RNA and DNA are rapidly emerging as targets for therapeutics and as potential frameworks for nanotechnology. Accurate methods for predicting and designing structures and dynamics of nucleic acids would accelerate progress in these and other applications. Suitable approximations for modeling nucleic acids are being developed but require validation against disparate experimental observations. Here, nuclear magnetic resonance spectra for RNA and DNA single strands, CAAU and UCAAUC, are used as benchmarks to test molecular dynamics simulations with AMBER force fields OL3 and ROC-RNA for RNA and BSC1 for DNA. A detailed scheme for making comparisons is also presented. The results reflect recent progress in approximations and reveal remaining challenges.

RNA folding free energy change nearest neighbor parameters are widely used to predict folding stabilities of secondary structures. They were determined by linear regression to datasets of optical melting experiments on small model systems. Traditionally, the optical melting experiments are analyzed assuming a two-state model, i.e. a structure is either complete or denatured. Experimental evidence, however, shows that structures exist in an ensemble of conformations. Partition functions calculated with existing nearest neighbor parameters predict that secondary structures can be partially denatured, which also directly conflicts with the two-state model. Here, a new approach for determining RNA nearest neighbor parameters is presented. Available optical melting data for 34 Watson–Crick helices were fit directly to a partition function model that allows an ensemble of conformations. Fitting parameters were the enthalpy and entropy changes for helix initiation, terminal AU pairs, stacks of Watson–Crick pairs and disordered internal loops. The resulting set of nearest neighbor parameters shows a 38.5% improvement in the sum of residuals in fitting the experimental melting curves compared to the current literature set.

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some two-by-two nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with two-by-two nucleotide internal loops closed by GU pairs were investigated. Surprisingly, NMR revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some two-by-two nucleotide internal loops closed by GU pairs, non-self-complementary sequences were designed that preclude formation of hairpins. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

RNA thermodynamics play an important role in determining the 2D and 3D structures of RNA. Internal loops of the sequence, 5’-GMNU/3’-UNMG, are relatively unstable thermodynamically. Here, five GU flanked 2×2 nucleotide internal loops were structurally investigated in order to reveal determinants for their instability. The internal loops investigated are: 5’-GCAU/3’-UACG, 5’-UUCG/3’-GCUU, 5’-GCUU/3’-UUCG, 5’-GUCU/3’-UCUG, and 5’-GCCU/3’-UCCG. Two-dimensional NMR spectra indicate the absence of GU wobble base pairing in 5’-GCUU/3’-UUCG, 5’-GUCU/3’-UCUG and 5’-GCCU/3’-UCCG. The 5’-GCUU/3’-UUCG loop has an unusual conformation of the GU base pairs, in which U’s O2 carbonyl forms a bifurcated hydrogen bond with G’s amino and imino protons. The internal loop of 5’-GUCU/3’-UCUG displays a shifted configuration in which GC pairs flank a U-U pair and several U’s are in fast exchange between positions inside and outside the helix. In contrast, 5’-GCAU/3-UACG and 5’-UUCG/3’-GCUU both have the expected GU wobble base pairs flanking the internal loop. Evidently, GU base pairs flanking internal loops are more likely to display atypical structures relative to Watson-Crick base pairs flanking internal loops. This appears to be more likely when the G of the GU pair is 5’ to the loop. Such unusual structures could serve as recognition elements for biological function and as benchmarks for structure prediction methods.

Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we describe the coarse topography of the chicken ASIC1 intracellular domains determined by FRET, measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and C tail from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments towards the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.