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. 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. This aptamer was created using Systematic Evolution for Ligands by EXponential enrichment, or SELEX, which is also known as in vitro evolution.

The aptamer was designed to be an RNA mimic of green fluorescent protein (GFP); similar to GFP for proteins, Spinach can be used for the fluorescently labeling RNA and tracking it in vivo. A method for inserting the Spinach sequence after an RNA sequence of interest is readily available.

GFP’s fluorophore is made up of three cyclized amino acids within the beta-barrel structure: Serine65-Tyrosine66-Glycine67. This structure, 4-hydroxybenzlidene imidazolinone (HBI) was the basis for the synthetic analogue used in the SELEX studies. 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.