Bifunctional cross-linking approaches for mass spectrometry-based investigation of nucleic acids and protein-nucleic acid assemblies

Methods : a Companion to Methods in Enzymology
M ScalabrinDaniele Fabris

Abstract

With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differe...Continue Reading

Citations

Oct 24, 2020·Current Pathobiology Reports·Rachel CarterAmanda Haymond
Jun 15, 2021·Nucleic Acids Research·Alice SosicBarbara Gatto
Oct 1, 2021·Chemical Reviews·Eric LargyValérie Gabelica
Dec 24, 2021·Mass Spectrometry Reviews·Thomas Kenderdine, Dan Fabris

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