Molecular interactions and reactions in living cells occur with high concentrations of background molecules and ions. Many research studies have shown that intracellular molecules have characteristics different from those obtained using simple aqueous solutions. To better understand the behavior of biomolecules in intracellular environments, biophysical experiments were conducted under cell-mimicking conditions in a test tube. It has been shown that the molecular environments at the physiological level of macromolecular crowding, spatial confinement, water activity and dielectric constant, have significant effects on the interactions of DNA and RNA for hybridization, higher-order folding, and catalytic activity. The experimental approaches using in vitro model systems are useful to reveal the origin of the environmental effects and to bridge the gap between the behaviors of nucleic acids in vitro and in vivo. This paper highlights the model experiments used to evaluate the influences of intracellular environment on nucleic acid interactions.

Water-soluble neutral cosolutes can be used to quantify biomolecular properties in the particular molecular environment occurring in a cell. We studied the conformation and the thermal stability of DNA and RNA structures in the presence of PEG [poly(ethylene glycol)] and smaller cosolutes of glycerol, ethylene glycol, 1,3-propanediol, 2-methoxyethanol, and 1,2-dimethoxyethane. Although the neutral cosolutes destabilized the oligonucleotide duplex and the hairpin structures, the left-handed Z-form duplex was more energetically favored in the cosolute-containing solutions. These observations were due to the contribution of water molecule on the nucleotide structure formations because the cosolutes act as an osmolyte to reduce the water activity of a solution. Moreover, the sodium ion condensation for the duplex and the hairpin formations was reduced in the presence of PEG, while that for the transition from the B-form to the Z-form was unaltered. The CD (circular dichroism) and EPR (electron paramagnetic resonance) spectra demonstrated that the cosolutes changed the helical conformation of the unstructured oligonucleotides, but not those of the ordered structures. The results of the favorable formation of the noncanonical nucleotide structures, and minimized conformational and thermal perturbations of the ordered nucleotide structures in the cosolute-containing solutions implicate the significance of the intracellular environment on DNA and RNA structures in a cell.