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

Lauren M. Aufdembrink, Tanner G. Hoog, Matthew R. Pawlak, Benjamin F. Bachan, Joseph M. Heili, Aaron E. Engelhart

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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 260 nm 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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsAmanda E. Hargrove
PublisherAcademic Press Inc.
Pages23-43
Number of pages21
ISBN (Print)9780128168318
DOIs
StatePublished - 2019

Publication series

NameMethods in Enzymology
Volume623
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

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Keywords

  • Aptamer
  • DNA
  • Fluorescence
  • RNA
  • Thermal denaturation
  • UV-vis
  • qPCR

PubMed: MeSH publication types

  • Journal Article
  • Research Support, U.S. Gov't, Non-P.H.S.

Cite this

Aufdembrink, L. M., Hoog, T. G., Pawlak, M. R., Bachan, B. F., Heili, J. M., & Engelhart, A. E. (2019). Methods for thermal denaturation studies of nucleic acids in complex with fluorogenic dyes. In A. E. Hargrove (Ed.), Methods in Enzymology (pp. 23-43). (Methods in Enzymology; Vol. 623). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2019.05.029