No abstract

The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under molecular crowding condition of 40% polyethylene glycol with an average molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (Tm) of the DNA duplexes in the crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.

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.

The relationship of i-motif DNAs with cancer has prompted the development of specific ligands to detect and regulate their formation. Some plant flavonols show unique fluorescence and anti-cancer properties, which suggest the utility of the theranostics approach to cancer therapy related to i-motif DNA. We investigated the effect of the plant flavonol, fisetin (Fis), on the physicochemical property of i-motif DNAs. Binding of Fis to the i-motif from the promoter region of the human vascular endothelial growth factor (VEGF) gene dramatically induced the excited state intramolecular proton transfer (ESIPT) reaction that significantly enhanced the intensity of the tautomer emission band of Fis. This unique response was due to the coincidence of the structural change from i-motif to the hairpin-like structure which is stabilized via putative Watson-Crick base pairs between some guanines within the loop region of the i-motif and cytosines in the structure. As a result, the VEGF i-motif did not act as a replication block in the presence of fis, which indicates the applicability of fis for the regulation of gene expression of VEGF. The fluorescence and biological properties of Fis may be utilised for theranostics applications for cancers related to a specific cancer-related gene, such as VEGF. Besides the canonical right-handed DNA double helix, DNA sequences can adopt non-canonical structures, including G-quadruplexes (G4s) and i-motifs, that are stabilised by non-Watson-Crick base pairing 1. G4 structures are formed from guanine (G)-rich sequences, whereas i-motifs are formed from the cytosine (C)-rich sequences. The i-motif structure consists of two parallel duplexes that intercalate with each other in an antiparallel orientation (Fig. 1A). The parallel duplexes are held together via hydrogen bonding between a neutral cytosine (C) and a protonated cytosine (C +) (Fig. 1B) 2. The formation of an i-motif structure occurs more readily in an acidic condition because protonation of cytosine is required to form a hemiprotonated CC + base pair. However, there is increasing evidence to suggest that the i-motif structures can also form at neutral pH 3,4 and even in the nuclei of living mammalian cells 5,6. The i-motif forming sequences are found in or near the promoter regions of >40% of all human genes, which can regulate the expressions of genes like Bcl2 7,8 and HRAS 9. Furthermore, the i-motif along the cancer-related DNA sequences strongly inhibits DNA replication 10. Therefore, i-motif DNAs are attractive targets for the diagnosis of cancer risk and to treat cancer by the modulation of gene transcription and replication. Considering the use of specific targeted therapy based on specific targeted test (i.e., theranostics) for both diagnosis and therapeutics, some ligands that both emit a fluorescent signal upon binding to a specific i-motif and regulate the transcription or replication of the target gene are required. Currently, no ligands have such dual functions for i-motif DNAs. Flavonols and other related compounds o...

Recent advancement in nucleic acid techniques inside cells demands the knowledge of the stability of nucleic acid structures in molecular crowding. The nearest-neighbor model has been successfully used to predict thermodynamic parameters for the formation of nucleic acid duplexes, with significant accuracy in a dilute solution. However, knowledge about the applicability of the model in molecular crowding is still limited. To determine and predict the stabilities of DNA duplexes in a cell-like crowded environment, we systematically investigated the validity of the nearest-neighbor model for Watson–Crick self-complementary DNA duplexes in molecular crowding. The thermodynamic parameters for the duplex formation were measured in the presence of 40 wt% poly(ethylene glycol)200 for different self-complementary DNA oligonucleotides consisting of identical nearest-neighbors in a physiological buffer containing 0.1 M NaCl. The thermodynamic parameters as well as the melting temperatures ( T m ) obtained from the UV melting studies revealed similar values for the oligonucleotides having identical nearest-neighbors, suggesting the validity of the nearest-neighbor model in the crowding condition. Linear relationships between the measured Δ G ° 37 and T m in crowding condition and those predicted in dilute solutions allowed us to predict Δ G ° 37 , T m and nearest-neighbor parameters in molecular crowding using existing parameters in the dilute condition, which provides useful information about the thermostability of the self-complementary DNA duplexes in molecular crowding.