iSpinach is an improved version of Spinach, an aptamer which can bind to fluorescent protein and increase their fluorescent properties after forming a complex.
Generation of iSpinach
RNA aptamers or short oligonucleotide RNA sequences have been isolated which can bind to fluorescent proteins and improve their properties.
One example of such an aptamer is Spinach which can bind to 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), a dye which is commercially available and resembles native GFP. This dye is non-toxic, permeable to cell membranes, has low interaction with native components inside a cell, and low fluorescence when unbound to any compound. Spinach is a DFHBI-binding aptamer which can enhance its fluorescence by 200 times. However, the first Spinach aptamer was thermally unstable and had limited folding efficiency. This led to the development of a newer version of Spinach which was termed iSpinach.
Structure of iSpinach
iSpinach consists of a G-quadraplex structure which has helical structures with guanine tetrads. The G-quadraplex structure can form either one or more strands. The recognition of DFHBI is achieved by a pocket consisting of the G-quartet, a base triple, and an unpaired G residue. As this structure is stabilized by potassium ions, presence or absence of ions can change the fluorescence associated with iSpinach.
Performance of iSpinach
Increased size and fluorescence
iSpinach is 1.3 times smaller than the previous versions of Spinach aptamer (Spinach and Spinach2). This leads to increased permeability inside cells. Also, the fluorescence of iSpinach is 1.4 times higher than its previous aptamer versions. This increased fluorescence is attributed to two main properties: iSpinach has two times higher affinity towards DFHBI, and its folding efficiency has also improved dramatically when compared to previous versions.
The melting point of the complex formed between iSpinach and DFHBI has increased by 8°C, compared to previous Spinach versions. This led to increased thermal stability in iSpinach.
Presence of ions
It is known than potassium can increase the stability of Spinach aptamers, however, iSpinach is more stable in the presence of other ions, such as lithium, cesium, and sodium. Also, sodium increases the fluorescence of the iSpinach by two times.
The folding efficiency of iSpinach is 60%, which is higher than Spinach and Spinach2. The increased folding efficiency also leads to increased binding with DFHBI.
Applications of iSpinach
The improved fluorescence and folding properties of iSpinach could make it a valuable tool as a fluorescent probe inside cells. iSpinach has been used to visualised ribozymes, and it was found that iSpinach is a better contender than Spinach or other DFHBI-binding proteins. It could measure the ribozyme activity without altering the activity levels. There was no fluorescence observed when Spinach2 was used. Thus, iSpinach can perform even in vivo conditions and maintains the activity of the enzyme.
The improved folding and florescence properties make it a good candidate as a biosensor.
Probes for ionic microenvironment
Several probes and previous Spinach aptamers only fluoresce in high potassium conditions. However, iSpinach can work even in cellular ionic levels and this has been tested experimentally.
Limitations of iSpinach
One of the main limitations of iSpinach is that its affinity towards DFHBI can be improved. One of the ways to resolve this is to have more stringent selection criteria for binding when iSpinach aptamers are isolated. iSpinach can also dissociate in the presence of light, therefore using stronger light source can help in identifying mutant with less light-dependent dissociation rates.