The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3′-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3′-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.
We here report on the synthesis of the first mimic of the DNA binding domain of the c-Myc/Max-bHLH-ZIP transcription factor able to selectively recognize its cognate E-box sequence 5'-CACGTG-3' through the major groove of the double-stranded DNA. The designed peptidosteroid conjugate was shown to be effective as DNA binder in the presence of excess competitor DNA.
Locked nucleic acid (LNA) oligonucleotides bind DNA target sequences forming Watson-Crick and Hoogsteen base pairs, and are therefore of interest for medical applications. To be biologically active, such an oligonucleotide has to efficiently bind the target sequence. Here we used molecular dynamics simulations and electrophoresis mobility shift assays to elucidate the relation between helical structure and affinity for LNA-containing oligonucleotides. In particular, we have studied how LNA substitutions in the polypyrimidine strand of a duplex (thus forming a hetero duplex, i . e . a duplex with a DNA polypurine strand and an LNA/DNA polypyrimidine strand) enhance triplex formation. Based on seven polypyrimidine single strand oligonucleotides, having LNAs in different positions and quantities, we show that alternating LNA with one or more non-modified DNA nucleotides pre-organizes the hetero duplex toward a triple-helical-like conformation. This in turn promotes triplex formation, while consecutive LNAs distort the duplex structure disfavoring triplex formation. The results support the hypothesis that a pre-organization in the hetero duplex structure enhances the binding of triplex forming oligonucleotides. Our findings may serve as a criterion in the design of new tools for efficient oligonucleotide hybridization.
Pseudoisocytidine (ΨC) is a synthetic cytidine analogue that can target DNA duplex to form parallel triplex at neutral pH. Pseudoisocytidine has mainly two tautomers, of which only one is favorable for triplex formation. In this study, we investigated the effect of sequence on ΨC tautomerization using λ-dynamics simulation, which takes into account transitions between states. We also performed in vitro binding experiments with sequences containing ΨC and furthermore characterized the structure of the formed triplex using molecular dynamics simulation. We found that the neighboring methylated or protonated cytidine promotes the formation of the favorable tautomer, whereas the neighboring thymine or locked nucleic acid has a poor effect, and consecutive ΨC has a negative influence. The deleterious effect of consecutive ΨC in a triplex formation was confirmed using in vitro binding experiments. Our findings contribute to improving the design of ΨC-containing triplex-forming oligonucleotides directed to target G-rich DNA sequences.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.