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Although genetically encoded light-up RNA aptamers have become promising tools for visualizing and tracking RNAs in living cells, aptamer/ligand pairs that emit in the far-red and near-infrared (NIR) regions are still rare. In this work, we developed a light-up RNA aptamer that binds silicon rhodamines (SiRs). SiRs are photostable, NIR-emitting fluorophores that change their open-closed equilibrium between the non-colored spirolactone and the fluorescent zwitterion in response to their environment. This property is responsible for their high cell permeability and fluorogenic behavior. Aptamers binding to SiR were in vitro selected from a combinatorial RNA library. Sequencing, bioinformatic analysis, truncation and mutational studies revealed a 50-nucleotide minimal aptamer, SiRA, which binds with nanomolar affinity to the target SiR. In addition to silicon rhodamines, SiRA binds structurally related rhodamines and carborhodamines, making it a versatile tool spanning the far-red region of the spectrum. Photophysical characterization showed that SiRA is remarkably resistant to photobleaching and constitutes the brightest far-red light-up aptamer system known to date owing to its favorable features: a fluorescence quantum yield of 0.98 and an extinction coefficient of 86,000 M-1cm-1. Using the SiRA system, we visualized the expression of RNAs in bacteria in no-wash live-cell imaging experiments, and also report stimulated emission depletion (STED) super-resolution microscopy images of aptamer-based, fluorescently labeled mRNA in live cells. This work represents, to our knowledge, the first application of the popular SiR dyes, and of intramolecular spirocyclization as a means of background reduction in the field of aptamer-based RNA imaging. We anticipate a high potential for this novel RNA labeling tool to address biological questions.
This article was published in the following journal.
Name: Journal of the American Chemical Society
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A trace element that constitutes about 27.6% of the earth's crust in the form of SILICON DIOXIDE. It does not occur free in nature. Silicon has the atomic symbol Si, atomic number 14, and atomic weight 28.09.
Inorganic compounds that contain silicon as an integral part of the molecule.
Silicon polymers that contain alternate silicon and oxygen atoms in linear or cyclic molecular structures.
A broad family of synthetic organosiloxane polymers containing a repeating silicon-oxygen backbone with organic side groups attached via carbon-silicon bonds. Depending on their structure, they are classified as liquids, gels, and elastomers. (From Merck Index, 12th ed)
Heterodimeric transcription factors containing a DNA-binding alpha subunits, (CORE BINDING FACTOR ALPHA SUBUNITS), along with a non-DNA-binding beta subunits, CORE BINDING FACTOR BETA SUBUNIT. Core Binding Factor regulates GENETIC TRANSCRIPTION of a variety of GENES involved primarily in CELL DIFFERENTIATION and CELL CYCLE progression.
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. During DNA sequencing, the bases of a small fragment of DNA are sequentially identified from signals emitted as each fragment is re-synthesized from a ...