II. Thesis


Molecular biophysics is a scientific discipline that studies biomolecules. This discipline has experienced a revolution thanks to the development of single-molecule techniques. These techniques allows us to obtain new and valuable information that complements the traditional bulk assays. Optical tweezers is an experimental technique that uses the radiation pressure of light to exert forces on a tiny dielectric microsphere. Biomolecules can be bonded to such microspheres in order to perform pulling experiments at the single-molecule level. Minitweezers is a dual counter-propagating laser tweezers instrument that measures the force exerted on the microsphere by conservation of light momentum. The instrument has high stability and resolution (0.1 pN in force and 0.5 nm in distance) in the measurements. The properties of the DNA molecule can be studied with the Minitweezers at the single-molecule level. The DNA is a biomolecule that forms a double helix that stores the genetic information of the cells. DNA unzipping experiments consist in pulling apart the two strands of DNA by exerting mechanical forces on the extremities of the molecule. In such process, base-pairs (bp) are disrupted sequentially, showing a succession of cooperative unzipping regions (CUR) of different sizes (between 1-100 bp). In a DNA unzipping experiment, we measure the force vs. distance curve (FDC) of the molecule, which has a characteristic sawtooth-like shape that is sequence-dependent. The FDC is analyzed with a Bayesian approach to infer the size distribution of the CURs. The experimental accuracy does not allow to observe CURs of sizes below 10 bp. Furthermore, the unzipping of one bp at a time can only be achieved by having an optical trap stiffness value higher than 0.1 N/m, which corresponds to the stiffness of a single nucleotide of DNA. This has been deduced from a toy model specifically introduced to study the CUR size distributions. In addition, the FDCs are theoretically predicted by the nearest-neighbor (NN) model adapted to unzipping experiments. The NN model describes the hybridization reaction of two strands of DNA. By fitting the experimental FDCs to the model, the unique 10 NN bp free energies are obtained with 0.1 kcal$ \cdot $mol$ ^{-1}$ precision between 10 mM-1 M of monovalent salt concentration. The results show that the unzipping FDCs and the melting temperatures of oligos are correctly described with a specific salt correction for each of the 10 NN bp free energies. Differently from the previous experiments, the unzipping of DNA can also be performed at controlled force. These last type of experiments exhibit large hysteresis and irreversibility. The free energy landscape is a tool that helps to understand the unzipping at controlled force. Finally, the work presented in this thesis can be extended to find practical applications of DNA unzipping, such as sequencing of DNA by force, and measurement of thermodynamic properties of biomolecules in conditions not accessible by bulk methodologies.

List of abbreviations
AD anti-digoxigenin
ADC analog to digital converter
AFM atomic force microscopy
API application programming interface
CF controlled force
CUR cooperative unzipping region
DAC digital to analog converter
DFC distance versus force curve
DNA deoxyribonucleic acid
dsDNA double-stranded DNA
FBG fiber Bragg grating
FDC force versus distance curve
FDC$ _f$ force versus distance curve at controlled force
FDC$ _x$ force versus distance curve at controlled position
FEC force versus extension curve
FJC freely jointed chain
FRET fluorescence resonance energy transfer
FFT fast Fourier transform
FT fluctuation theorem
GLMT generalized Lorentz-Mie theory
HOT holographic optical tweezers
LOT laser optical tweezers
MC Monte Carlo
MT magnetic tweezers
NA numerical aperture
NN nearest neighbor
NNBP nearest neighbor base pair
PBS polarizing beam-splitter
PIC programmable interface controller
PSD position sensitive detector
PWM pulse width modulation
RNA ribonucleic acid
SA streptavidin
SD steepest descent
SLM spatial light modulator
SME single-molecule experiments
SPI serial peripheral interface
ssDNA single-stranded DNA
TE transverse electric
TEM transverse electromagnetic
TM transverse magnetic
UO unified oligonucleotide
WLC worm-like chain

JM Huguet 2014-02-12