Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.
Nucleic acid aptamers can be selected from pools of random-sequence oligonucleotides to bind a wide range of biomedically relevant proteins with affinities and specificities that are comparable to antibodies. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry. Despite these properties, aptamers have been slow to reach the marketplace, with only one aptamer-based drug receiving approval so far. A series of aptamers currently in development may change how nucleic acid therapeutics are perceived. It is likely that in the future, aptamers will increasingly find use in concert with other therapeutic molecules and modalities.
Aptamers are ligand-binding nucleic acids whose affinities and selectivities can rival those of antibodies. They have been adapted to analytical applications not only as alternatives to antibodies, but as unique reagents in their own right. In particular, aptamers can be readily site-specifically modified during chemical or enzymatic synthesis to incorporate particular reporters, linkers, or other moieties. Also, aptamer secondary structures can be engineered to undergo analyte-dependent conformational changes, which, in concert with the ability to specifically place chemical agents, opens up a wealth of possible signal transduction schemas, irrespective of whether the detection modality is optical, electrochemical, or mass based. Finally, because aptamers are nucleic acids, they are readily adapted to sequence- (and hence signal-) amplification methods. However, application of aptamers without a basic knowledge of their biochemistry or technical requirements can cause serious analytical difficulties.
We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to 'print' complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.
Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. When Ϸ800 yeast strains expressing GFP-tagged proteins were grown to stationary phase, a surprising number of proteins involved in intermediary metabolism and stress response were observed to form punctate cytoplasmic foci. The formation of these discrete physical structures was confirmed by immunofluorescence and mass spectrometry of untagged proteins. The purine biosynthetic enzyme Ade4-GFP formed foci in the absence of adenine, and cycling between punctate and diffuse phenotypes could be controlled by adenine subtraction and addition. Similarly, glutamine synthetase (Gln1-GFP) foci cycled reversibly in the absence and presence of glucose. The structures were neither targeted for vacuolar or autophagosome degradation nor colocalized with P bodies or major organelles. Thus, upon nutrient depletion we observe widespread protein assemblies displaying nutrient-specific formation and dissolution.aggregation ͉ metabolism ͉ microscopy ͉ proteomics ͉ quiescence
Signal amplification is a key component of molecular detection. Enzyme-free signal amplification is especially appealing for the development of low-cost, point-of-care diagnostics. It has been previously shown that enzyme-free DNA circuits with signal-amplification capacity can be designed using a mechanism called ‘catalyzed hairpin assembly’. However, it is unclear whether the efficiency and modularity of such circuits is suitable for multiple analytical applications. We have therefore designed and characterized a simplified DNA circuit based on catalyzed hairpin assembly, and applied it to multiple different analytical formats, including fluorescent, colorimetric, and electrochemical and signaling. By optimizing the design of previous hairpin-based catalytic assemblies we found that our circuit has almost zero background and a high catalytic efficiency, with a kcat value above 1 min−1. The inherent modularity of the circuit allowed us to readily adapt our circuit to detect both RNA and small molecule analytes. Overall, these data demonstrate that catalyzed hairpin assembly is suitable for analyte detection and signal amplification in a ‘plug-and-play’ fashion.
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