Abstract
G-quadruplex-forming oligonucleotides containing modified nucleotide chemistries have demonstrated promising pharmaceutical potential. In this work, we systematically investigate the effects of sugar-modified guanosines on the structure and stability of a (4+0) parallel and a (3+1) hybrid G-quadruplex using over 60 modified sequences containing a single-position substitution of 2′-O-4′-C-methylene-guanosine (LNAG), 2′-deoxy-2′-fluoro-riboguanosine (FG) or 2′-deoxy-2′-fluoro-arabinoguanosine (FANAG). Our results are summarized in two parts: (I) Generally, LNAG substitutions into ‘anti’ position guanines within a guanine-tetrad lead to a more stable G-quadruplex, while substitutions into ‘syn’ positions disrupt the native G-quadruplex conformation. However, some interesting exceptions to this trend are observed. We discover that a LNAG modification upstream of a short propeller loop hinders G-quadruplex formation. (II) A single substitution of either FG or FANAG into a ‘syn’ position is powerful enough to perturb the (3+1) G-quadruplex. Substitution of either FG or FANAG into any ‘anti’ position is well tolerated in the two G-quadruplex scaffolds. FANAG substitutions to ‘anti’ positions are better tolerated than their FG counterparts. In both scaffolds, FANAG substitutions to the central tetrad layer are observed to be the most stabilizing. The observations reported herein on the effects of LNAG, FG and FANAG modifications on G-quadruplex structure and stability will enable the future design of pharmaceutically relevant oligonucleotides.
Highlights
G-quadruplexes are four-stranded nucleic acid structures composed of stacked layers of guanine tetrads, stabilized by Hoogsteen hydrogen bonds and coordinating cations [1,2]
circular dichroism (CD) spectroscopy was used to probe the G-quadruplex folding topology based on well-characterized patterns in CD spectra [66]
Our current study demonstrates that FANAG are generally better tolerated than FG when substituted into the G-tetrad core of G-quadruplex DNA
Summary
G-quadruplexes are four-stranded nucleic acid structures composed of stacked layers of guanine tetrads, stabilized by Hoogsteen hydrogen bonds and coordinating cations [1,2]. Guanine-rich G-quadruplex-forming sequences are present in some critical regions of the human genome, and the formation of these structures has been shown to play important roles in various biological processes [3,4,5,6,7,8,9,10]. Many engineered G-quadruplex–forming sequences show high affinity towards biologically important protein targets. Native DNA chemistry is prone to enzymatic digestion. The incorporation of alternative nucleic acid chemistries can enhance the lifetime and other pharmacological properties of G-quadruplex-based drugs
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