Guanine-adenine (GA) base pairs play important roles in determining the structure, dynamics, and stability of RNA. In RNA internal loops, GA base pairs often occur in tandem arrangements and their structure is context and sequence dependent. Calculations reported here test the thermodynamic integration (TI) approach with the amber99 force field by comparing computational predictions of free energy differences with the free energy differences expected on the basis of NMR determined structures of the RNA motifs (5'-GCGGACGC-3')(2), (5'-GCiGGAiCGC-3')(2), (5'-GGCGAGCC-3')(2), and (5'-GGiCGAiGCC-3')(2). Here, iG and iC denote isoguanosine and isocytidine, which have amino and carbonyl groups transposed relative to guanosine and cytidine. The NMR structures show that the GA base pairs adopt either imino (cis Watson-Crick/Watson-Crick A-G) or sheared (trans Hoogsteen/Sugar edge A-G) conformations depending on the identity and orientation of the adjacent base pair. A new mixing function for the TI method is developed that allows alchemical transitions in which atoms can disappear in both the initial and final states. Unrestrained calculations gave DeltaG degrees values 2-4 kcal/mol different from expectations based on NMR data. Restraining the structures with hydrogen bond restraints did not improve the predictions. Agreement with NMR data was improved by 0.7 to 1.5 kcal/mol, however, when structures were restrained with weak positional restraints to sample around the experimentally determined NMR structures. The amber99 force field was modified to partially include pyramidalization effects of the unpaired amino group of guanosine in imino GA base pairs. This provided little or no improvement in comparisons with experiment. The marginal improvement is observed when the structure has potential cross-strand out-of-plane hydrogen bonding with the G amino group. The calculations using positional restraints and a nonplanar amino group reproduce the signs of DeltaG degrees from the experimental results and are, thus, capable of providing useful qualitative insights complementing the NMR experiments. Decomposition of the terms in the calculations reveals that the dominant terms are from electrostatic and interstrand interactions other than hydrogen bonds in the base pairs. The results suggest that a better description of the backbone is key to reproducing the experimental free energy results with computational free energy predictions.

• Publication type
• Journal Article MeSH

The formation of a cation-stabilized guanine quadruplex (G-DNA) stem is an exceptionally slow process involving complex kinetics that has not yet been characterized at atomic resolution. Here, we investigate the formation of a parallel stranded G-DNA stem consisting of four strands of d(GGGG) using molecular dynamics simulations with explicit inclusion of counterions and solvent. Due to the limitations imposed by the nanosecond timescale of the simulations, rather than watching for the spontaneous formation of G-DNA, our approach probes the stability of possible supramolecular intermediates (including two-, three-, and four-stranded assemblies with out-of-register base pairing between guanines) on the formation pathway. The simulations suggest that "cross-like" two-stranded assemblies may serve as nucleation centers in the initial formation of parallel stranded G-DNA quadruplexes, proceeding through a series of rearrangements involving trapping of cations, association of additional strands, and progressive slippage of strands toward the full stem. To supplement the analysis, approximate free energies of the models are obtained with explicit consideration of the integral cations. The approach applied here serves as a prototype for qualitatively investigating other G-DNA molecules using molecular dynamics simulation and free-energy analysis.

• MeSH
• Time Factors MeSH
• DNA chemistry   MeSH
• G-Quadruplexes MeSH
• Guanine chemistry   MeSH
• Ions MeSH
• Cations MeSH
• Kinetics MeSH
• Nucleic Acid Conformation MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Oligonucleotides chemistry   MeSH
• Sodium chemistry   MeSH
• Software MeSH
• Temperature MeSH
• Thermodynamics MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• DNA MeSH
• Guanine MeSH
• Ions MeSH
• Cations MeSH
• Oligonucleotides MeSH
• Sodium MeSH

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80 ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries of the three non-Watson-Crick basepairs that differ from the consensus bacterial sequence. The deep groove of Loop E motifs provides unique sites for cation binding. Binding of Mg(2+) rigidifies Loop E and stabilizes its major groove at an intermediate width. In the absence of Mg(2+), the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations that, in contrast to Mg(2+), penetrate into the most negative regions inside the deep groove. The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with the bacterial consensus. Structures with a narrow deep groove essentially collapse around a string of Na(+) cations with long coordination times. The Loop E non-Watson-Crick basepairing is complemented by highly specific hydration sites ranging from water bridges to hydration pockets hosting 2 to 3 long-residing waters. The ordered hydration is intimately connected with RNA local conformational variations.

• MeSH
• RNA, Bacterial chemistry   MeSH
• Base Pair Mismatch MeSH
• Nucleic Acid Denaturation MeSH
• Species Specificity MeSH
• Escherichia coli chemistry   MeSH
• Magnesium chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Macromolecular Substances MeSH
• Models, Molecular * MeSH
• Base Pairing * MeSH
• Computer Simulation MeSH
• Motion MeSH
• RNA, Ribosomal, 5S chemistry   MeSH
• RNA, Plant chemistry   MeSH
• Solvents chemistry   MeSH
• Sodium chemistry   MeSH
• Spinacia oleracea chemistry   MeSH
• Binding Sites MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Comparative Study MeSH
• Validation Study MeSH
• Names of Substances
• RNA, Bacterial MeSH
• Magnesium MeSH
• Macromolecular Substances MeSH
• RNA, Ribosomal, 5S MeSH
• RNA, Plant MeSH
• Solvents MeSH
• Sodium MeSH
• Water MeSH