Probing the transition state for nucleic acid hybridization using phi-value analysis.
Genetic regulation by noncoding RNA elements such as microRNA and small interfering RNA (siRNA) involves hybridization of a short single-stranded RNA with a complementary segment in a target mRNA. The physical basis of the hybridization process between the structured nucleic acids is not well understood primarily because of the lack of information about the transition-state structure. Here we use transition-state theory, inspired by phi-value analysis in protein folding studies, to provide quantitative analysis of the relationship between changes in the secondary structure stability and the activation free energy. Time course monitoring of the hybridization reaction was performed under pseudo-steady-state conditions using a single fluorophore. The phi-value analysis indicates that the native secondary structure remains intact in the transition state. The nativelike transition state was confirmed via examination of the salt dependence of the hybridization kinetics, indicating that the number of sodium ions associated with the transition state was not substantially affected by changes in the native secondary structure. These results propose that hybridization between structured nucleic acids undergoes a transition state leading to formation of a nucleation complex and then is followed by sequential displacement of preexisting base pairings involving successive small energy barriers. The proposed mechanism might provide new insight into physical processes during small RNA-mediated gene silencing, which is essential to selection of a target mRNA segment for siRNA design.
Kim
Jandi
J
Department of Biotechnology, Yonsei University, Shinchon-Dong 134, Seodaemun-Gu, Seoul 120-749, Korea.
Shin
Jong-Shik
JS
D001483:Base Sequence; D002151:Calorimetry; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D005786:Gene Expression Regulation; D007700:Kinetics; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics; D008962:Models, Theoretical; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification; D013816:Thermodynamics
D035683:MicroRNAs; D009838:Oligodeoxyribonucleotides; D004247:DNA