Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Thermal Behavior of Retrovirus RNA Dimers and Its Stabilization by Paromomycin Using Fluorescence Melting
Retroviral RNA undergoes a series of structural rearrangements, comprising dimerization of two strands RNA in the course of packaging in a virus particle. The dimerization includes the formation of kissing loop (KD) and extended (ED) RNA dimers. The fluorescence-based melting is used in this study to determine thermodynamic characteristics of the RNA dimers. For this purpose 2-aminopurine (2-AP) was incorporated in the loop of short fragment HIV-1 RNA and the loop hairpin of avian leukosis virus RNA (ALV).
The fluorescence melting of the loop-loop region reveals dramatic difference between the two types of dimer structures, KD and ED, for both HIV-1 and ALV RNAs. The temperature curve for the KD RNA is biphasic while ED RNA dimer is characterized by single temperature transition. We suggest that the first transition in the melting curves of KD RNA (temperature range 20°C to 55°C for HIV-1 RNA and 30°C to 55°C for ALV RNA) corresponds to disruption of the loop-loop interaction. The refolding to the extended RNA dimers occurs at 55°C to 65°C. Further melting of the newly formed extended dimers (HIV-1 and ALV) is observed in the temperature range from 65°C to 75°C.
It is known that the structure of the kissing loop RNA dimer is stabilized by aminoglycoside antibiotic paromomycin. Using the fluorescence melting, we also investigated the effect of the aminoglycoside antibiotic paromomycin on the stability of the kissing loop RNA dimer. The melting temperature of the kissing loop dimer HIV-1 RNA is increased by approximately 6°C upon interaction with paromomycin. The influence of paromomycin on stability of the kissing loop dimer of ALV RNA by was not significant. Our study also showed that 2-aminopurine fluorescence can be useful in testing novel ligands, which influence the dimerization of retroviral RNA.
Dmitry N. Kaluzhny
Engelhardt Inst. of Molecular Biology