Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures.
This report illustrates the approaches employed to investigate critical aspects of the activity of crosslinking reagents toward nucleic acid substrates, which should be evaluated to identify candidate probes for mass spectrometric 3D (MS3D) investigations of biomolecules and macromolecular complexes. Representative members of different classes of bifunctional reagents were taken into consideration, including bikethoxal and phenyl-diglyoxal [bis-(1,2-dicarbonyls)], cisplatin (coordinative binding agents), chlorambucil and nitrogen mustard [bis-(2-chloroethyl)amines], and sym-triazine trichloride (triazines). Nanospray-Fourier transform mass spectrometry (FTMS) was applied without desalting or separation procedures to characterize the covalent products obtained by probing dinucleotide and trinucleotide substrates under a variety of experimental conditions in vitro. The carefully controlled composition of these substrates enabled us to obtain valid comparisons of probe activity toward individual nucleotides and evaluate possible base-specific effects, including the stability of the different adducts in solution under the selected reaction conditions. The gas-phase behavior of the observed products was investigated using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) to obtain valuable information for guiding the design of sequencing experiments and helping the data interpretation. Structured RNA substrates, such as HIV-1 stemloop 1, were finally employed to investigate the structural determinant of adduct formation and highlight the different nature of the spatial information provided by the various candidate probes.
Zhang
Qingrong
Q
University of Maryland Baltimore County, Baltimore, Maryland, USA.
Yu
Eizadora T
ET
University of Maryland Baltimore County, Baltimore, Maryland, USA.
Kellersberger
Katherine A
KA
University of Maryland Baltimore County, Baltimore, Maryland, USA.
Crosland
Elizabeth
E
Villa Julie College, Stevenson, Maryland, USA.
Fabris
Daniele
D
Marlene and Stewart, Greenbaum Cancer Center, University of Maryland Baltimore County and University of Maryland, Baltimore, Maryland, USA. fabris@umbc.edu.
D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry*; D030561:Databases, Nucleic Acid; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry; D015347:RNA Probes / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared
D003432:Cross-Linking Reagents; D015342:DNA Probes; D015347:RNA Probes; D012313:RNA; D004247:DNA