Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Conformation Change of the Loop Adenine of Avian Leukosis Virus RNA Upon Antibiotic Binding Revealed by 2-Aminopurine Fluorescence
Structural dynamics and local changes in the dimerization site of avian retroviral RNA were studied. Our task was to identify the state of adenine located in the loop of RNA hairpin under the dimerization and understand the effects of various factors on the local RNA conformation. To this end we replaced the unpaired adenine in the hairpin loop of avian leukosis virus RNA with a fluorescent probe 2-aminopurine (2-AP). This approach permitted us to discriminate between the local conformation of the unpaired adenine in several RNA states during dimerization dimerization: hairpin, a kissing loop dimer (KD) and an extended duplex dimer (ED). It was shown that fluorescence intensity of 2-AP in the monomer hairpin RNA has higher than that of the both RNA dimers. Observed fluorescence quenching on RNA dimerization can be explained by interaction of unpaired adenine to neighboring bases of the RNA loop upon dimerization. It has also been found that the intensity of 2-AP fluorescence for the two RNA dimers is different. Significantly lower intensity of fluorescence for extended dimer can be attributed to its more overall compact RNA structure.
It is known, that kissing loop dimers of retroviral RNA are unstable intermediates in the process of retroviral RNA dimerization. The study the KD to ED transition of the RNA is important since it is an extended stable dimer to be packed in the viral particle. Effect of RNA ligands, including aminoglycoside antibiotics, on the KD structure applies to this task. We studied the conformational change of the unpaired loop adenine of kissing loop dimer RNA upon the interaction with aminoglycoside antibiotics (AMG): paromomycin, neomycin, tobramycin and kanamycin B. It turned out that only tobramycin increases nearly three times the intensity of fluorescence 2-AP of kissing loop dimer. This result implies that 2-AP loses the initial intra-loop interactions in the structure KD on interaction with tobramycin, becoming more exposed into the solution that is reflected in its fluorescence increase. Probably, the fluorescence of 2-aminopurine can be successfully used to detect the binding of ligands to different structures of RNA.