Tianbing Xia

Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson-Crick base pairs. To test end or base composition effects, new sets of duplexes are included that have identical nearest neighbors, but different base compositions and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of DeltaG degrees37 from a nearest-neighbor model. A physical model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total number of Watson-Crick hydrogen bonds on the base composition of a duplex. On average, the new parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM within 3.2%, 6.0%, 6.8%, and 1.3 degreesC, respectively. These predictions are within the limit of the model, based on experimental results for duplexes predicted to have identical thermodynamic parameters.

Myotonic dystrophy is caused by an expanded CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is approximately 15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.

Water molecules in the DNA grooves are critical for maintaining structural integrity, conformational changes, and molecular recognition. Here we report studies of site- and sequence-specific hydration dynamics, using 2-aminopurine (Ap) as the intrinsic fluorescence probe and with femtosecond resolution. The dodecamer d[CGCA(Ap)ATTTGCG]2 was investigated, and we also examined the effect of a specific minor groove-binding drug, pentamidine, on hydration dynamics. Two time scales were observed: approximately 1 ps (bulk-like) and 10-12 ps (weakly bound type), consistent with layer hydration observed in proteins and DNA. However, for denatured DNA, the cosolvent condition of 40% formamide hydration is very different: it becomes that of bulk (in the presence of formamide). Well known electron transfer between Ap and nearby bases in stacked assemblies becomes inefficient in the single-stranded state. The rigidity of Ap in the single strands is significantly higher than that in bulk water and that attached to deoxyribose, suggesting a unique role for the dynamics of the phosphate-sugar-base in helix formation. The disparity in minor and major groove hydration is evident because of the site selection of Ap and in the time scale observed here (in the presence and absence of the drug), which is different by a factor of 2 from that observed in the minor groove-drug recognition.

Arginine-rich peptide motifs (ARMs) capable of binding unique RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. Bacteriophage ARMs necessary for transcription antitermination bind to distinct boxB RNA hairpin sequences with a characteristic induced alpha-helical structure. Characterization of ARMs from lambdoid phages reveals that the dissociation constant of the P22 bacteriophage model-antitermination complex (P22(N21)-P22boxB) is 200 +/- 56 pM in free solution at physiologic concentrations of monovalent cation, significantly stronger than previously determined by gel mobility shift and polyacrylamide gel coelectophoresis, and 2 orders of magnitude stronger than the tightest known native ARM-RNA interaction at physiological salt. Here, we use a reciprocal design approach to enhance the binding affinity of two separate alpha-helical ARM-RNA interactions; one derived from the native lambda phage antitermination complex and a second isolated using mRNA display selection experiments targeting boxB RNA.

Over the past several years, there has been a renewed interest in complex orthogonal designs for their application in space-time block coding. Motivated by the success of this application, this paper generalizes the definition of complex orthogonal designs by introducing orthogonal designs over the quaternion domain. This paper builds a theory of these novel quaternion orthogonal designs, offers examples, and provides several construction techniques. These theoretical results, along with the results of preliminary simulations, lay the foundation for developing applications of these designs as orthogonal space-time-polarization block codes.