Abstract
G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).
Highlights
Nucleic acids constitute the fundamental biomolecular machinery to transfer genetic information, but are involved in the regulation of gene expression [1]
The objective of the present study is to build a database of G4 sequences with sufficient stability and validated folds in 1 mM KCl + 100 mM trimethylammonium acetate (TMAA) assay conditions
The stock oligonucleotide solutions were diluted to 100 M in 100 mM TMAA supplemented with 1 mM KCl in water and Nuclear magnetic resonance (NMR) buffer (Supplementary Table S1)
Summary
Nucleic acids constitute the fundamental biomolecular machinery to transfer genetic information, but are involved in the regulation of gene expression [1]. G-quadruplexes (G4s) have been the subject of intense structural and biological research, given their roles in gene regulation and other related cellular processes [3,4]. G4s have important biological effects in replication, transcription, translation, mutagenesis, genome damage repair, telomere maintenance, or RNA splicing [3,5,6]. Their key role in different cellular processes makes them crucial drug targets for diseases [6,7,8]. G4s have numerous other applications in theranostics, supramolecular chemistry, or nanotechnology [6,9,10,11,12]
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