1. 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

2. Outline Introduction Optical tweezers Experiments Model Results Conclusions

3. Outline Introduction Optical tweezers Experiments Model Results Conclusions

4. Structure of DNA 4 different types of complementary bases (A) Adenine (C) CytosineA - T (G) GuanineC - G (T) Thymine... ATGCTGCGAAACTTTGGCTGA... 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)

5. Motivation Can we infer the DNA sequence by force? F F UNZIPPING We need a suitable experimental setup to perform DNA unzipping

6. Outline Introduction Optical tweezers Experiments Model Results Conclusions

7. Optical tweezers A focused laser beam produces an optical trap Light is deflected when a force is applied We can apply and measure forces and distances 0-100 pN0.1 pN resolution 0-10  m 5 nm resolution 1 m1 m Ashkin, A. "Phys. Rev. Lett. 24, 156-159", (1970)

8. Outline Introduction Optical tweezers Experiments Model Results Conclusions

9. Experimental setup UNZIPPING: Pulling apart both strands of dsDNA from the same end Molecule Experimental setup 1m1m Bockelmann et. al., Biophys. J.:82:1537-1553 (2002)

10. 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 1 2 3 1 2 3

11. Outline Introduction Optical tweezers Experiments Model Results Conclusions

12. 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 ssDNA – Freely Jointed Chain dsDNA – Worm Like Chain Bead in the trap – harmonic potential S. Cocco et. al., PNAS:98:8608-8613 (2002)

13. 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-- --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 The energy of DNA determined by the sequence where  i is the energy to open the i th base pair Total energy of the system (energy landscape) Equilibrium FDC

14. Unzipping mechanism DNA energy Elastic energy DNA energy Elastic energy When elastic energy is too high, new base pairs are open

15. Outline Introduction Optical tweezers Experiments Model Results Conclusions

16. Comparison with experiments 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) Direct Reverse 6 kbps

17. Comparison with experiments 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) --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-- --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

18. Correcting the energies -Correction of the 10 DNA stacking energies -Similar values, but corrected up to 10% -Corrections have different signs Només MFOLD!

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

20. Avalanche detection

21. Avalanche analysis

22. Detection of intermediate states

23. We want to know the number of open base pairs at each experimental measure. How to detect states in such noise data?

24. Avalanche analysis More Zoom...

25. Detection of intermediate states All possible states of the system are caracterized by the total distance and the number of open basepairs (x tot, n) x tot 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.

26. Detection of intermediate states

27. Avalanche analysis

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

29. 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)