RNA Aptamers and Epitranscriptomics: Charting Unexplored Territories in RNA Biology

1. Introduction

In recent years, RNA biology has undergone a remarkable transformation, deriven by discoveries that have expanded our understanding of RNA beyond its traditional roles in coding, decoding, and catalysis. Two areas at the forefront of this shift, including RNA aptamer technology [1,2,3,4] and epitranscriptomics [5,6,7], have independently opened new avenues for molecular control, therapeutic development, and systems-level regulation of gene expression. However, despite their rapid advancement, these domains have largely evolved in parallel, with limited investigation into how they might conceptually or mechanistically intersect.

RNA aptamers, known as synthetic, short, and structured RNA oligonucleotide molecules selected through in vitro evolution techniques such as Systematic Evolution of Ligands by EXponential enrichment (SELEX), have been extensively utilized for their high affinity and specificity toward a broad range of molecular targets [3,8]. Their application in diagnostics [1], molecular imaging [9,10], targeted therapy [11,12], and biosensing [13,14] has been well documented. By adopting complex three-dimensional conformations, aptamers are capable of mimicking antibody-like behavior, yet retain the versatility and programmability inherent to nucleic acids [15].

Over the past decades, SELEX has been refined into several advanced variants, such as Cell-SELEX, Toggle-SELEX, High-Throughput SELEX (HT-SELEX), and Microfluidic SELEX, which is designed to improve specificity, speed, or physiological relevance of selection process [16]. These innovative approaches have enabled aptamer discovery for challenging and complex targets, including whole cells, small molecules, and membrane proteins, and facilitated integration with next-generation sequencing (NGS) and computational modeling [17,18,19]. Furthermore, novel non-SELEX selection methods, such as in vivo functional selection, which uses riboswitch-based reporters or survival assays, and machine learning-guided in silico aptamer prediction, are also emerging as promising tools for the rapid discovery and rational design of functional RNA binders [20,21]. These approaches offer alternatives to classical selection methods, particularly in contexts where functional output of the aptamers is prioritized. It is worth noting that RNA aptamer functions are critically dependent on RNA folding dynamics, secondary structure stability, and sequence-specific interactions, all of which are properties now known to be influenced by chemical modifications within the RNA itself [22]. In parallel, the field of epitranscriptomics has revealed that RNA molecules are subject to extensive and reversible chemical modifications that can impact their structure, localization, stability, and translational efficiency [7,23,24,25]. Numerous types of RNA modifications have been identified to date [26,27], and importantly, these modifications have been shown to influence RNA structure and interactions in both predictable and context-dependent ways [7,28,29].

Despite their structural impact, epitranscriptomic marks have received little attention regarding their effects on synthetic RNAs like aptamers. Conversely, the idea that aptamers could be designed to recognize specific RNA modifications or modulate modification-dependent functions remains underexplored. This gap presents an opportunity to integrate aptamer design with the chemical logic of the epitranscriptome to gain mechanistic insights and develop novel RNA tools (see Figure 1 and Figure 2). This perspective does not claim established links between aptamers and epitranscriptomics but highlights the theoretical basis and emerging evidence for their convergence. By examining how modifications influence aptamer function, or how aptamers might detect such states, new research directions may emerge. Framing this intersection aims to stimulate collaboration and uncover regulatory links and functional synergies that remain largely unexplored.

2. RNA Aptamers: Synthetic Precision in Targeting and Sensing

RNA aptamers have emerged as one of the most versatile synthetic tools in molecular biology and biotechnology [30,31]. They are typically generated through SELEX, the most widely used in vitro selection method. SELEX involves repeated rounds of binding, partitioning (which separates sequences bound to the target from unbound sequences), elution, and amplification to isolate high-affinity aptamers from large, randomized RNA libraries. During each SELEX cycle, sequences that bind to a specific molecular target are separated from non-binders, then reverse-transcribed, amplified, and transcribed back into RNA, progressively enriching the pool for functional aptamer candidates [32]. These short, single-stranded RNAs are capable of folding into well-defined three-dimensional structures that enable them to bind their targets with high specificity and affinity [33]. A wide array of molecules, including proteins, small metabolites, ions, and even other nucleic acids, can be targeted using aptamers, making them powerful agents for molecular recognition, sensing, and therapeutic modulation [34].

