Biology subjects

Izgu, E. C.

Publications and source records attributed to Izgu, E. C..

3 recordsLinked to original sources

bioRxiv2022

MicroRNA Detection in Biological Media Using a Split Aptamer Platform

Intercellular microRNA (miRNA)-based communication has been implicated in a wide array of functional and dysfunctional biological processes. This has raised attention to the potential use of miRNAs as biomarkers for disease diagnosis and prognosis and produced interest in their detection. Though the list of clinically significant miRNA biomarkers is rapidly expanding, it remains challenging to adapt current tools to investigate new targets in biological environments. Systematic approaches for the rapid development of miRNA biosensors are valuable to reduce this disparity. We describe here a methodology for developing aptamer-based fluorescent biosensors that can specifically detect miRNAs in biological environments, including culture medium from HeLa cells, human serum, and human plasma. This methodology includes the semi-rational design of the hybridization between a pair of split DNA aptamer oligonucleotides and the miRNA target to build a pool of potential sensor designs, and the screening of this pool for designs with high signal-to-background ratio and sequence selectivity. The method uses natural oligonucleotides without chemical modification, and is effective in buffer, 10%, and 30% (v/v) biological media. Following this approach, we developed sensors that detect three miRNA targets (miR-19b, miR-21, and miR-92a) at concentrations as low as 5 nM without amplification and are selective against single-nucleotide mutants. This work expands upon the current design principles of nucleic acid-based biosensors and provides a method to rapidly develop diagnostic tools for novel and niche miRNA targets of interest.

biochemistry↗

bioRxiv2021

Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugarphosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9{degrees}N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3[->]P5 phosphoramidate linkages in the presence of 3-amino-2,3-dideoxynucleoside 5-triphosphate (3-NH2-ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9{degrees}N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9{degrees}N-NRQS) that incorporated 3-NH2-ddNTPs into a 3-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

biochemistry↗

bioRxiv2021

Bioorthogonal Functionalization of Material Surfaces with Bioactive Molecules

The functionalization of material surfaces with biologically active molecules is crucial for enabling technologies in life sciences, biotechnology, and medicine. However, achieving biocompatibility and bioorthogonality with current synthetic methods remains a challenge. We report herein a novel surface functionalization method that proceeds chemoselectively and without a free transition metal catalyst. In this method, a coating is first formed via the tyrosinase-catalyzed putative polymerization of a tetrazine-containing catecholamine (DOPA-Tet). One or more types of molecule of interest containing trans-cyclooctene are then grafted onto the coating via tetrazine ligation. The entire process proceeds under physiological conditions and is suitable for grafting bioactive molecules with diverse functions and structural complexities. Utilizing this method, we functionalized material surfaces with enzymes (alkaline phosphatase, glucose oxidase, horseradish peroxidase), a cyclic peptide (cyclo[Arg-Gly-Asp-D-Phe-Lys], or c(RGDfK)), and an antibiotic (vancomycin). Colorimetric assays confirmed the maintenance of the biocatalytic activities of the grafted enzymes on the surface. We established the mammalian cytocompatibility of the functionalized materials with fibroblasts. Surface functionalization with c(RGDfK) showed improved fibroblast cell adhesion and cytoskeletal organization. Microbiological studies with Staphylococcus aureus indicated that surfaces coated using DOPA-Tet inhibit the formation of biofilms. Vancomycin-grafted surfaces additionally display significant inhibition of planktonic S. aureus growth.

biochemistry↗