The Evolution and Application of a Novel DNA Aptamer Targeting Bone Morphogenetic Protein 2 for Bone Regeneration.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

Directed Evolution of a G-Quadruplex Peroxidase DNAzyme and Application in Proteomic DNAzyme-Aptamer Proximity Labeling.

DNAzymes have been limited in application by their low catalytic rates. Here, we evolved a new peroxidase DNAzyme mSBDZ-X-3 through a directed evolution method based on the capture of self-biotinylated DNA catalyzed by its intrinsic peroxidase activity. The mSBDX-X-3 DNAzyme has a parallel G-quadruplex structure and has more favorable catalytic properties than all previously reported peroxidase DNAzyme variants. We applied mSBDZ-X-3 in an aptamer-coupled proximity-based labeling proteomic assay to determine the proteins that bind to cell surface cancer biomarkers EpCAM and nucleolin. Confocal microscopy, western blot analysis, and LC-MS/MS showed that the hybrid DNAzyme aptamer-coupled proximity assay-labeled proteins associated with EpCAM and nucleolin within 6-12 min in fixed cancer cells. The labeled proteins were identified by mass spectrometry. This study provides a highly efficient peroxidase DNAzyme, a methodology for selection of such variants, and a method for its application in spatial proteomics using entirely nucleic acid-based tooling.

Molecular epidemiology of Vibrio cholerae associated with flood in Brahamputra River valley, Assam, India.

Cholera is often caused when drinking water is contaminated through environmental sources. In recent years, the drastic cholera epidemics in Odisha (2007) and Haiti (2010) were associated with natural disasters (flood and Earthquake). Almost every year the state of Assam India witnesses flood in Brahamputra River valley during reversal of wind system (monsoon). This is often followed by outbreak of diarrheal diseases including cholera. Beside the incidence of cholera outbreaks, there is lack of experimental evidence for prevalence of the bacterium in aquatic environment and its association with cholera during/after flood in the state. A molecular surveillance during 2012-14 was carried out to study prevalence, strain differentiation, and clonality of Vibrio cholerae in inland aquatic reservoirs flooded by Brahamputra River in Assam. Water samples were collected, filtered, enriched in alkaline peptone water followed by selective culturing on thiosulfate bile salt sucrose agar. Environmental isolates were identified as V. cholerae, based on biochemical assays followed by sero-grouping and detailed molecular characterization. The incidence of the presence of the bacterium in potable water sources was higher after flood. Except one O1 isolate, all of the strains were broadly grouped under non-O1/non-O139 whereas some of them did have cholera toxin (CT). Surprisingly, we have noticed Haitian ctxB in two non-O1/non-O139 strains. MLST analyses based on pyrH, recA and rpoA genes revealed clonality in the environmental strains. The isolates showed varying degree of antimicrobial resistance including tetracycline and ciprofloxacin. The strains harbored the genetic elements SXT constins and integrons responsible for multidrug resistance. Genetic characterization is useful as phenotypic characters alone have proven to be unsatisfactory for strain discrimination. An assurance to safe drinking water, sanitation and monitoring of the aquatic reservoirs is of utmost importance for combating the impending epidemic threat in the flood affected areas. Further, the management of flood through multi-prong approaches and sustainable utilization of environmental resources would be effective in disease management.