Experimental approaches for measuring pKa's in RNA and DNA

Pallavi Thaplyal, Philip C. Bevilacqua

Research output: Chapter in Book/Report/Conference proceedingChapter

26 Citations (Scopus)

Abstract

RNA and DNA carry out diverse functions in biology including catalysis, splicing, gene regulation, and storage of genetic information. Interest has grown in understanding how nucleic acids perform such sophisticated functions given their limited molecular repertoire. RNA can fold into diverse shapes that often perturb pKa values and allow it to ionize appreciably under biological conditions, thereby extending its molecular diversity. The goal of this chapter is to enable experimental measurement of pKa's in RNA and DNA. A number of experimental methods for measuring pKa values in RNA and DNA have been developed over the last 10 years, including RNA cleavage kinetics; UV-, fluorescence-, and NMR-detected pH titrations; and Raman crystallography. We begin with general considerations for choosing a pKa assay and then describe experimental conditions, advantages, and disadvantages for these assays. Potential pitfalls in measuring a pKa are provided including the presence of apparent pKa's due to a kinetic pKa or coupled acid- and alkali-promoted RNA unfolding, as well as degradation of RNA, precipitation of metal hydroxides and poor baselines. Use of multiple data fitting procedures and the study of appropriate mutants are described as ways to avoid some of these pitfalls. Application of these experimental methods to RNA and DNA will increase the number of available nucleic acid pKa values in the literature, which should deepen insight into biology and provide benchmarks for pKa calculations. Future directions for measuring pKa's in nucleic acids are discussed.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages189-219
Number of pages31
EditionC
DOIs
StatePublished - 2014

Publication series

NameMethods in Enzymology
NumberC
Volume549
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

RNA
DNA
Nucleic Acids
Hydroxides
RNA Folding
RNA Cleavage
Benchmarking
Assays
Crystallography
Information Storage and Retrieval
RNA Stability
Alkalies
Catalysis
Kinetics
Fluorescence
Metals
Titration
Gene expression
Acids
Nuclear magnetic resonance

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Thaplyal, P., & Bevilacqua, P. C. (2014). Experimental approaches for measuring pKa's in RNA and DNA. In Methods in Enzymology (C ed., pp. 189-219). (Methods in Enzymology; Vol. 549, No. C). Academic Press Inc.. https://doi.org/10.1016/B978-0-12-801122-5.00009-X
Thaplyal, Pallavi ; Bevilacqua, Philip C. / Experimental approaches for measuring pKa's in RNA and DNA. Methods in Enzymology. C. ed. Academic Press Inc., 2014. pp. 189-219 (Methods in Enzymology; C).
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Thaplyal, P & Bevilacqua, PC 2014, Experimental approaches for measuring pKa's in RNA and DNA. in Methods in Enzymology. C edn, Methods in Enzymology, no. C, vol. 549, Academic Press Inc., pp. 189-219. https://doi.org/10.1016/B978-0-12-801122-5.00009-X

Experimental approaches for measuring pKa's in RNA and DNA. / Thaplyal, Pallavi; Bevilacqua, Philip C.

Methods in Enzymology. C. ed. Academic Press Inc., 2014. p. 189-219 (Methods in Enzymology; Vol. 549, No. C).

Research output: Chapter in Book/Report/Conference proceedingChapter

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Thaplyal P, Bevilacqua PC. Experimental approaches for measuring pKa's in RNA and DNA. In Methods in Enzymology. C ed. Academic Press Inc. 2014. p. 189-219. (Methods in Enzymology; C). https://doi.org/10.1016/B978-0-12-801122-5.00009-X