Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure

Abstract

Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures.

A Dynamic G-Quadruplex Region Regulates the HIV-1 Long Terminal Repeat Promoter

“…Two G-N7 alkylators were separately employed: DMS, the standard reactant to highlight G bases involved in G-quartets, and clerocidin (CL), endowed with finely tunable reactivity 27, 28 . In the CL protection assay, tracts 2, 3, 4 and 5 in the LTR-II sequence were all protected (Fig.…”

Structure-Based Peptide Design to Modulate Amyloid Beta Aggregation and Reduce Cytotoxicity

“…This is similar to bulge bending in DNA helices, where the mismatch of a single base pair causes a kink or bulge in the helix. Interestingly, in DNA, these bulges serve as recognition sites for ligands[ 43 – 44 ], raising the possibility that a similar kink in Aβ 1–42 fibrils could produce an accessible binding site to facilitate the clearance of Aβ deposits.…”

The Size of the Internal Loop in DNA Hairpins Influences Their Targeting with Partially Complementary Strands

“…Among these noncanonical DNA structures are stem-loop motifs that are involved in several biological functions. − Hence, targeted modulation of their properties have significant diagnostic and therapeutic potential. Besides a variety of compounds, such as the natural product Clerocidin, nucleic acid oligonucleotides (ODNs) can also be used to target noncanonical DNA structures and RNA molecules. This latter approach is very selective since ODNs offer single-base specificity .…”