Molecular dynamics correctly models the unusual major conformation of the GAGU RNA internal loop and with NMR reveals an unusual minor conformation.

The RNA "GAGU" duplex, (5'GACGUCA), contains the internal loop (5'-GAGU-3') , which has two conformations in solution as determined by NMR spectroscopy. The major conformation has a loop structure consisting of -Watson-Crick/Hoogsteen GG pairs, A residues stacked on each other, U residues bulged outside the helix, and all sugars with a C2'- conformation. This differs markedly from the internal loops, (5'-GC-3'), (5'-AU-3'), and (5'-UAGG-3'), which all have -Watson-Crick/Watson-Crick AG "imino" pairs flanked by -Watson-Crick/Watson-Crick canonical pairs resulting in maximal hydrogen bonding. Here, molecular dynamics was used to test whether the Amber force field (ff99 + bsc0 + OL3) approximates molecular interactions well enough to keep stable the unexpected conformation of the GAGU major duplex structure and the NMR structures of the duplexes containing (5'-GC-3'), (5'-AU-3'), and (5'-UG-3') internal loops. One-microsecond simulations were repeated four times for each of the duplexes starting in their NMR conformations. With the exception of (5'-UAGG-3'), equivalent simulations were also run starting with alternative conformations. Results indicate that the Amber force field keeps the NMR conformations of the duplexes stable for at least 1 µsec. They also demonstrate an unexpected minor conformation for the (5'-GAGU-3') loop that is consistent with newly measured NMR spectra of duplexes with natural and modified nucleotides. Thus, unrestrained simulations led to the determination of the previously unknown minor conformation. The stability of the native (5'-GAGU-3') internal loop as compared to other loops can be explained by changes in hydrogen bonding and stacking as the flanking bases are changed.