Spinach aptamer

The need for fluorescently tracking RNA rose as its roles in complex cellular functions has grown to not only include mRNA, rRNA, and tRNA, but also RNAi, siRNA, snoRNA, and lncRNA, among others.

[1][2] Spinach is a synthetically derived RNA aptamer born out of the need for a way of studying the role of RNAs at the cellular level.

Many derivatives of this structure were screened using SELEX, but the chosen fluorophore, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), showed the best selective fluorescence with high quantum yield (0.72) when bound to the RNA sequence 24-2, deemed Spinach.

As the fluorophore of GFP and its derivatives are covalently bound to/a part of the protein, free exchange cannot happen and thus photobleaching results.

An adapted structure, which includes two binding sites, limits fluorescence of the aptamer to (1) the fluorophore and (2) the protein or small molecule.

The SELEX method used for the design of the RNA aptamer Spinach.
The SELEX method.
RNA aptamer Spinach. PDB: 4KZD
Crystal structure of Spinach PDB: 4KZD [1]