In order to better elucidate RNA structure-function relationships and to improve design of pharmaceutical agents that target specific RNA motifs, an understanding of RNA primary, secondary, and tertiary structure is necessary. The prediction of RNA secondary structure from sequence is an intermediate step in predicting RNA three-dimensional structure. RNA secondary structure is typically predicted using a nearest neighbor model based on free energy parameters. The current free energy parameters for 2×3 nucleotide loops are based on a dataset of 23 2×3 loops and internal loops of other sizes. A database of representative RNA secondary structures was searched to identify 2×3 nucleotide loops that occur in nature. Seventeen of the most frequent 2×3 nucleotide loops in this database were studied by optical melting experiments. Fifteen of these loops melted in a two-state manner, and the associated experimental ΔG°37,2×3 values are, on average, 0.6 and 0.7 kcal/mol different from the values predicted for these internal loops using the predictive models proposed by Lu, Turner, and Mathews [Lu, Z. J., Turner, D. H., and Mathews, D. H. (2006) Nucleic Acids Res. 34, 4912–4924] and Chen and Turner [Chen, G. and Turner, D. H. (2006) Biochemistry 45, 4025–4043], respectively. These new ΔG°37,2×3 values can be used to update the current algorithms that predict secondary structure from sequence. In order to improve free energy calculations for duplexes containing 2×3 nucleotide loops that still do not have experimentally-determined free energy contributions, an updated predictive model was derived. This new model resulted from a linear regression analysis of the data reported here combined with 31 previously studied 2×3 nucleotide internal loops. Most of the values for the parameters in this new predictive model are within experimental error of the previous models, suggesting that approximations and assumptions associated with the derivation of the previous nearest neighbor parameters were valid. The updated predictive model predicts free energies of 2×3 nucleotide internal loops within 0.4 kcal/mol, on average, of the experimental free energy values. Both the experimental values and the updated predictive model can be used to improve secondary structure prediction from sequence.

Acetone is one of the most abundant volatile organic compounds (VOC) in human breath and high acetone levels result in ketoacidosis. Many acetone sensors have been developed; however, many disadvantages are present; therefore, there is a need for a more specific, biologically friendly, and simpler biosensor for breath acetone detection for diabetics. This study outlines a procedure for isolation, purification, and characterization of NADPH-dependant carbonyl reductase enzyme for use in an amperometric acetone sensor. Carbonyl reductase was extracted from Saccharomyces pastorianus yeast cells and purified using column chromatography. Carbonyl reductase activity was evaluated using activity assays and was also studied electrochemically. Furthermore, this study outlines a procedure for a flow injection method used for detecting aqueous acetone in the presence of carbonyl reductase. Lastly, various immobilization techniques were tested in order to improve current response and the selectivity towards acetone.

Immobilization is not only useful for preserving enzyme activity, but also to adhere an enzyme to a surface, such as an electrode, so that the enzyme does not leach into solution during testing. Current immobilization approaches do not readily allow for adjustments to the distance between the enzyme and the electrode or other enzymes. The ability to control the distance of enzymes relative to each other on an electrode can allow for optimal placement and improved current responses. In this report, we investigate the use of comb-branched DNA for enzyme immobilization. A DNA foundation strand was covalently attached to multiwalled carbon nanotubes on a glassy carbon electrode. Comb-branched DNA was then successfully formed using a previously-identified deoxyribozyme to attach DNA strands at specific locations on this foundation strand. By changing the foundation strands, the placement of the DNA strands can be adjusted, allowing for distance changes between the enzyme and the electrode surface. Using standard bioconjugation methods, alcohol dehydrogenase and glucose dehydrogenase were attached to these comb-branched DNA structures, resulting in enzyme immobilization on electrode surfaces. Amperometric analysis revealed both distance and DNA foundation strand length dependence for current response of these enzymes in the presence of their appropriate substrates.