Toward multiplexing the application of solvent accessibility probes for the investigation of RNA three-dimensional structures by electrospray ionization-Fourier transform mass spectrometry.
Multiple solvent accessibility probes can be applied simultaneously to investigate the three-dimensional structure of complex RNA substrates when electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS) is employed in place of polyacrylamide gel electrophoresis (PAGE). We show that classic chemical probes, such as dimethylsulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, can be combined in probing mixtures designed to assess the full spectrum of base pairing and steric protection for the most abundant ribonucleotides included in RNA. After probe-independent hydrolysis of the alkylated substrate, the mixture of oligonucleotide products is mass mapped by ESI-FTMS analysis, which enables the unambiguous identification of probed bases from the unique mass signatures provided by the different chemical modifiers. In this bottom-up approach, any theoretical limit to the size of the possible target RNA will be determined by the effectiveness of the hydrolysis procedure rather than by the performance of the detection technique. Control experiments performed on the stem-loop 4 of human immunodeficiency virus type 1 have shown no adverse interactions between the reagents combined in the probing cocktails. No significant discrepancies between the alkylation patterns offered by the cocktails and the individual reagents could be detected, indicating that multiplexing the probe application does not necessarily lead to structural distortion but provides valid data on base accessibility and protection. To demonstrate the ruggedness of this approach, optimized cocktails were finally employed to assess the stability of the folded structure of mouse mammary tumor virus pseudoknot in the presence of different amounts of Mg2+. Multiplexing the probe application constitutes an essential step toward high-throughput applications, which will take advantage of a strategy that maximizes the information attainable from a single experiment, while minimizing time and sample consumption over PAGE-based methods.
Yu
Eizadora
E
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
Fabris
Daniele
D
D001483:Base Sequence; D005583:Fourier Analysis; D015497:HIV-1 / Q000235:genetics; D015335:Molecular Probes / Q000032:analysis* / Q000737:chemistry; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012997:Solvents / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D015335:Molecular Probes; D012997:Solvents; D012313:RNA