Single molecules FRET studies of short and long RNA samples

Abstract

In the classic view of the central dogma of biology, ribonucleic acids (RNA) only code for proteins. However, many exceptions to this dogma are now known as a result of genomic studies and spectroscopy and microscopy techniques at the single- molecule level. Now it is well know that some RNA molecules are involved in splicing, signal recognition of proteins and even have catalytic activity per se. Such roles have been identified to the shape that the RNA molecule can adopt. In this thesis we show the design, construction and calibration of an optical setup for measure at the single-molecule level, fluorescence resonance energy transfer (FRET) to study different physical and biological characteristics of short and long RNA molecules. The optical setup was made up of three main parts, a laser light source for excitation of dyes, a commercial epi-fluorescent microscope and an ultra sensitive single photon detectors. The calibration of the optical setup was made by using short DNA-labeled fragments with donor-acceptor dyes attached at opposite ends of the DNA molecules. A calibration curve for FRET was obtained. The calibration curve was used to measure for the first time the end-to-end distances of RNA molecules obtained from different biological sources. Our results prove that the two ends of the RNA molecules studied are in close proximity. These results migth indicate that the close proximity provides a so called “effective circularization” of RNA molecules that should facilitate translation. We also observed with smFRET that conserved RNA-targets sequences, located in the messenger RNA of the human immunodeficiency virus (HIV-1), adopt different structural changes by punctual mutations and temperature changes. Our results suggest the equilibrium coexistence of different RNA conformations, whose popula- tion percentage are easily changed by moderate temperature changes. To study if the observed RNA populations expose its RNA-target sequence, the RNA-Induced Silencing Complex (RISC), consisting in its core of an Argonaute protein (Thermus thermophilus Argonaute) and a short single-stranded interference RNA (siRNA) ei- ther Pol or Nef, was incubated with the above RNA-targets and FRET measurements were performed. Our results indicate that RNA-punctual mutations may help or af- fect to the RNA target to escape of cleaving by RISC complex.