This week, we hear from Adrien again on a particular RNA based aptamer, known as the Spinach aptamer. Adrien explains how this aptamer works, what makes it unique and the types of applications it can serve.
The most famous example of an aptamer-based biosensor is the Spinach aptamer. This aptamer mimics the Green Fluorescent Protein (GFP), one of the most-used tools in molecular biology because it is possible to target GFP to different parts of the cell, and, using a blue light, obtain visual information.
The target of the Spinach aptamer is DMHBI, a derivative (i.e. a compound made from) of the hydroxybenzlidene imidazolinone (HBI). HBI also happens to be the fluorophore of the Green Fluorescent Protein (GFP). DMHBI is non fluorescent under UV light. When the DMHBI molecule is bound by RNA, its is held very tightly. Normally, the atoms of a molecule shift or rotate along their bond. However, but in the RNA-bound state, DMBHI must release its excess shifting energy as fluorescence. Specifically, the compound emits a light at a wavelength of 529 nm (which is the wavelength corresponding to the color green).
The Spinach aptamer can be coupled to a second aptamer chosen to bind a target molecule of interest. This coupling is what makes the Spinach so uniquely useful: fluorescence is visible only if the second aptamer is bound to the target molecule. Another way to think about this concept is that the Spinach will be bright green only if the binding molecule is in the cell or test tube.
(a). Both DFHBI (green ball) and Spinach are non-fluorescent until binding occurs and activates the fluorescence of the Spinach-DFHBI complex. Stem loop 3 of Spinach can tolerate insertion of additional sequences, and it is the region that is modified to generate sensors. (b) In Spinach-based sensors, Spinach is modified to include a transducer region (magenta) and a recognition module (cyan). (c) In the absence of DFHBI and ligand (orange hexagon), the Spinach-based sensor displays minimal fluorescence. However, upon target binding, the recognition module of the sensor folds and induces folding of the Spinach portion of the sensor. The Spinach-based sensor is then able to bind DFHBI and activate fluorescence. From Strack et al. (2014).
In the last two weeks, we have covered the basic concepts of aptamer binding, SELEX, and the Spinach aptamer. To conclude, aptamers are developed for a lot of purposes, such as making riboswitches, detection of cell metabolites, live tracking of RNA molecules, simultaneous detection of mRNA and protein, etc. The diversity of function which originates from RNA structure is at the heart of aptamer development. It is a field of research both fascinating and full of opportunity to develop a plethora of novel “nano-tools”.