No abstract

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O− P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wildtype enzyme: catalyzing the formation of internucleotidyl N−P linkages using 3′-NH 2 -ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity. MD simulations provided insights into how a single substitution enhances the productive positioning of a 3′-amino nucleophile in the active site. Further remodeling of the protein−nucleic acid interface in the finger subdomain yielded a quadruple-mutant variant (9°N-NRQS) displaying DNA-dependent NP-DNA polymerase activity. In addition, the engineered promiscuity of 9°N-NRQS was leveraged for one-pot synthesis of DNA�NP-DNA copolymers. This work sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

Peroxynitrite (ONOO−), a redox-active species generated under nitrosative stress conditions, can promote oxidative damage of lipids. Herein, we report the development and multifaceted utilization of a novel, activity-based phospholipid probe, PLP-ONOO–, for imaging lipid environments that are targeted by ONOO− in biomimetic and biological systems. Using PLP-ONOO– and the natural lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, we built synthetic protocell membranes in the form of giant vesicles (GVs). These GVs responded to ONOO− by lighting up and displayed excellent selectivity against other redox-active species, introducing an unprecedented function to biomimetic membrane architectures. Live HeLa cells containing PLP-ONOO– were investigated under cellular nitrosative stress induced by either external administration of peroxynitrite or endogenous stimulation with interferon-γ / lipopolysaccharide / phorbol myristate acetate. Both conditions enhanced the coumarin fluorescence within cells, providing insight into intracellular lipid environments potentially prone to peroxynitrite-mediated oxidative damage. This work offers a chemical reactivity-dependent function for synthetic vesicles and opens a possibility for investigating nitrosative stress at subcellular levels.

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 sugar-phosphate 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°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°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°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.

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.