Nuclease dead Cas9 is a programmable roadblock for DNA replication

Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein-DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9-guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro

Development of Fluorogens for the Detection of Physiological Analytes

The phenomenon of fluorescence has enabled us to gain a deeper understanding of biological structures and processes. Labeling of biomolecules using fluorescence tools such as fluorescent proteins, organic fluorophores, inorganic complexes, and quantum dots allows for these biomolecules to be visualized spatiotemporally for increasing our understanding of their function. To augment our understanding of biological processes there is a growing interest in the detection and quantification of the concentrations of various cellular analytes as they play a key role in the regulation of cellular behavior. Thisthesis focusses on the functionalization of existing fluorescence tools to enable them to detect and quantify various cellular analytes. The fluorescence tool described here is the Fluorogen Activating Protein (FAP) technology as it provides spatiotemporal resolution and ease of labeling both at the cellular and organismal levels. This technology is able to activate the fluorescence of a dark (non-fluorescent) dye molecule upon non-covalent binding to a cognate, targeted protein partner that can be linked to a protein of interest. The fluorogenic dyes can be modified to obtain desired properties, but are limited, therefore a tandem dye approach was initiated. This thesis summarizes the outcomes of the development of a new tandem dye and demonstration of its localization ability and potential in analyte detection. This tandem dye is a coumarin-Malachite Green (MG) conjugate (donor-acceptor), in which the MG binds to a FAP, dL5**, and emits in the farred region where autofluorescence of cells is limited. 7-hydroxycoumarins were coumarins of choice since there is a vast literature to show that the fluorescence of 7-hydroxycoumarin depends on whether it has been capped or is in its phenolate form. As a first step, to demonstrate the ability of 7-hydroxycoumarins to act as donors for MG dL5**, we rationally selected two coumarins and determined that a coumarin containing a hydroxy group in the 7th position and a carboxylic acid in the 3rd position are importantfor any energy transfer to take place in the tandem dye. Further, we concluded that it was essential for the 7-hydroxy group to have a low pKa for the coumarin to be in its phenolate form under physiological conditions. Pacific blue was determined to be a suitable donor and the cell-permeable property of Pacific Blue-Malachite Green (BluR2) tandem dye has been used in conjunction with a cell-excluded tandem dye, HCM, to visualize anddistinguish protein pools inside and outside the cell using different colors. To demonstrate the ability of the 7-hydroxycoumarin-MG tandem dye as a functional probe, a 7- hydroxycoumarin analogous to Pacific Blue was conjugated to MG to enable the detection of pH (hydrogen ion concentration) changes in cells. 6-chloro 7-hydroxycoumarin-3- carboxylic (6ClC) is structurally and spectroscopically similar to Pacific Blue, except thatthe 7-hydroxy group on the former has a lower pKa compared to the latter since it is flanked by one weak-electron withdrawing chlorine (in 6ClC) instead of two strongelectron-withdrawing fluorines (in Pacific Blue). This difference elevated the pKa from 3.7 to 6.3 and the elevated pKa displayed a systematic enhancement in the coumarinexcitation cross-section and the energy transfer between 6ClC to MG-dL5** with an increase in alkalinity of buffers from pH 4.0 to 7.7. This in vitro experiment is significantas this systematic change in fluorescence can be visualized during pH changes in cellular processes like lysosomal trafficking and exocytosis. The availability of a FAP targeted to the lysosomal protein makes this measurement possible, thus making 6ClC-MG (BluRpH) a genetically targeted, far-red, ratiometric pH sensor. To expand the repertoire of analyte detecting tandem dyes with MG-dL5** as an acceptor, we chose to develop new tandem dye probes to detect reactive oxygen species. As a first step, we chose to convert the Cy3-MG tandem dye pair into Cy3[H]MG, as reduced Cy3[H], also known as hydrocyanines. Hydrocyanines are well-characterized superoxide anion sensors and display superior selectivity to superoxide anion compared to existing commercial probes like hydroethidine and mitosox. Also, the modularity of synthesis allowed us tofunctionalize hydrocyanines to label another genetically encodable tag, a Halo tag, by conjugating a suitable linker that can be recognized by the haloalkane dehalogenaseenzyme (Halo tag). In pursuance of our original goal of developing more analyte-detecting tandem dyes using the BluR platform – coumarin-MG tandem dyes, building blocks were developed for the detection of hydrogen peroxides. For this, a 7-hydroxycoumarin-3-ethyl ester was converted into 7-Bpincoumarin-3-ethyl ester, which can then be conjugated to MG. The coumarin will become fluorescent and transfer energy when it detects hydrogen peroxide as it converts Bpin into a hydroxy group. This mechanism is well-established using other fluorophores containing a phenol as part of their structure. In conclusion, this thesis has shown that the FAP platform can be functionalized to detect small molecule analytes in the cell. The combined use of tandem dyes and a genetically targetable tag (FAP) is a wonderful recipe provides many advantages for unravelling the mysteries of biology. <br

Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells

Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics

High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon’s ON-TARGET<i>plus</i> SMARTpool siRNA Kinase library (715 target kinases) with and without 10 μM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment