Identification of regions in multiple sequence alignments thermodynamically suitable for targeting by consensus oligonucleotides: application to HIV genome

BACKGROUND: Computer programs for the generation of multiple sequence alignments such as "Clustal W" allow detection of regions that are most conserved among many sequence variants. However, even for regions that are equally conserved, their potential utility as hybridization targets varies. Mismatches in sequence variants are more disruptive in some duplexes than in others. Additionally, the propensity for self-interactions amongst oligonucleotides targeting conserved regions differs and the structure of target regions themselves can also influence hybridization efficiency. There is a need to develop software that will employ thermodynamic selection criteria for finding optimal hybridization targets in related sequences. RESULTS: A new scheme and new software for optimal detection of oligonucleotide hybridization targets common to families of aligned sequences is suggested and applied to aligned sequence variants of the complete HIV-1 genome. The scheme employs sequential filtering procedures with experimentally determined thermodynamic cut off points: 1) creation of a consensus sequence of RNA or DNA from aligned sequence variants with specification of the lengths of fragments to be used as oligonucleotide targets in the analyses; 2) selection of DNA oligonucleotides that have pairing potential, greater than a defined threshold, with all variants of aligned RNA sequences; 3) elimination of DNA oligonucleotides that have self-pairing potentials for intra- and inter-molecular interactions greater than defined thresholds. This scheme has been applied to the HIV-1 genome with experimentally determined thermodynamic cut off points. Theoretically optimal RNA target regions for consensus oligonucleotides were found. They can be further used for improvement of oligo-probe based HIV detection techniques. CONCLUSIONS: A selection scheme with thermodynamic thresholds and software is presented in this study. The package can be used for any purpose where there is a need to design optimal consensus oligonucleotides capable of interacting efficiently with hybridization targets common to families of aligned RNA or DNA sequences. Our thermodynamic approach can be helpful in designing consensus oligonucleotides with consistently high affinity to target variants in evolutionary related genes or genomes

Optimization of Duplex Stability and Terminal Asymmetry for shRNA Design

Prediction of efficient oligonucleotides for RNA interference presents a serious challenge, especially for the development of genome-wide RNAi libraries which encounter difficulties and limitations due to ambiguities in the results and the requirement for significant computational resources. Here we present a fast and practical algorithm for shRNA design based on the thermodynamic parameters. In order to identify shRNA and siRNA features universally associated with high silencing efficiency, we analyzed structure-activity relationships in thousands of individual RNAi experiments from publicly available databases (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/​siRNA/si_shRNA_selector/ ). Using this statistical analysis, we found free energy ranges for the terminal duplex asymmetry and for fully paired duplex stability, such that shRNAs or siRNAs falling in both ranges have a high probability of being efficient. When combined, these two parameters yield a ~72% success rate on shRNAs from the siRecords database, with the target RNA levels reduced to below 20% of the control. Two other parameters correlate well with silencing efficiency: the stability of target RNA and the antisense strand secondary structure. Both parameters also correlate with the short RNA duplex stability; as a consequence, adding these parameters to our prediction scheme did not substantially improve classification accuracy. To test the validity of our predictions, we designed 83 shRNAs with optimal terminal asymmetry, and experimentally verified that small shifts in duplex stability strongly affected silencing efficiency. We showed that shRNAs with short fully paired stems could be successfully selected by optimizing only two parameters: terminal duplex asymmetry and duplex stability of the hypothetical cleavage product, which also relates to the specificity of mRNA target recognition. Our approach performs at the level of the best currently utilized algorithms that take into account prediction of the secondary structure of the target and antisense RNAs, but at significantly lower computational costs. Based on this study, we created the si-shRNA Selector program that predicts both highly efficient shRNAs and functional siRNAs (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/​siRNA/si_shRNA_selector/ ).National Institutes of HealthNational Library of Medicin

Hazardous alcohol drinking and premature mortality in Russia: a population based case-control study.

BACKGROUND: The reason for the low life expectancy in Russian men and large fluctuations in mortality are unknown. We investigated the contribution of alcohol, and hazardous drinking in particular, to male mortality in a typical Russian city. METHODS: Cases were all deaths in men aged 25-54 years living in Izhevsk occurring between Oct 20, 2003, to Oct 3, 2005. Controls were selected at random from the city population and were frequency matched to deaths by age. Interviews with proxy informants living in the same household as cases were done between Dec 11, 2003, and Nov, 16 2005, and were obtained for 62% (1750/2835) of cases and 57% (1750/3078) of controls. We ascertained frequency and usual amount of beer, wine, and spirits consumed and frequency of consumption of manufactured ethanol-based liquids not intended to be drunk (non-beverage alcohol), and markers of problem drinking. Complete information on markers of problem drinking, frequency of alcohol consumption, education, and smoking was available for 1468 cases and 1496 controls. FINDINGS: 751 (51%) cases were classed as problem drinkers or drank non-beverage alcohol, compared with 192 (13%) controls. The mortality odds ratio (OR) for these men, compared with those who either abstained or were non-problematic beverage drinkers, was 6.0 (95% CI 5.0-7.3) after adjustment for smoking and education. The mortality ORs for drinking non-beverage alcohol in the past year (yes vs no) was 9.2 (7.2-11.7) after adjustment for age. Adjustment for volume of ethanol consumed from beverages lowered the OR to 8.3 (6.5-10.7), and further adjustment for education and smoking reduced it to 7.0 (5.5-9.0). A strong direct gradient with mortality was seen for frequency of non-beverage alcohol drinking independent of volume of beverage ethanol consumed. 43% of mortality was attributable to hazardous drinking (problem drinking or non-beverage alcohol consumption, or both) adjusted for smoking and education. INTERPRETATION: Almost half of all deaths in working age men in a typical Russian city may be accounted for by hazardous drinking. Our analyses provide indirect support for the contention that the sharp fluctuations seen in Russian mortality in the early 1990s could be related to hazardous drinking as indicated by consumption of non-beverage alcohol