Methods for thermal denaturation studies of nucleic acids in complex with fluorogenic dyes

Thermal denaturation is a common technique in the biophysical study of nucleic acids. These experiments are typically performed by monitoring the increase in absorbance (hyperchromism) of a sample at 260nm with temperature (Mergny & Lacroix, 2003; Puglisi & Tinoco, 1989). This wavelength is chosen as nucleic acids of mixed sequence typically exhibit their maximum absorbance here. Exceptions exist, however, some noncanonical nucleic acid structures exhibit differing spectral changes with temperature, resulting in other wavelengths being convenient reporters of secondary structure. In the case of nucleic acids that bind visible light-absorbing ligands, such as fluorogenic aptamers, another wavelength can be a convenient reporter of secondary structure stability and RNA-ligand recognition. As it can be difficult, if not impossible, to know which wavelength to employ a priori, we have developed a system for obtaining the full UV-visible spectrum of a sample at each wavelength, allowing for the subsequent extraction of the absorbance-temperature profile at the desired wavelength. Here, we describe the apparatus and software used to do so. We also describe another technique for the use of a qPCR instrument for measuring secondary structure stability of fluorescent nucleic acid-ligand complexes.