Full length HIV-1 genomic RNA was gently purified from NL4-3 virions (Genbank AF324493). The RNA was equilibrated in a native buffer [50 mM Hepes (pH 8.0), 200 mM potassium acetate (pH 8.0), 3 mM MgCl2] at 37 °C for 15 min and treated with 1M710 (link). Sites of 2′-hydroxyl modification were identified over read lengths spanning several hundred nucleotides using 31 primer extension reactions resolved by fluorescence-detected capillary electrophoresis6 (link),11 (link). Pairing probabilities were determined using RNA-Decoder13 (link) and secondary structure models were developed by incorporating SHAPE reactivities as a pseudo-free energy change term, in conjunction with nearest-neighbor parameters, in an accurate thermodynamics-based prediction algorithm22 (link),23 (link).
Protocol detail
Structural Probing of HIV-1 Genomic RNA
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Buffer
Capillary
Fluorescence
Genomic
Hepes
Hiv 1
Hydroxyl
Mgcl2
Nucleotides
Potassium acetate
Primer
Virions
Corresponding Organization :
Other organizations : University of North Carolina at Chapel Hill, Science Applications International Corporation (United States)
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Variable analysis
independent variables
- Treatment with 1M710
- Sites of 2'-hydroxyl modification
- Pairing probabilities
- Secondary structure models
- Native buffer [50 mM Hepes (pH 8.0), 200 mM potassium acetate (pH 8.0), 3 mM MgCl2]
- Temperature at 37 °C
- Incubation time of 15 min
- No positive or negative controls are explicitly mentioned in the input protocol.
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