Central to their utility is the structural adaptability of RNA. Aptamers function by adopting complex secondary and tertiary structures, such as pseudoknots, stem-loops, and G-quadruplexes, which are essential for creating binding pockets or interaction surfaces that mimic those of natural receptors. The specificity of binding is not merely a function of nucleotide sequence, but also of precise spatial orientation enabled by the conformational plasticity of the RNA backbone [35,36]. Consequently, any factor that modulates RNA structure, either locally (e.g. nucleotide modifications) or globally (e.g. ionic conditions, folding motifs), can influence aptamer performance [34,37]. Recent advancements have enabled the design of aptamers with enhanced stability and function, including the use of chemical modifications (e.g., 2'-fluoro, 2'-O-methyl substitutions) to improve resistance to nucleases or to fine-tune binding characteristics [38,39]. While these modifications are typically introduced artificially during in vitro synthesis, there is growing interest in understanding how naturally occurring RNA modifications, such as those studied in epitranscriptomics, might influence aptamer behavior, particularly in endogenous or in vivo contexts.

Applications of aptamers have been diverse, ranging from diagnostic biosensors for infectious pathogens and cancer biomarkers to targeted therapeutics that inhibit specific protein-protein interactions or deliver drug payloads [33,40,41]. Aptamers have also been engineered into synthetic circuits for gene expression control and RNA imaging platforms, highlighting their modularity [42,43]. Despite this progress, aptamer design has largely proceeded independently of insights gained through epitranscriptomic research, a disconnect that may obscure subtle but impactful interactions between synthetic RNAs and the chemical environment of the transcriptome [44,45]. Given that RNA aptamers are, ultimately, RNA molecules functioning within complex cellular environments, a deeper understanding of how naturally occurring RNA modifications alter their folding and interaction dynamics may provide crucial insights. This could become particularly relevant as aptamers are increasingly used in cellulo and in vivo diagnostic, and therapeutic applications (theranostics), where they are exposed to endogenous RNA-modifying enzymes and RNA-binding proteins [8,46]. This potential intersection calls for closer investigation into the compatibility between aptamer function and the modified cellular transcriptome.

3. Epitranscriptomics: Decoding the Chemical Language of RNA Modifications

Epitranscriptomics refers to the study of biochemical modifications on RNA molecules that alter their fate and function without changing the underlying sequence [7,23,24,25]. To date, over 170 modifications have been identified, and they are now recognized as key regulators of post-transcriptional gene expression, affecting processes such as splicing, translation, localization, degradation, and structural dynamics [26,27]. Among the most studied modifications are N⁶-methyladenosine (m⁶A), 5-methylcytosine (m⁵C), pseudouridine (Ψ), and N¹-methyladenosine (m¹A), each of which can be deposited, interpreted, or removed by dedicated enzymatic machineries termed writers, readers, and erasers [6]. The dynamic and reversible nature of these modifications enables cell to fine-tune gene expression in response to developmental cues, environmental stressors, or disease states [47,48,49,50,51,52,53,54,55]. For example, m⁶A has been implicated in the regulation of mRNA stability and translation efficiency, often through interactions with reader proteins such as YTHDF1-3 [56]. Likewise, Ψ has been shown to enhance base-pairing stability and promote translational fidelity [57,58], while m⁵C can influence RNA export and transcript stability [59]. These modifications can reshape both local and global RNA structure, sometimes inducing conformational switches that affect binding by proteins or ribonucleoprotein complexes [60,61]. The structural consequences of RNA modifications are of particular relevance to both natural and synthetic RNAs. Modifications can either stabilize or destabilize secondary structure motifs depending on their chemical nature and position within the transcript [62]. For instance, m⁶A is known to weaken A–U base pairs and destabilize local duplexes, facilitating the formation of single-stranded regions that may be recognized by RNA-binding proteins or non-coding RNAs [60,63,64]. This structural flexibility raises compelling questions about how modifications might similarly affect the folding and function of synthetic RNA tools such as aptamers.

