Difference between revisions of "CH391L/S14/Spinach RNA"

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===Comparitive technologies===
 
===Comparitive technologies===
 
FISH/RISH
 
FISH/RISH
 +
 
Malachite green
 
Malachite green

Revision as of 13:19, 3 March 2014

Spinach is an RNA aptamer which binds to the small molecule 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI), which is structurally similar to the fluorophore in GFP. Upon binding, the aptamer-small molecule complex becomes highly fluorescent, similar to GFP fluorescence. A variety of small molecules have been developed which fluoresce upon binding and provide a range of excitation and emission wavelengths. Spinach allows for high resolution monitoring of mRNA transcripts, providing information on transcription rates, and localization of mRNAs in vivo. Spinach was originally reported in Science Magazine in July 2011, and has since been cited by 120 articles.

Contents

Theory

The natural fluorophore of Green Fluorescent Protein (GFP) is formed through the auto-cyclization of three adjacent amino acids, Ser65, Tyr66, and Gly67, which react under oxidizing conditions to form hydroxybenzlidene imidazolinone (HBI). HBI itself is not significantly fluorescent; contacts made in GFP prevent intramolecular motions, which restrict energy dissipation pathways, leaving fluorescence as the most common. It was hypothesized by the Jaffrey lab that restricting motions of HBI using an RNA aptamer instead of proteinogenic amino acids could result in a similar fluorescence to GFP. The group suspected that a modular, fluorescent RNA aptamer would facilities RNA studies and RNA technologies, similar to the effect GFP has had on protein biochemistry and biotechnology.

Development and Physical Properties

The group synthesized HBI derivatives and tested their hypothesis, first by exploring the fluorescence of HBI derivative in vivo, to ensure that HBI was not fluorescent under normal conditions in a cell. No significant fluorescence was detected in the HBI derivatives, and they proceeded with selected for aptamers against the derivatives using SELEX (systematic evolution of ligands by exponential enrichment). After five rounds of selection, an increase in fluorescence was detected, and subsequent rounds of selection saw increases in fluorescence up to round ten. Individual sequences were screened for aptamers that accounted for the fluorescence, and the aptamer which gave the highest fluorescence was identified and named 13-2. They used truncations to determine a minimal aptamer sequence, which resulted in an increase in quantum yield. The aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an ex citation peak of 398 nm. Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate. Similar to work on GFP which has enhanced its quantum yield and tuned its excitation wavelengths, Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP, as opposed to the phenol form of HBI seen in wildtype GFP (wtGFP). The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI). Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence. The resulting complex shows 53% of the molar brightness of EGFP. This aptamer, 24-2, was given the name “spinach”.

Uses

Spinach is useful as a modular appendage to native RNA transcripts in the cell. The addition of the 60 bp nucleotide to the end of coding and noncoding transcripts in cells has been shown to allow for monitoring of RNA localization in vivo. The Jaffrey lab demonstrated this in the orginal spinach paper by appending spinach to the 3’ end of 5S ribosomal RNA. By growing human cells with this modified gene in the presence of DFHBI, similar fluorescent patterns were seen as other experiments who had monitored 5S localization. Additionally, when placed under stress, the cell responded by forming RNA granules colocalized with T-cell intracellular antigen-1-related protein, as was expected of the stress conditions.

Comparitive technologies

FISH/RISH

Malachite green