title: Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment.
abstract: BACKGROUND
  G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.
  
  
  METHODS
  Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.
  
  
  RESULTS
  The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.
  
  
  CONCLUSIONS
  CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.
  
  
  CONCLUSIONS
  Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Nadai
authors.forename: Giovanna
authors.initials: G
authors.lastname: Sattin
authors.forename: Giorgio
authors.initials: G
authors.lastname: Palù
authors.forename: Manlio
authors.initials: M
authors.lastname: Palumbo
authors.forename: Sara N
authors.initials: SN
authors.lastname: Richter
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D018983:DNA Footprinting; D017931:DNA Primers; D004224:Diterpenes / Q000494:pharmacology; D054856:G-Quadruplexes; D009690:Nucleic Acid Conformation
chemical_list: D017931:DNA Primers; D004224:Diterpenes; C039027:clerocidin
keywords: Alkylating agent; Clerocidin; DNA conformation; Footprinting; G-quadruplex; Protection assay