Download Towards DNA sequencing by force

Towards DNA sequencing by force
Josep Maria Huguet, Núria Forns, Fèlix Ritort
Small Biosystems Lab, Facultat de Física, UB
http://www.ffn.ub.es/ritort/
Steve B. Smith, Carlos Bustamante
Bustamante 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
Bases
DNA double helix
Strands
Genetic information
Cell needs access to genetic information
4 bases
... ATGCTGCGAAACTTTGGCTGA
-Transcription & translation (syntesis of proteins)
-Replication (duplication of DNA)
64 codons
... ATG CTG CGA AAC TTT GGC TGA
20 aminoacids
... Met Leu Arg Asn Phe Gly Stop
Protein
Structure, function
Watson J. & Crick F. (1953). Nature 171 (4356)
Both strands of DNA must be separated
to get to the bases
UNZIPPING
Motivation
UNZIPPING
F
F
Can we infer the DNA sequence by force?
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 trap
Light is deflected when a force is applied

F  E 2  I
 

pi  po  pb
1 mm
We can apply and measure forces
and distances
0-100 pN
0-10 mm
0.1 pN resolution
5 nm resolution
Ashkin, A. "Phys. Rev. Lett. 24, 156-159", (1970)
Outline
Introduction
Optical tweezers
Experiments
Model
Results
Conclusions
Experimental setup
UNZIPPING: Pulling apart both strands of dsDNA from the same end
Molecule
Experimental setup
1mm
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
Sawtooth-like shape
1
3
Averaged data (Running Average)
3
1
2
2
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
1 2
kx
2
Bead in the trap – harmonic potential
E ( x) 
dsDNA – Worm Like Chain
F ( x) 
ssDNA – Freely Jointed Chain

 bx  k BT 
1  x 
 
x( F )   coth 

 k BT  bx  Y 

k BT 
1
1 x


 
l p  41  x / L 2 4 L 
S. Cocco et. al., PNAS:98:8608-8613 (2002)
NN model and FDC
The energy of DNA
determined by the
sequence
n
EDNA n     i
i 0
where i is the energy
to open the i th base
pair
Total energy of the system (energy landscape)
E xtot , n   Eb xb   Eh xh   Es xs , n   EDNA n 
xtot  xb  xh  xs
DNA parameters
--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--
Equilibrium FDC
Z ( xtot )   exp  E ( xtot , n) 
n
Feq ( xtot )  
1 d
ln Z xtot 
 dxtot
Outline
Introduction
Optical tweezers
Experiments
Model
Results
Conclusions
Comparison with experiments
DNA parameters
--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--
M-fold is a web server that provides the stacking energies of DNA
http://frontend.bioinfo.rpi.edu/zukerm/dna/credit.html
J. Santalucia, Jr., PNAS:95:1460-1465 (1998)
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)