Non-canonical structures of nucleic acids inhibit or dysregulate biological reactions related to diseases. To control the reactions, methods for inducing the non-canonical structures have been developed using small molecules and oligonucleotides.

Cancer and neurodegenerative diseases are caused by genetic and environmental factors. Expression of tumour suppressor genes is suppressed by mutations or epigenetic silencing, whereas for neurodegenerative disease-related genes, nucleic acid-based effects may be presented through loss of protein function due to erroneous protein sequences or gain of toxic function from extended repeat transcripts or toxic peptide production. These diseases are triggered by damaged genes and proteins due to lifestyle and exposure to radiation. Recent studies have indicated that transient, non-canonical structural changes in nucleic acids in response to the environment can regulate the expression of disease-related genes. Non-canonical structures are involved in many cellular functions, such as regulation of gene expression through transcription and translation, epigenetic regulation of chromatin, and DNA recombination. Transcripts generated from repeat sequences of neurodegenerative disease-related genes form non-canonical structures that are involved in protein transport and toxic aggregate formation. Intracellular phase separation promotes transcription and protein assembly, which are controlled by the nucleic acid structure and can influence cancer and neurodegenerative disease progression. These findings may aid in elucidating the underlying disease mechanisms. Here, we review the influence of non-canonical nucleic acid structures in disease-related genes on disease onset and progression.

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 (T_m) 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. Significance The intracellular environment is occupied by a high concentration of various types of biomolecules that lead to a situation known as molecular crowding, which affects important biological reactions such as replication and transcription by altering the stability of nucleic acids. Because intracellular crowded conditions are heterogeneous, a general method is needed to predict the stability of nucleic acids in such conditions. Here, we predicted the stability of DNA duplexes under crowded conditions induced by a large amount of a cosolute at a physiological salt concentration using the nearest-neighbor model. We also extended the parameters for predicting DNA stability under conditions imposed by different cosolutes that mimicked a localized intracellular crowded region.

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 T_m 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.