Technologies for mapping RNA modifications have rapidly evolved, ranging from antibody-based immunoprecipitation methods (e.g., m⁶A-seq) to direct detection using nanopore sequencing or chemical labeling approaches [65,66]. However, functional studies on how these modifications modulate interactions with structured RNAs, such as aptamers, remain relatively scarce. It remains unclear, for example, whether endogenous modification patterns are sufficiently conserved or dynamic to impact aptamer-based assays or therapeutics deployed in vivo [67,68]. Furthermore, the cellular context in which aptamers function often includes active RNA-modifying enzymes that could potentially install modifications onto synthetic RNAs, especially when they are delivered exogenously or transcribed intracellularly [69,70]. The interplay between such modifications and aptamer behavior, whether enhancing, impairing, or modulating their function, has yet to be systematically explored. Thus, the epitranscriptome is not merely a background feature of the cellular environment but may serve as a regulatory layer with implications for any RNA-based technology [44]. A more integrated understanding of how this chemical language intersects with engineered RNAs like aptamers could illuminate novel dimensions of RNA control and inspire innovative approaches in biotechnology and therapeutics.

4. Structural Plasticity and Modification Sensitivity: A Conceptual Bridge

The structural adaptability of RNA molecules is essential to their diverse functions [71]; RNA aptamers, in particular, rely on stable and predictable folding to exert their binding capabilities. These structures are determined not only by base-pairing rules but also by the conformational preferences encoded in the nucleotide sequence [39]. Increasingly, it has become evident that chemical modifications, such as those observed in the epitranscriptomic landscape, can influence RNA structure in ways that may be relevant to both endogenous and synthetic RNAs, including aptamers [70].

Post-transcriptional modifications often affect local folding by altering hydrogen-bonding potential, base stacking, or backbone flexibility [72,73,74]. For instance, N⁶-methyladenosine (m⁶A) tends to destabilize RNA duplexes and promote single-stranded configurations; this can lead to shifts in the secondary structure that modulate access to binding sites or regulatory motifs [60,63,75]. Pseudouridine, by contrast, increases the rigidity of local regions; its ability to form additional hydrogen bonds can stabilize helices and influence long-range interactions [57,76,77]. These structural effects can propagate through an RNA molecule, creating subtle but functionally significant changes in tertiary architecture [77,78].

In the context of RNA aptamers, even minor structural disturbances can disrupt binding affinity or specificity. Typically, an aptamer’s function depends on the precise orientation of nucleotides within a folded domain. This folding creates complex three-dimensional structures formed by base-paired secondary elements (such as loops, stems, bulges, hairpins, and G-quadruplexes) [79], that enable highly specific molecular recognition through non-covalent interactions including hydrophobic and van der Waals forces, hydrogen bonding, electrostatic interactions, base stacking, and shape complementarity [80]. Thus, if a naturally occurring modification alters that geometry, either directly or allosterically, the aptamer’s ability to recognize its target could be compromised or, under certain conditions, enhanced [35]. Such a scenario suggests that epitranscriptomic marks could act as silent regulators of aptamer behavior, particularly in cellular environments where endogenous modifying enzymes are active. This idea is supported by previous studies demonstrating m⁶A-modified aptamers can exhibit changes in binding affinity or fluorescence upon enzymatic demethylation by RNA-modifying enzymes in cells [81,82].

The concept of a "modification-sensitive aptamer" has not yet been fully developed; however, several studies have indirectly hinted at the relevance of chemical context for RNA binding functionality [67,83,84]. Structural probing techniques such as SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension) have demonstrated that m⁶A and other modifications can shift RNA reactivity profiles; this implies a broader influence on folding pathways [85,86]. In the realm of aptamers, such effects could alter loop closure, pseudoknot formation, or other motifs necessary for high-affinity binding [87,88]. Experimental models for modification-sensitive aptamers could be developed by comparing aptamer performance in modified versus unmodified RNA backgrounds [22,89]. Synthetic systems could also be engineered to test how insertion of specific marks at key structural positions influences function. These models would allow for systematic evaluation of how epitranscriptomic environments modulate aptamer efficacy [90,91,92]. The significance of such interactions extends beyond structural interest; it suggests a potential bidirectional influence in which aptamers could be either regulated by, or responsive to, chemical signatures embedded in the transcriptome. Exploring these structural bridges can set the foundation for understanding compatibility, predictability, and robustness of aptamer-based applications in vivo [67].

