No abstract

We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem-loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem-loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.

We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem-loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem-loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.

Few examples of pseudomerohedrally twinned macromolecular crystals have been described in the literature. This unusual phenomenon arises when a fortuitous unit-cell geometry makes it possible for twinning to occur in a space group that ordinarily does not allow twinning. Here, the crystallization, structure determination and re®nement of the cocaine hydrolytic antibody 15A10 at 2.35 A Ê resolution are described. The crystal belongs to space group P2 1 , with two molecules in the asymmetric unit and unit-cell parameters a = 37.5, b = 108.4, c = 111.3 A Ê and fortuitously near 90; the re®ned twinning fraction is = 0.43. Interestingly, the noncrystallographic symmetry (NCS) and twin operators are nearly parallel, which appears to be a relatively frequent situation in protein crystals twinned by merohedry or pseudomerohedry.

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Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

We report herein the first homogeneous assays based on the ribonuclease activity of a deoxyribozyme. The previously reported deoxyribozyme was covalently modified with biotin and used to assay biotin-binding interactions through changes in fluorescence upon substrate turnover. Deoxyribozymes with fluorescence-based reporting have the potential to serve as general analytical tools.

We surveyed our collection of psychrophilic bacteria to determine the types of phosphatases they produce and whether any had heat-labile activities with potential applications. Assays at different temperatures showed that the activity from one isolate was optimal at 45؇C and decreased dramatically above 55؇C. This isolate, D10, had the rod-coccus morphological cycle and cell wall amino acids associated with members of the Arthrobacter genus. Interestingly, we found that this strain made two extracellular phosphatases that could be separated by ammonium sulfate fractionation and migration during polyacrylamide gel electrophoresis. One enzyme, designated D10A, hydrolyzed both X-phos (5-bromo-4-chloro-3-indolyl phosphate) and para-nitrophenyl phosphate as substrates and had activity over a broad pH range of 7 to 11. The second enzyme, D10B, lacked activity against X-phos and had a narrow pH range of about 8 to 9. In addition, the D10B enzyme required calcium for activity. The levels of activity of both enzymes decreased for cells grown in media containing more than 100 M P i. These results not only demonstrate the existence of different enzymes from one Arthrobacter strain but also suggest ways in which other studies may have missed phosphatases with unknown requirements.