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In parallel to the nucleic acid nanotechnology field, scientific attention has been drawn to nucleic acid aptamer development and their implementations. A nucleic acid aptamer consists of RNA and/or DNA oligonucleotides that bind to a specific target moiety (ligand) with high affinity and specificity. A wide range of various chemical and biological entities can serve as ligands ranging from a small molecule to a mammalian cell [47]. The development of aptamer oligonucleotides that bind to specific ligands was engineered through repeated rounds of in vitro selection also known as SELEX (systematic evolution of ligands by exponential enrichment) in the early 1990s in two independent groups of Larry Gold and Jack Szostak [48,49]. Since then, numerous different types of aptamers were engineered in academic research labs and found applications in various biotechnological fields (Figure 5.2a). Several recent reviews provide a critical evaluation of nucleic acid aptamer technology with their applications in vitro and in vivo [50–54].

Figure 5.2 (a) Examples of the diverse application of nucleic acid aptamers. (b) Schematic 2D representation of fluorogenic RNA aptamer YES gated function. (c) Examples of known light‐up RNA 3D structures of MG‐binding aptamer (red), Spinach RNA aptamer (green), and Mango RNA aptamer (gold) with corresponding fluorophore ligands.

Source: (Panel a) From Iliuk et al. [50]. Reproduced with the permission of American Chemical Society.

In this chapter, we are primarily focusing on fluorogenic RNA aptamers, also known as light‐up RNA aptamers. In particular, discussions will cover fluorogenic RNA aptamers as potential candidates to be used in biocomputing applications. Utilizing light‐up aptamers in bioanalytical disciplines for an analyte sensing in vivo has several advantages. For instance, RNA aptamers can be genetically fused to genes coding for cellular RNAs of interest that in turn can be advantageous in endogenous synthesis of the targeting sequence [55–58]. Inserting light‐up aptamers directly into the target endogenous RNA can be restricted by the labor‐intensive genetics. However, these aptamers, even when used in multiple copies, do not significantly increase the size of the cellular RNA. Only the imaging dye needs to be introduced exogenously, and usually small nonpolar organic dyes can be passively diffused into cells. The major concerns of applying unmodified RNA aptamers in vivo are their susceptibility to degradation by nucleases, making it difficult to intracellularly express them. To tackle this problem, aptamer molecules are expressed by being inserted into a complex noncoding RNA strand, like tRNA, or into an RNA junction to protect the aptamer from degradation and induce folding. Such insertion of the RNA aptamer sequence into endogenous RNA not only allows transcription to be monitored in vitro but can also be used to regulate gene expression in a logical manner [59,60].

In a fluorogenic RNA aptamer, the ligand is typically a small organic dye that can emit fluorescent light upon binding to its aptamer host molecule (otherwise, it is in a nonfluorescent state). This feature is very attractive to develop binary ON and OFF systems where a fluorescence state is ON and a nonfluorescence state is OFF as shown in Figure 5.2b. Over the past two decades, several RNA fluorogenic aptamers were developed, and all can be potentially implemented for fabrication of a nanocomplex with a simple YES logic operation with fluorescence outputs ON and OFF. The examples of such RNA aptamers and ligands as well as their properties are summarized in Table 5.1, and structures of the three most common RNA aptamers are illustrated in Figure 5.2c. The interaction between a ligand and a binding pocket of the host aptamer is usually non‐covalent. Therefore, the interaction can be easily reversed by introducing denaturing agents or certain conditions that will disrupt the correct pocket conformation. Such disruption prevents ligand binding and thus serves as input signals. Once the ligands are in an unbound state (free in solution), their intrinsic fluorescence emission is diminished.

Table 5.1 List of some common RNA aptamers with corresponding fluorogenic ligands and properties.

Fluorogen dye	Light‐up aptamer	KD (nM)	Ex/Em (nm)	ℰ (M−1/cm)	Φ a)	Fluorogen structure	PDB ID b)	References
OTB	DiR2s‐Apt	662	380/421	73 000	51		6DB9	[61]
DFHBI	Spinach	540	469/501	24 300	72		4TS0	[62]
DFHBI‐1T	Spinach2	560	482/505	31 000	94		6B3K	[63]
DFHBI‐2T	Spinach2	1300	500/523	29 000	12		6B3K	[63]
TO‐1	Mango	3	510/535	77 500	14		5V3F	[64]
DFHO	Corn	70	505/545	29 000	25		6E80	[65]
DIR	DIR apt	86	600/646	134 000	26		3T0W	[66]
Mal. Green	MG aptamer	117	630/650	150 000	19		1Q8N	[67]
DIR‐pro	DIR2s‐Apt	252	600/658	164 000	33		6D89	[61]
TO‐3	Mango	6–8	637/658	9300	N/A		5V3F	[64]

^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 (SAH) [82], and thiamine pyrophosphate (TPP) [83]. Also, the development of these aptamers has simplified RNA imaging in mammalian, yeast, and bacterial cells [44,84–87].