5. RNA Modifications as Modulators or Targets of Aptamer-Based Tools

The recognition that RNA modifications influence not only the fate of transcripts but also their molecular interactions raises an important question: can these modifications be directly targeted by aptamer-based tools? While most aptamers are currently selected against proteins, small molecules, or ions, the concept of developing aptamers to recognize modified RNA bases remains largely unexplored, yet conceptually sound [67,93,94]. RNA modifications represent chemically distinct features that could, in principle, serve as epitopes for aptamer recognition [3,95,96]. The presence of a methyl group on adenosine (as in m⁶A) or an isomerized uridine (as in Ψ) introduces differences in hydrogen bonding patterns and steric properties that could be selectively bound by a structured RNA aptamer [82,97,98,99,100]. In theory, aptamers could be designed to distinguish between modified and unmodified bases within otherwise identical sequence contexts; such specificity would have profound implications for diagnostic and mechanistic applications. For instance, a selected RNA aptamer so called A-Pepper exhibits fluorescence changes depending on m⁶A status, and Broccoli-based assays similarly differentiate modified from unmodified RNA, demonstrating that aptamer-based tools can, resolve chemical differences arising from epitranscriptomic modifications [82,101].

Although few studies have directly attempted direct aptamer selection against RNA modifications, some conceptual precedents exist from DNA aptamer research, where modified nucleotides have been targeted successfully. For instance, DNA aptamers have been selected to bind specifically to methylated DNA sequences (e.g. methylated CpG dinucleotide sites) [102,103]. These examples suggest that, in principle, selective detection of modified versus unmodified bases is feasible. Expanding such efforts into RNA aptamers could provide valuable tools for detecting or isolating modified transcripts. These aptamers could be incorporated into biosensors or affinity capture platforms, enabling studies of RNA modification dynamics in live cells or complex mixtures [104].

Beyond sensing, there is also interest in whether RNA modifications could act as endogenous regulators of aptamer activity. Aptamers that are transcribed within cells, either from expression constructs or viral vectors, may acquire modifications from endogenous writer enzymes. If these modifications occur at functionally relevant positions, they could alter binding capacity or structural stability as shown in experimental studies and molecular dynamics (MD) simulations where specific chemical substitution in thrombin-binding aptamer significantly changed its structural stability and target affinity [105,106]. While this is often considered an undesirable side effect, it could also be repurposed as a control mechanism: aptamers could be designed to remain inactive until modified, thereby acting as on/off switches or conditional regulators. For instance, MD simulations of a thrombin-binding aptamer incorporating 5-furyl-2′-deoxyuridine revealed that the position of the modification within the sequence critically determines its impact on the aptamer–protein interaction sensitivity by modulating structural dynamics at the modification site, altering solvent exposure, influencing stacking with neighboring bases, and affecting interactions with thrombin, all without disrupting the global conformation of the aptamer [107].The design of such responsive systems would require a deeper understanding of the sequence preferences of modifying enzymes and the structural outcomes of various marks. It would also necessitate improved methods for site-specific installation of modifications in vitro, as well as the ability to map and verify modification status in cellular RNA populations at high resolution [108,109]. These tools are under active development in the epitranscriptomics field; their integration into aptamer design workflows could facilitate a new class of chemically-responsive RNA devices. In this conceptual framework, RNA modifications are not just obstacles to be accounted for, but potential control points or targets for aptamer-based strategies. Whether used to sense, respond to, or exploit the modified state of transcripts, aptamers could offer precise and programmable solutions for probing the epitranscriptome.

