Benzylic Trifluoromethyl Accelerates 1,6-Elimination Toward Rapid Probe Activation.

Activity-based detection of hydrogen sulfide in live cells can expand our understanding of its reactivity and complex physiological effects. We have discovered a highly efficient method for fluorescent probe activation, which is driven by H2S-triggered 1,6-elimination of an α-CF3-benzyl to release resorufin. In detecting intracellular H2S, 4-azido-(α-CF3)-benzyl resorufin offers significantly faster signal generation and improved sensitivity compared to 4-azidobenzyl resorufin. Computed free energy profiles for the 1,6-elimination process support the hypothesis that a benzylic CF3 group can reduce the activation energy barrier toward probe activation. This novel probe design allows for near-real-time detection of H2S in HeLa cells under stimulation conditions.

Direct assessment of nitrative stress in lipid environments: Applications of a designer lipid-based biosensor for peroxynitrite.

Lipid membranes and lipid-rich organelles are targets of peroxynitrite (ONOO-), a highly reactive species generated under nitrative stress. We report a membrane-localized phospholipid (DPPC-TC-ONOO-) that allows the detection of ONOO- in diverse lipid environments: biomimetic vesicles, mammalian cell compartments, and within the lung lining. DPPC-TC-ONOO- and POPC self-assemble to membrane vesicles that fluorogenically and selectively respond to ONOO-. DPPC-TC-ONOO-, delivered through lipid nanoparticles, allowed for ONOO- detection in the endoplasmic reticulum upon cytokine-induced nitrative stress in live mammalian cells. It also responded to ONOO- within lung tissue murine models upon acute lung injury. We observed nitrative stress around bronchioles in precision cut lung slices exposed to nitrogen mustard and in pulmonary macrophages following intratracheal bleomycin challenge. Results showed that DPPC-TC-ONOO- functions specifically toward iNOS, a key enzyme modulating nitrative stress, and offers significant advantages over its hydrophilic analog in terms of localization and signal generation.

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, and 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 morphology 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.

A Small-Molecule Approach to Bypass In Vitro Selection of New Aptamers: Designer Pre-Ligands Turn Baby Spinach into Sensors for Reactive Inorganic Targets.

Fluorescent light-up aptamer (FLAP) systems are promising biosensing platforms that can be genetically encoded. Here, we describe how a single FLAP that works with specific organic ligands can detect multiple, structurally unique, non-fluorogenic, and reactive inorganic targets. We developed 4-O-functionalized benzylidene imidazolinones as pre-ligands with suppressed fluorescent binding interactions with the RNA aptamer Baby Spinach. Inorganic targets, hydrogen sulfide (H2S) or hydrogen peroxide (H2O2), can specifically convert these pre-ligands into the native benzylidene imidazolinones, and thus be detected with Baby Spinach. Adaptation of this approach to live cells opened a new opportunity for top-down construction of whole-cell sensors: Escherichia coli transformed with a Baby Spinach-encoding plasmid and incubated with pre-ligands generated fluorescence in response to exogenous H2S or H2O2. Our approach eliminates the requirement of in vitro selection of a new aptamer sequence for molecular target detection, allows for the detection of short-lived targets, thereby advancing FLAP systems beyond their current capabilities. Leveraging the functional group reactivity of small molecules can lead to cell-based sensors for inorganic molecular targets, exploiting a new synergism between synthetic organic chemistry and synthetic biology.

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, 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 wild-type enzyme: catalyzing the formation of internucleotidyl N-P linkages using 3'-NH2-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.

MicroRNA detection in biologically relevant media using a split aptamer platform.

MicroRNA (miRNA)-based intercellular communication has been implicated in many functional and dysfunctional biological processes. This has raised interest in the potential use of miRNAs as biomarkers for diagnosis and prognosis. Though the list of clinically significant miRNA biomarkers is expanding, it remains challenging to adapt current chemical tools to investigate miRNAs in complex environments native to cells and tissues. We describe here a methodology for rapidly developing aptamer-based fluorescent biosensors that can specifically detect miRNAs in biologically relevant media (10-30% v/v), including medium collected from cultured HeLa cells, human serum, and human plasma. This methodology involves the semi-rational design of the hybridization between DNA oligonucleotides and the miRNA target to build a pool of potential aptamers, and the screening of this pool for high signal-to-background ratio and target specificity. The DNA oligonucleotides are readily available and require no chemical modification, rendering these chemical tools highly adaptable to any novel and niche miRNA target. Following this approach, we developed sensors that detect distinct oncogenic 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 provides a systematic approach toward the development of miRNA biosensors that are easily accessible and can perform in biological environments with minimal sample handling.

Surgically induced astigmatism after laser in situ keratomileusis for spherical myopia.

PURPOSE: To study risk factors for surgically induced astigmatism (SIA) after laser in situ keratomileusis (LASIK). METHODS: In a retrospective case control study of 104 eyes (52 patients) that underwent LASIK for myopia (spherical ablation alone), two groups were studied: 42/104 eyes with SIA, and controls (62/104 eyes). The main variables studied were preoperative refraction, corneal thickness, preoperative keratometric power, amount of ablation, ablation zone diameter, flap thickness, flap size, and the presence of complications. The effect of SIA on visual performance was also evaluated. RESULTS: The mean myopia for which LASIK was undertaken was -4.50 +/- 2.04 D. Mean scalar astigmatism induced was 0.35 +/- 0.50 D at 1 month, 0.33 +/- 0.40 D at 3 months, and 0.16 +/- 0.60 D at 6 months. SIA based on refractive cylinder was 0.66 +/- 0.29 D at 1 month, 0.54 +/- 0.32 D at 3 months, and 0.49 +/- 0.34 D at 6 months. Mean axis of vector induced astigmatism was 82.5 degrees +/- 57 degrees at 1 month, 98.86 degrees +/- 52.4 degrees at 3 months, and 113.9 degrees +/- 62.6 degrees at 6 months. Risk factors associated with the occurrence of SIA were preoperative keratometric power of >44 D [OR (95% CI); 1.97 (0.62 to 6.26)], ablation zone diameter of <6 mm [OR (95 % CI) 2.76; (0.6 to 12.6)], and suction ring diameter of 8.5 mm [OR (95% CI) 12.46; (2.0 to 77.38)]. The occurrence of SIA had no significant effect on uncorrected Snellen high contrast visual acuity, contrast sensitivity, and glare in comparison with controls. CONCLUSION: Surgically induced astigmatism was more likely to occur with the use of smaller suction rings of 8.5 mm and in ablation zones less than 6 mm. Parameters for visual performance were not affected by the presence of surgically induced astigmatism.