrections have different signs

Detection of intermediate states

Detection of intermediate states

We want to know the number of open base pairs at each experimental measure.

How to detect states in such noise data?

Detection of intermediate states

All possible states of the system are caracterized by the total distance and the number of open basepairs (xtot, n)

xtot is given the point.

We select the most probable state (n) for each experimental point. The most probable state is the theoretical state that passes closest to the experimental point.

Detection of intermediate states

Avalanche analysis

Avalanche distribution function

Experimentally we cannot see avalanches smaller than 10 base pairs.The sequencing by force is still an experimental challenge.

Conclusions

•We have inferred DNA thermodynamics using optical tweezers and performing single molecule experiments.

•The NN model is useful to extract information about the intermediate states from the experimental noise measurements.

•Sequencing DNA by force is not possible yet•Cooperative avalanches (intrinsic mechanism)•Experimental resolution (10 bps)