Luminescent platforms for monitoring changes in the solubility of amylin and huntingtin in living cells

2016 ◽  
Vol 12 (10) ◽  
pp. 2984-2987 ◽  
Author(s):  
Jia Zhao ◽  
Quyen Vu ◽  
Cliff I. Stains
Keyword(s):  
Small Molecules ◽  
Living Cells

Cell-based assays for amylin and huntingtin solubility, capable of reporting on the influence of mutations and small molecules, are reported.

scholarly journals Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

2010 ◽  
Vol 107 (5) ◽  
pp. 1870-1875 ◽  
Author(s):  
Alex K. Shalek ◽  
Jacob T. Robinson ◽  
Ethan S. Karp ◽  
Jin Seok Lee ◽  
Dae-Ro Ahn ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Silicon Nanowires ◽  
Mammalian Cells ◽  
Living Cells ◽  
Diverse Range ◽  
Viral Packaging ◽  
Neuronal Progenitor ◽  
Efficient Delivery ◽  
Surface Modified

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell’s membrane and subsequently release surface-bound molecules directly into the cell’s cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology’s potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

A Heavy Metal Tracer Method for Demonstrating Fluid and Ion-Accessible Channels in Epithelia

1981 ◽  
Vol 39 ◽  
pp. 640-641
Author(s):  
J. A. V. Simson ◽  
S. Wilmeth
Keyword(s):  
Heavy Metal ◽  
Small Molecules ◽  
Cell Biology ◽  
Inorganic Ions ◽  
Living Cells ◽  
Ruthenium Red ◽  
Tracer Method ◽  
Tracer Studies ◽  
Permeable Barrier ◽  
Gain Access

The use of enzymes such as horseradish peroxidase or of metal-containing macromolecules such as ferritin and iron dextran have been extremely valuable in tracer studies designed to determine the fate of exogenous macromolecules during endocytosis and in exocytosis/endocytosis coupling during secretion. An important unanswered question in the cell biology of secretion is: How do small molecules and inorganic ions gain access to the lumina of secretory epithelium during secretion? Macro molecular tracers cannot be used to answer this question since they are larger than the pore sizes involved, and most heavy-metal-containing small ionic tracers (such as lanthanum and ruthenium red) have been used only in the presence of fixatives to determine the existence of channels that remain patent in fixed cells and tissues. These tracers have only rarely been used to examine functioning fluid-permeable channels because of their toxicity to living cells (see Hayat, 1975). It is clear, however, that the equilibration of very small ions and molecules across a permeable barrier (e.g., fenestrated capillaries) occurs very rapidly, in a matter of perhaps 2-4 seconds.

scholarly journals Modulating protein activity using tethered ligands with mutually exclusive binding sites

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Alberto Schena ◽  
Rudolf Griss ◽  
Kai Johnsson
Keyword(s):  
Small Molecules ◽  
Binding Sites ◽  
Protein A ◽  
Living Cells ◽  
Protein Activity ◽  
Research Areas ◽  
Specific Effector ◽  
Tethered Ligands ◽  
General Method

Abstract The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand–protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brent Townshend ◽  
Joy S. Xiang ◽  
Gabriel Manzanarez ◽  
Eric J. Hayden ◽  
Christina D. Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

scholarly journals Imaging Small Molecules in Living Cells with a Tiny Tag and Raman Microscopy

2012 ◽  
Vol 52 (1) ◽  
pp. 034-035
Author(s):  
Katsumasa FUJITA ◽  
Mikiko SODEOKA
Keyword(s):  
Small Molecules ◽  
Living Cells ◽  
Raman Microscopy
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