6. Potential for Synthetic Biology and Theranostics Integration

The growing interest in RNA technologies has positioned both RNA aptamers and epitranscriptomics as promising components of future synthetic biology systems. In this context, aptamers can be viewed as programmable elements capable of sensing and controlling molecular events [110,111], while epitranscriptomic modifications serve as regulatory inputs that shape RNA behavior [61,112]. When brought into conceptual proximity, these two systems suggest the potential for creating hybrid regulatory frameworks that are both chemically tunable and highly modular. RNA aptamers have already been incorporated into a variety of synthetic circuits; examples include small-molecule-responsive riboswitches [113,114], aptamer-controlled gene expression switches [115], and ligand-triggered RNA scaffolds for signal integration [116,117]. In many cases, the response of these systems relies on conformational changes that occur upon target binding [114,118]. This inherent sensitivity to structural transitions makes them potentially responsive to endogenous cues introduced by epitranscriptomic marks. For instance, modifications such as base methylation can work as internal signals by altering the secondary structure of RNA [60,119]. Typically, the addition of N⁶A-methyladenosine (m⁶A) on an RNA can change the base pairing and refold the RNA locally, a phenomenon known as the m⁶A-switch [63]. These modifications can make the previously hidden sequences accessible, or vice versa, in a way that resembles the conformational changes caused by ligand binding in aptamers [63,120]. Therefore, if an aptamer or its adjacent sequence is modified by a chemical mark through epitranscriptomic processes, the resulting conformational change could potentially modulate the aptamer’s binding or regulatory output [83,121]. In therapeutic contexts, aptamers have been explored as alternatives to antibodies for targeted delivery, inhibition of disease-associated proteins, and modulation of signaling pathways [122,123]. Several RNA aptamers have reached clinical evaluation stages, such as Pegaptanib a Food and Drug Administration (FDA) approved RNA aptamer medication to treat neovascular age-related macular degeneration by binding and inhibiting vascular endothelial growth factor (VEGF) [122,124]. However, in vivo functionality of aptamers often depends on both resistance to degradation and maintenance of structural fidelity, which are influenced by the cellular environment. In fact, aptamers selected in vitro sometimes fail to perform in vivo due to rapid nuclease digestion, suboptimal ionic conditions, or competition with other biomolecules [122].In this setting, RNA chemical modifications may either support or interfere with aptamer efficacy depending on the location and type of chemical marks introduced. For instance, incorporating 2′-fluoropyrimidines or 2′-O-methyl groups at the ribose 2′-position greatly increases nuclease resistance and serum stability without compromising target binding affinity [38,125]. However, addition of a locked nucleic acid (LNA) nucleotide into the loop of a thrombin-binding DNA aptamer was shown to stop its ability to bind thrombin [126]. Therefore, if the relationship between aptamer activity and RNA modification status can be better understood, opportunities may arise for engineering theranostic systems that are conditional upon the epitranscriptomic state of the target cell. For instance, aptamers could be engineered to function only in cells with elevated levels of m⁶A or other modifications, introducing a form of endogenous selectivity and improving therapeutic precision [81]. Conversely, aptamers could be used to block or enhance the activity of reader proteins, creating downstream effects on the stability or translation of modified transcripts. For example, a short RNA decoy corresponding to HIV’s TAR sequence was shown to bind and sequester the viral Tat protein, thereby inhibiting Tat’s downstream effects on gene expression [127].

Synthetic biology platforms could also benefit from aptamers that are sensitive to RNA modification states. In such designs, RNA circuits could include structural domains whose behavior depends on the presence or absence of specific marks, allowing cells to respond to physiological or pathological conditions in real time [81,82,128,129,130]. Regulatory switches could thus be activated or silenced based on the modification profile, introducing a novel axis of control that bridges molecular sensing and functional output [131,132]. The success of such applications will depend on the ability to map modification patterns accurately, identify modification hotspots within aptamer sequences, and predict structure-function outcomes [133,134,135]. While these requirements present technical challenges, advances in direct RNA sequencing [136,137], structural modeling [135], and in vivo RNA tracking [138] offer potential solutions. By incorporating RNA modifications as variables in aptamer design and deployment, synthetic biology and therapeutic systems may gain an additional layer of control that is both dynamic and biologically grounded.

