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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]
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