Melting of Model HIV-1 Stem-Loop 1 RNA Dimers Monitored by 2-Aminopurine Fluorescence

Abstract

Viral maturation of HIV-1 involves refolding of its genomic RNA, which is believed to include a rearrangement of the SL1 stem-loop from a metastable conformation called kissing loop dimer (KD) to a stable one termed extended dimer (ED). To investigate this rearrangement in vitro we have studied the thermal melting of the RNA dimers formed by slightly modified 23-nucleotide SL1 RNA of HIV-1 Mal. Local structural changes in the RNA dimers during the melting were monitored by changes in the fluorescence of 2-aminopurine (2AP) incorporated in predetermined positions of RNA. We have shown that the stem regions of both preformed KD and ED melt in the temperature interval from 75°C to 90°C. Kissing loop interface of the KD RNA is found to be disrupted at lower temperatures from 20°C to 55°C, at which the stem regions remain intact. Conversion of the preformed KD to ED overcoming the kinetic barrier occurs between 55°C and 65°C. The melting of “loop-loop” regions in both preformed and newly formed EDs takes place around 70°C. Our finding that thermoinduced KD-to-ED conversion is preceded by transient dissociation of loop-loop interface disagrees with a common idea of strand exchange without disruption of loop-loop-contact.