Abstract
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 (Tm) 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 ΔG°37 and Tm in crowding condition and those predicted in dilute solutions allowed us to predict ΔG°37, Tm 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.
Highlights
Estimation of the stability of nucleic acid duplexes is important in various molecular biology techniques, such as polymerase chain reaction (PCR) [1], hybridization-induced sequencing [2] and antigene targeting in gene therapy [3]
The minimum and maximum frequencies of occurrence of nearest-neighbor were observed for dTA/dAT and dCG/dGC with 4.2% and 16.7% of the total frequency, respectively. These extremum values are in proximity to the minimum and maximum frequencies of 4.8% and 14.9% for dTA/dAT and dAA/dTT, respectively, with the total nearest-neighbors set reported by SantaLucia et al for calculating improved nearest-neighbor parameters for DNA duplex formation [14]
Similar magnitudes of differences (7.7%, 8.2%, 6.5% and 2.3◦C for H◦, T S◦, G◦37 and Tm, respectively) were observed for pairs of RNA/DNA hybrid sequences with identical nearest-neighbors in the absence of cosolute [14]. These results clearly suggest that the nearest-neighbor model is valid even in the molecular crowding condition, since the model predicts that the pairs with identical nearest neighbors will have identical thermostability and thermodynamic parameters for duplex formation
Summary
Estimation of the stability of nucleic acid duplexes is important in various molecular biology techniques, such as polymerase chain reaction (PCR) [1], hybridization-induced sequencing [2] and antigene targeting in gene therapy [3]. The nearest-neighbor model developed by Tinoco et al is one of the extensively used methods to predict the thermostability of Watson–Crick nucleic acid duplexes, assuming a two-state melting behavior of the duplexes [8,9] According to this model, the thermodynamic values ( H◦, S◦ and G◦37) for a duplex formation consists of three terms: (i) a free energy change for helix initiation to form a first base pair in the double helix, (ii) a free energy change for helix propagation as the sum of each subsequent base pair and (iii) a free energy change of mixing entropy term for self-complementary strands. The nearest-neighbor parameters were developed by some groups, including us, and have been commonly used to predict the stabilities of different types of DNA–DNA, RNA– RNA, RNA–DNA duplexes, and duplexes formed by peptide nucleic acid (PNA) and DNA [10,11,12,13,14,15,16,17,18]
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