In 1990, a woman was wrongly convicted of poisoning her infant son and was sentenced to life in prison. Her conviction was based on laboratory work that wrongly identified ethylene glycol as present in her son’s blood and in the formula he drank prior to his death. The actual cause of the infant’s death, a metabolic disease, was eventually disclosed as a result of analytical work done by scientists who believed the mother was wrongly convicted. On the basis of the scientists’ work, the conviction was overturned. This real-life case serves as the launching point for a biochemistry laboratory experiment, which uses polymerase chain reaction. Students design their own primers, amplify two biological samples, and analyze the results by gel electrophoresis to determine if their patient has a genetic mutation. This genetic mutation causes the metabolic disease, which if present can be used to “solve” the case.

RNA secondary structure prediction using free energy minimization is one method to gain an approximation of structure. Constraints generated by enzymatic mapping or chemical modification can improve the accuracy of secondary structure prediction. We report a facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The lowest free energy structure without constraints has only 27% of the base pairs present in the phylogenetic structure. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNAPhe, which is accurately predicted in the absence of constraints (95%). Although RNase H mapping does not improve secondary structure prediction, it does eliminate all other suboptimal structures predicted within 10% of the lowest free energy structure. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions. Moreover, it can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules.

Influenza A is a negative-sense RNA virus with an eight-segment genome. Some segments code for more than one polypeptide product, but how the virus accesses alternate internal open reading frames (ORFs) is not completely understood. In segment 2, ribosomal scanning produces two internal ORFs, PB1-F2 and N40. Here, chemical mapping reveals a Mg2+ dependent pseudoknot structure that includes the PB1-F2 and N40 start codons. The results suggest that ribosome interactions with the pseudoknot may affect the level of translation for PB1-F2 and N40.

The prediction of RNA 3D structure from sequence alone is a long-standing goal. High resolution, experimentally determined structures of simple noncanonical pairings and motifs are critical to the development of prediction programs. Here, we present the NMR structure of the duplex, (5′CCAGAAACGGAUGGA)2, which contains an 8x8 nucleotide internal loop flanked by three Watson-Crick pairs on each side. The loop is comprised of a central 5′AC/3′CA nearest neighbor flanked by two 3RRs motifs, a known stable motif consisting of three consecutive sheared GA pairs. Hydrogen bonding patterns between base pairs in the loop, all-atom RMSD for the loop, and deformation index were used to compare the structure to automated predictions by MC-sym, RNA FARFAR, and RNAComposer.

Influenza A kills hundreds of thousands of people globally every year and has potential to generate more severe pandemics. Influenza A’s RNA genome and transcriptome provide many potential therapeutic targets. Here, nuclear magnetic resonance (NMR) experiments suggest that one such target could be a hairpin loop of eight nucleotides in a pseudoknot that sequesters a 3' splice site in canonical pairs until a conformational change releases it into a dynamic 2X2 nucleotide internal loop. NMR experiments reveal that the hairpin loop is dynamic and able to bind oligonucleotides as short as pentamers. A 3D NMR structure of the complex contains four and likely five base pairs between pentamer and loop. Moreover, a hairpin sequence was discovered that mimics the equilibrium of the influenza hairpin between its structure in the pseudoknot and upon release of the splice site. Oligonucleotide binding shifts the equilibrium completely to the hairpin secondary structure required for pseudoknot folding. The results suggest this hairpin can be used to screen for compounds that stabilize the pseudoknot and potentially reduce splicing.