AbstractsBiology & Animal Science

Single molecule studies in a temperature-jump optical trap

by Sara de Lorenzo Ros

Institution: Universitat de Barcelona
Year: 2015
Keywords: Biofísica; Biophysics; Termodinàmica; Termodinámica; Thermodynamics; ADN; DNA; Temperature jump; Salt de temperatura; Salto de temperatura; Optical tweezers; Pinza óptica; Pinça òptica; Ciències Experimentals i Matemàtiques
Record ID: 1125539
Full text PDF: http://hdl.handle.net/10803/286174


In the field of biophysics, the study of the thermodynamic characteristics of biomolecules, such as DNA, RNA or proteins, allows us to understand more about the building blocks of life. The thermodynamic characterization of the biomolecule gives us clues as to their functions and capabilities inside every living organism. The thermodynamic characterization of nucleic acids describes how temperature affects the stability and the structure of double stranded DNA. The melting temperature of DNA (T(M)) is defined as the temperature at which half of the DNA strands in a bulk solution experiment are in the double stranded DNA (dsDNA) or random coil configuration and half of the DNA are in the single-stranded DNA (ssDNA) configuration. Using T(M), it has been possible to experimentally determine the thermodynamic parameters of Delta-G, Delta-H and Delta-S. Viceversa, when the thermodynamic parameters of a given nucleic acid sequence are known, the TM can be predicted. This effect has important applications for biomolecule techniques such as PCR (Polymerase chain reaction) or sequencing. Traditionally thermodynamic properties of DNA have been determined using bulk techniques such as calorimetry or UV absorbance. In both cases the melting temperature has been determined by changing the temperature or pH of the entire sample. Over the past two decades single molecule force spectroscopy has been established as a powerful, accurate and bulk-complementary method of characterizing the thermodynamics of nucleic acids. Optical trapping is an experimental technique which allows force to be exerted on a micrometric particle by using the radiation pressure of light. The miniTweezers (mT) is the newest generation of optical tweezers instruments. This instrument can be used to exert and measure forces in a range between 1-200 pN and has unprecedented resolution (0.1 pN in force an around 1 nm in distance) with very high thermal and noise stability. Optical trapping is very useful in the field of molecular biology because it allows forces to be exerted on single biomolecules bonded to the micrometric particle. This technique is used to carry out pulling experiments on single molecules allowing us to study the mechanical, thermodynamic and kinetic properties of the molecule. Mechanical melting or unzipping is a process that consists of pulling apart the two strands of the dsDNA until the base pairs are disrupted and the molecule converts into ssDNA. In this case, and in contrast to other techniques, force, rather than temperature or pH, is used to open the molecule. Past experiments have shown that better resolution can be obtained using single molecule techniques than can be obtained using bulk experiments. Although force unzipping provides a direct estimation of Delta-G at room temperature, extracting the value of TM always requires the determination of the Delta-H and Delta-S contributions and until now has not been reliable accomplished.; En el campo de la biofísica, el estudio de las características termodinámicas de las…