Nearest-neighbor parameters for predicting DNA duplex stability in diverse molecular crowding conditions

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.

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

“…For example, 5′rAG3′/5′dCT3′ is represented as rAG/dCT. The NN parameters were determined (Table 2 ) using a linear least-squares computer program (Supporting Information) ( 41 ). To determine appropriate parameters, the selection of the hybrid sequences were done carefully so that all the potential 16 NN base pairs present in an almost equal number of frequencies ( 16 ).…”

Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates

“…Next, we investigated the effect of molecular crowding on the primer extension described above. In this study, PEG 200 was used as a crowder molecule, because PEG 200 is broadly used to study the effect of molecular crowding on not only the stability of DNA duplexes but also polymerase reactions [ 15 , 17 , 20 , 22 ]. The decrease in water activity significantly destabilizes the Watson–Crick base pairs [ 17 ].…”

“…In this study, PEG 200 was used as a crowder molecule, because PEG 200 is broadly used to study the effect of molecular crowding on not only the stability of DNA duplexes but also polymerase reactions [ 15 , 17 , 20 , 22 ]. The decrease in water activity significantly destabilizes the Watson–Crick base pairs [ 17 ]. Furthermore, the decrease in dielectric constant results in the enhancement of stacking interactions between the primer and an incorporated nucleotide in the reaction of primer extension using T7 RNA polymerase [ 23 ].…”

“…Nucleic acids are among the materials most sensitive to crowding conditions. With the use of crowder molecules such as poly(ethylene glycol)s (PEGs) to mimic intracellular crowding conditions, it has been clarified that DNA duplex thermal stabilities decrease due to a decrease in the water activity [ 15 , 16 , 17 ]. Molecular crowding also decreases the dielectric constant of the solution, which can increase the ribozyme activities [ 18 , 19 ].…”

Molecular crowding induces primer extension by RNA polymerase through base stacking beyond Watson–Crick rules

“…18 The base pairs of a DNA duplex, RNA duplex, and DNA/RNA hybrid are destabilised by molecular crowding, but the magnitude of destabilisation is different between them. [19][20][21] Furthermore, the instability caused by mismatched base pairs can become very slight compared to that by matched pairs. 22 These altered properties can affect forces for substrate recognition by polymerases.…”