CH391L/S14/Spinach RNA

Spinach is an RNA aptamer which binds to the small molecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), which is structurally similar to the fluorophore in green fluorescent protein (GFP), hydroxybenzylidene imidazolinone (HBI). Upon binding, the aptamer-small molecule complex becomes highly fluorescent, similar to GFP fluorescence. A variety of HBI derivatives with corresponding aptamers 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.

Theory
The natural fluorophore of 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, but becomes fluorescent when it is vibration restricted Meech2009. Side chain contacts made with HBI in GFP prevent intramolecular motions, which restrict the energy dissipation pathways available for HBI after excitation by a photon, this restriction leaves fluorescence as the most readily available and common pathway for relaxation. 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 Jaffrey2011. An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection Ellington1990. The group suspected that a modular, fluorescent RNA aptamer would facilitate 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 cellular conditions Jaffrey2011. No significant fluorescence was detected from the HBI derivatives, and they proceeded by selecting for aptamers against the HBI derivative 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) using SELEX (systematic evolution of ligands by exponential enrichment). After five rounds of selection, an increase in fluorescence was detected, and each subsequent round of selection saw an increase in fluorescence, up to the tenth round of selection. Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2. The Jaffrey lab then used truncations to determine a minimal aptamer sequence, which resulted in an increase in quantum yield of fluorescence. The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation 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 Jaffrey2011.

The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP. The EGFP fluorophore is most often in the phenolate form while wild type GFP is most commonly in the phonol form Tsien1998  Heim1995, this phenolate from is suspected to be the reason for EGFPs significant improvement in fluorescence. Following that logic, the 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. 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” Jaffrey2011.

Uses
Spinach is useful as a modular appendage to RNA transcripts in the cell. The addition of the 60 bp spinach aptamer to the 5' or 3' 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 Jaffrey2011. 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.

Jaffrey was also able to develop a spinach variant which is capable of detecting a second molecule, specifically S-Adenosyl-Methionine (SAM) Jaffrey2012. The aptamer binds to both DFHBI and SAM, and only when bound to both small molecules does the complex fluoresce. This technology could be expanded to a variety of small molecules, and used as a sensitive detection method.

Comparitive technologies

 * FISH - Fluorescent in situ hybridization is a technique used to probe for the presence of a DNA or RNA sequence. A small oligonucleotide primer is synthesized with a tag for an antibody.  The primer is added to a sample of cell and hybridizes with RNA or DNA.  An antibody or marker protein is then added to the cell, which binds to the tag and can be marked with a fluorophore, allowing visualization.  RNA FISH involves RNA-RNA hybridization, which often signals for degradation.

iGEM
In 2012 the 2012 Carnegie Mellon iGEM team used spinach and fluorogen activating protein (FAP) together to monitor transcription and translation rates simultaneously. They have used this to evaluate T7-lac promoters. The team has expressed interested in expanding this to evaluate other parts, including RBS and other promoters. Additionally, they hope this technology will be used to study metabolic burden, gene regulation, and synthetic circuits.