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.
TheoryThe 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). 2]. 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. An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection. 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. 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 1]. 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, this phenolate 1].
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. 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). 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.
- 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.
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.
Error fetching PMID 1697402:
Error fetching PMID 9759496:
Error fetching PMID 7854443:
Error fetching PMID 19771336:
Error fetching PMID 22403384:
- Error fetching PMID 21798953:
The spinach paper
- Error fetching PMID 19771336:
- Error fetching PMID 1697402:
The original paper describing aptamers
- Error fetching PMID 9759496:
A GFP review
- Error fetching PMID 7854443:
- Error fetching PMID 22403384:
The spinach-SAM detection aptamer