Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

Abstract

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na+ or K+ in the presence or absence of Mg2+). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°37 and Tm values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg2+, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.

Mechanisms underlying sequence-dependent DNA hybridisation rates in the absence of secondary structure

“…associated with the formation of any DNA duplex can be estimated from the sum of the free energy of all 2 contiguous base-pairing interactions, as well as additional parameters to account for the relative stabilities of the ends of the duplex (32,33). Given that there are only 10 unique combinations of 2-base sequences, nearest neighbour models can be parameterised experimentally including in different buffer conditions (34)(35)(36)(37)(38)(39)(40)(41), and algorithmic implementations predict hybridisation 𝑇 ! reasonably well (42,43).…”

Optimized design of antisense oligomers for targeted rRNA depletion

“…At the time of writing, Banerjee et al. published improved RNA-DNA parameters ( 34 ), which may yield slightly different predicted T m s compared to the old parameters used here; future software updates will incorporate these parameters. Once the entire target sequence is tiled, a second refinement phase adjusts each oligo position within the window defined by the upstream and downstream gaps, to yield maximized distances from upstream and downstream oligos within the ideal T m range.…”

Transcriptome screening followed by integrated physicochemical and structural analyses for investigating RNA-mediated berberine activity

“…coptisine (COP), palmatine (PAM) and epiberberine (EBRB) (Table 2 )) were also used to investigate the roles of the dimethoxy and dioxolane groups in BRB on RNA stabilization. A buffer containing 10 mM Na 2 HPO 4 , 100 mM NaCl and 1 mM EDTA, which was used as a standard buffer with physiological ionic strength ( 55 ), was used for the melting assay. Based on the stock solution of the ligands, the buffer contained 0.1% DMSO.…”