Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities.

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.

Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine.

The ability to detect and visualize cellular events and their associated target biological analytes through use of cell-permeable profluorogenic probes is dependent on the availability of activatable probes that respond rapidly and selectively to target analytes by production of fluorescent reporting molecules whose excitation and emission energies span a broad range. Herein is described a new probe, DCM-Cys, that preferentially reacts with cysteine to form a dicyanomethylene-4H-pyran (DCM) reporter whose red-energy fluorescence can be stimulated by two-photon, near-infrared excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-to-background ratio. These aforementioned characteristics and the ability of DCM-Cys to provide selective, nanomolar-level in vitro cysteine detection, as demonstrated by its lack of significant response to other thiols and potential interfering agents from biological environments, are attributed to the molecular designs of the DCM-Cys probe and DCM reporter. Attachment of an acryl moiety to the DCM reporter via a self-eliminating, electron-withdrawing benzyl alcohol-carbamate linker offers a probe having selective, sensitive reaction with cysteine to rapidly produce a reporter whose energies of excitation and emission (λabsreport = 480 nm, λemisreport = 640 nm) are red-shifted from those of the DCM-Cys probe (λabsprobe = 440 nm, λemisprobe = 550 nm), thereby leading to low background signal from abundant probe and a large signal from the resulting reporter of cysteine presence.

Rapid, photoinduced electron transfer-modulated, turn-on fluorescent probe for detection and cellular imaging of biologically significant thiols.

There is a very limited number of existing probes whose fluorescence is turned on in the presence of the class of biological thiols made up of glutathione, cysteine, and homocysteine. The extant probes for this class of biological thiols commonly have poor aqueous solubility and long analyte response times, and they demand a very high probe/thiol ratio for decreased time of significant reporter signal generation; knowledge regarding their selectivity with respect to other sulfur-based analytes is unclear. Described here is a previously unreported photoinduced electron-transfer-quenched probe (HMBQ-Nap 1) that offers highly selective and rapid in vitro detection of this class of biologically important thiols at low concentrations and low probe/thiol ratio, and importantly, very rapid imaging of these biological thiols in human cells.