Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge.
Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells.
Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin-polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.nanopore sequencing | protein design | polymer-tagged nucleotides | single-molecule detection | integrated electrode array D NA sequencing is a fundamental technology in the biological and medical sciences (1). Advances in sequencing technology have enabled the growth of interest in individualized medicine with the hope of better treating human disease. The cost of genome sequencing has dropped by five orders of magnitude over the last decade but still remains out of reach as a conventional clinical tool (2, 3). Thus, the development of new, high-throughput, accurate, low-cost DNA-sequencing technologies is a high priority. Ensemble sequencing-by-synthesis (SBS) platforms dominate the current landscape. During SBS, a DNA polymerase binds and incorporates a nucleotide analog complementary to the template strand. Depending on the instrumentation, this nucleotide is identified either by its associated label or the appearance of a chemical by-product upon incorporation (4). These platforms take advantage of a high-fidelity polymerase reaction but require amplification and have limited read lengths (5). Recently, single-molecule strategies have been shown to have great potential to achieve long read lengths, which is critical for highly scalable and reliable genomic analysis (6-9). Pacific Biosciences' SMRT SBS approach has been used for this purpose but has lower throughput and higher cost compared with current secondgeneration technology (10).Since the first demonstration of single-molecule characterization by a biological nanopore two decades ago (11), interest has grown in using nanopores as sensors for DNA base discrimination. One appro...
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