Towards DNA sequencing by force
description
Transcript of Towards DNA sequencing by force
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)