7. Technological Convergence: Detecting, Mapping, and Manipulating Modifications

Technological advances in RNA research have brought aptamer-based platforms and epitranscriptomic mapping tools closer to potential integration [81,101,139]. This convergence offers practical opportunities for improving how RNA modifications are detected, quantified, and manipulated, especially in complex or living systems [94,134]. RNA aptamers, with their high specificity and tunable binding properties, can serve as customizable tools for addressing some of the limitations faced by current epitranscriptomic techniques. One of the main challenges in epitranscriptomics is the detection of modifications at single-nucleotide resolution [140]. Traditional antibody-based techniques, such as m⁶A-seq or MeRIP-seq, provide general information about modification enrichment but suffer from low resolution and cross-reactivity. Emerging methods such as direct RNA sequencing using nanopore technology offer site-specific information [141], yet they often require high RNA input and complex signal processing. In this landscape, RNA aptamers could fill a unique niche in epitranscriptomics. If aptamers are selected or engineered to bind specific modified nucleotides or the modified form of a transcript, they could be deployed as molecular probes for direct detection of epitranscriptomic states. This has been demonstrated a m⁶A-sensitive fluorescent RNA aptamer system, known as A-pepper, which reports m⁶A enzymes’ activity in vitro and in live cells based on fluorescent signal enhancement resulting from the methylation and demethylation of the aptamer [101]. Aptamers could also be used for affinity capture to isolate modified RNAs from a mixture, then enrichment could be followed by high-throughput sequencing to identify modification patterns. The feasibility of such systems has been shown by the selection of L-RNA aptamers that can selectively capture structured native RNA (D-RNA) [142,143]. Fluorescently labeled aptamers could also be used in imaging platforms to visualize modifications in fixed or live cells. For instance, fluorescent light-up aptamers (e.g., the Mango and Pepper families) have already demonstrated high-contrast RNA imaging in cells, indicating their potential for visualizing RNA modification dynamics [144,145,146]. In principle, an aptamer that binds a modified base, but not to the unmodified base, can be tagged with a fluorophore to serve as a fluorescent probe. For instance, DNA aptamers have already been selected to recognize specific post-translational modifications, such as particular histone acetylation with high selectivity and have been used in atomic force microscopy-based imaging of these modifications [147]. By extension, fluorescent RNA aptamer-based sensors could be developed to track RNA base modifications in cells, enabling researchers to track modification dynamics in response to stimuli or disease progression in real-time. Such applications would require aptamers that can discriminate between modified and unmodified bases, a level of specificity that remains technically challenging but conceptually feasible [81,101,148]. In this regard, aptamers have shown the ability to distinguish molecules differing by only a single methyl group. For example, researchers have developed an RNA aptamer that binds theophylline about 10,000-fold more tightly than caffeine, despite caffeine having just one extra methyl group, supporting their potential role in epitranscriptomic detection tools [149].

Another potential direction involves using aptamers to manipulate the activity of the enzymes responsible for writing, reading, or erasing RNA modifications. Aptamers that bind to m⁶A writers like METTL3 [150], readers like YTHDF proteins [93], or erasers like ALKBH5 and FTO [81,82] could be used to inhibit or redirect their activity in a programmable manner. This type of aptamer function would parallel the use of inhibitory antibodies or small molecules but would offer improved tunability and intracellular delivery potential, especially if expressed as part of an RNA-based therapeutic system [37,110]. Furthermore, aptamer-controlled systems could be designed to act as responsive elements in RNA editing tools [131,151,152,153]. For instance, aptamers could gate the activity of an RNA-editing enzyme by binding a modification or its corresponding reader protein; such gating could be used to control editing efficiency or spatial targeting [152,154]. These systems would be useful in precision medicine, where therapeutic interventions must be tightly controlled and context-specific. The technological merging of aptamer platforms and epitranscriptomic analysis tools represents an underdeveloped but promising area. For this potential to be realized, more research is needed to develop high-affinity aptamers for modified bases, optimize delivery systems for aptamer-based tools, and integrate these elements into existing transcriptomic workflows. If successful, such integration could yield a powerful suite of molecular instruments for observing and controlling the RNA modification landscape.

8. Challenges and Considerations for Future Research

While the conceptual link between RNA aptamers and epitranscriptomics offers promising avenues for innovation, several challenges must be addressed before meaningful integration can occur. These span technical limitations and gaps in fundamental understanding, each requiring careful consideration to avoid premature conclusions or inefficient designs. One major limitation is the lack of systematic data on how endogenous RNA modifications affect synthetic aptamer structure and function. Most aptamer studies are performed in vitro under defined conditions, which do not reflect the chemical complexity of cellular RNA environments [155]. Aptamers that function reliably in buffer may behave unpredictably in cells, where post-transcriptional modifications and RNA-binding factors abound [122]. Without experimental systems that simulate modified RNA contexts, aptamer compatibility with the epitranscriptome remains unclear. Moreover, intracellularly expressed aptamers could become substrates for RNA-modifying enzymes. Whether such modifications occur randomly or follow enzyme-specific sequence preferences is poorly understood, as this has not been systematically studied. If key positions in an aptamer are chemically altered, e.g., by m6A methylation, binding affinity or structure may be disrupted. For instance, a Broccoli aptamer modified with m6A lost fluorescence until a demethylase removed the mark [82]. To improve stability and pharmacokinetics, chemical modifications like 2′-fluorinated pyrimidines, 2′-O-methyl ribose, or PEGylation have been employed. However, these do not always prevent enzymatic targeting and may not be compatible with in vivo expression [156].

