Rolling circle amplification with fluorescently labeled dUTP-balancing the yield and degree of labeling.

Detection methods based on rolling circle amplification (RCA) have been applied to a large number of targets in molecular biology. The key feature of RCA-based methods as well as other nucleic acid amplification methods is their exceptional sensitivity, which allows the detection of molecules at low concentrations, achieved by signal amplification due to nucleic acid magnification and subsequent detection. Variations on the method, such as immuno-RCA, extend the range of potential targets that can be detected. Employing fluorescently labeled nucleotides for direct incorporation into an amplification product is an attractive method for RCA product detection. However, the effectiveness of this approach remains doubtful. In our study, we utilized different modified dUTPs, including sulfo-cyanine3-dUTP, sulfo-cyanine5-dUTP, sulfo-cyanine5.5-dUTP, BDP-FL-dUTP, and amino-11-dUTP, to investigate whether the properties of the fluorophore used for modification affected the reaction yield and effectiveness of incorporation of nucleotide analogs by phi29 DNA polymerase. Among the modified dUTPs, sulfo-cyanine3-dUTP demonstrated the highest incorporation effectiveness, equal to 4-9 labels per 1000 nucleotides. The mean length of the RCA product was estimated to be approximately 175,000 nucleotides. The total increase in fluorescence from a single target/product complex was 850 times. The results obtained in the study illustrate the possibility of successful application of nucleotide analogs for RCA detection and present quantitative characteristics of fluorescently labeled dUTPs to be incorporated into RCA products.

Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,ß-Hydrolase Superfamily Enzymes.

Comparative bioinformatic analysis is the cornerstone of the study of enzymes' structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements - the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,ß-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes' evolution and to create of a databank of enzyme active site structures.