Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Fluorescent based approach for monitoring HIV-1 RNA interaction with aminoglycosides

Dimerization of genomic RNAs is an essential process for retrovirus replication. Interaction between dimerization initiation sites (DIS) includes two steps: formation of intermediate kissing loop complex and subsequent maturation of the structure to form more stable linear dimer (1). DIS are conserved within different subtypes of human immunodeficiency virus type-1 (HIV-1) thereby can be considered a new potential target for antiviral drugs. Several aminoglycoside antibiotics were shown to bind specifically kissing dimer of HIV-1 RNA (2). We proposed a fluorescent-based technique to monitor the interaction between RNA DIS and aminoglycoside antibiotics directly in solution. Highly fluorescent probe 2-aminopurine was introduced into RNA chain at the position of unpaired adenine (A272).

The method was tested on several model RNA fragments corresponding to natural and mutated DIS of HIV-1 subtype A and aminoglycoside antibiotic paromomycin. 2-aminopurine responded to the ligand binding by increase in fluorescence, that allowed to determine standard characteristics of the RNA-drug binding. Paromomycin appeared to bind specifically both kissing loop and extended dimers RNA with similar affinities. Spermine and magnesium ions, while stabilizing the kissing loop dimer, competed with paromomycin for binding to RNA. Replacement of paromomycin with magnesium demonstrates reversible bulging out of unpaired adenine A272. Finally, due to different fluorescent intensity of the RNA dimer this technique is applicable for studies of transition kinetics from kissing loop to extended dimer RNA. The results assume further application of this fluorescent-based approach for screening aminoglycosides derivatives and other potential ligands of HIV-1 DIS RNA.

This research was supported by the Russian Foundation for Basic Research, project 05-04-49532

References and Footnotes
  1. Paillart et al., Nat. Rev. Microbiol. 2, 461-472 (2004)
  2. Ennifar et al., NAR 34, 2328-2339 (2006)

Elvira E. Minyat*
Dmitry N. Kaluzhny
Artemy D. Beniaminov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 199911, Russia

Email: eminyat@gmail.com