a Φ referred to quantum yield of the complex expressed in percentage.
b Protein Data Bank ID number.
A highly effective RNA‐based fluorogenic unit should possess specific features. The ideal dye needs to display a high absorption coefficient (ɛ) to ensure sensitive detection and to minimize fluorescence background. The fluorophore should show a low ratio of photons absorbed to photons emitted (quantum yield), meaning it should have a high fluorescence enhancement and brightness. The RNA–fluorophore interaction should be highly specific and occur with high affinity to make it possible to use low concentrations while still obtaining high contrast and keeping background fluorescence low. The aptamer–fluorophore complex also needs to be photostable to extend the ability for data acquisition. Often, these types of fluorogenic aptamers have superior characteristics over the electrochemical and colorimetric approach for sensing and imaging. For example, RNA strands do not require chemical conjugation, and the RNA aptamer provides high sensitivity and high speed of response while also exhibiting high spatial resolution [68,69].
Malachite green (MG)‐binding RNA aptamer was one of the earliest models of RNA light‐up aptamer and was extensively studied in laboratory settings [38–40,43,46,67,70,71]. Excitation of free triphenylmethane fluorophore (MG) in a solution results in low fluorescence due to easy vibrational de‐excitation (i.e. excess energy from the MG excited state is dissipated in the form of vibrational movement). When MG is bound to its RNA aptamer, MG is stabilized in a planar form, and vibrations are restricted, which results in a 2000‐fold increase of fluorescence [67]. In nucleic acid nanofabrication, fluorescent aptamers can potentially act as fluorescent reporter units that can be harbored into a larger complex nanoparticle by simple extension of individual strands. Several previous reports have shown that MG‐binding aptamers can be used for co‐transcriptional assembly verification [43] as well as monitoring of the dynamic behavior of interdependent RNA–DNA hybrids [35].
More recently, a novel and much less intracellular toxic RNA aptamer, as compared with MG RNA aptamer, Spinach RNA aptamer, was developed [62]. The Spinach RNA aptamer binds the green fluorescent protein (GFP) fluorophore analog DFHBI ((Z)‐4‐(3,5‐difluoro‐4‐hydroxybenzylidene)‐1,2‐dimethyl‐1H‐imidazol‐5(4H)‐one) [72]. The work on the Spinach aptamer has been extended to produce Spinach 2 that has much greater thermostability and brightness. However, Spinach 2 is more susceptible to degradation by nucleases [63,73]. Further effort has been made to develop yet another “vegetable” aptamer called Broccoli that binds DFHBI‐1T (derivative of the DFHBI dye) [74].
The interaction between the aptamer and its target often causes slight structural rearrangement in favor of stabilization of the RNA–ligand complex. This feature can be used to control RNA–ligand binding allosterically, where the allosteric site (sensing module) can be connected to the aptamer region (reporting module) through a communication module. This strategy was developed by Ronald Breaker using ribozymes as reporting modules [75,76], and later a similar strategy was implemented using MG‐binding RNA aptamer as a reporter unit [77,78]. Allosteric biosensors can also be used for protein detection for specific applications [79]. With these biosensors, target metabolite molecules as well as enzymes participating in an intracellular pathway can be identified. For instance, RNA biosensors are now commonly used to sense the presence of the following metabolites: cyclic AMP [80], cyclic di‐AMP [73], S‐adenosylmethionine (SAM) [81], FMN [78], S‐adenosyl‐L‐homocysteine