Another challenge lies in selecting aptamers that recognize modified nucleotides with high specificity. Modified and unmodified bases often differ subtly, and aptamer binding depends on more than base identity; factors like structural context, solvent accessibility, and competition from cellular components also play roles [68,157]. Addressing this may require adapting SELEX protocols, for example by incorporating modified RNA libraries (e.g., 2′-fluoro, 2′-O-methyl, base analogs) to broaden interaction profiles and improve discrimination [158]. Structurally biased libraries may further enrich aptamers with desired conformations, enhancing their ability to distinguish modified from unmodified bases [159].

Integration into epitranscriptomic studies also raises concerns about off-target effects. Even highly specific aptamers may bind unintended targets in cellular contexts, especially if altered by endogenous modifications or protein interactions [160,161]. A methylated RNA aptamer, for example, failed to bind its ligand until demethylated [82]. Similarly, the DNA aptamer AS1411, targeting nucleolin, was found to bind fibronectin 1 in serum, negating its intended in vivo function [160]. These examples emphasize the need for thorough characterization under physiological conditions [161]. On the epitranscriptomic side, accurately mapping RNA modifications at single-base resolution remains a challenge [162,163]. Without spatial and temporal precision, predicting aptamer–modification interactions is difficult. Advances in direct RNA sequencing and structure-aware detection are helping, but reliable maps are still lacking.

Ethical and safety issues may also emerge in therapeutic settings [164,165,166]. If aptamer activity depends on modification status, misregulated epitranscriptomic profiles could cause unintended effects. Rigorous validation and control systems will be essential for clinical applications. Nonetheless, these challenges are not insurmountable. Addressing them systematically may enable the next generation of RNA tools that function within the chemical logic of the epitranscriptome.

9. Conclusions

RNA aptamers and epitranscriptomics are powerful yet largely separate areas of RNA biology. Aptamers offer programmable specificity and structural adaptability, while epitranscriptomic modifications serve as a chemical language regulating RNA behavior. Though each field has advanced independently, their intersection may hold untapped potential for research and innovation. This perspective explores how RNA modifications might affect aptamer folding or interactions, and how aptamers could detect or respond to modified RNA states, forming feedback between recognition and regulation. Applications in synthetic biology and therapeutics are discussed, along with conceptual and technical challenges. Rather than a conclusion, this article invites researchers across RNA structure, sensing, and synthetic biology to explore these overlaps and develop tools to probe or exploit the modified transcriptome. Bridging these domains may deepen our understanding of post-transcriptional regulation, enable smarter therapeutic systems, and blur the line between endogenous RNA control and synthetic design, opening new directions for RNA research.

Table 1. Potential intersections between RNA aptamer function and epitranscriptomic mechanisms.

Feature	RNA Aptamers	Epitranscriptomics	Possible Intersection
Functional dependency	Structure-dependent binding	Structure-dependent regulation	RNA modifications may alter aptamer folding or stability
Selection/design methods	SELEX-based in vitro evolution	Genetic, enzymatic, or chemical modification	Modified RNA libraries could be used for aptamer selection
Key chemical elements	Unmodified or chemically stabilized nucleotides	m⁶A, Ψ, m⁵C, etc.	Aptamers could be sensitive to or recognize these modifications
Role in gene regulation	Target inhibition; molecular recognition	mRNA splicing, stability, translation	Combined tools may control gene expression in a condition-specific manner
Therapeutic use	Targeted drug delivery; inhibition of proteins	Cancer, neurological diseases, immune regulation	Aptamer-based delivery of epitranscriptome-modifying agents
Response to cellular environment	May be degraded or misfolded	Responds to stress, development, signaling	Aptamer activity may vary depending on local modification state
Potential for biosensing	High specificity, real-time detection	Biomarker potential (modification levels)	Aptamers as sensors for detecting RNA modification patterns
Compatibility with in vivo systems	Requires stabilization strategies	Naturally occurring mechanisms	Modified-state-responsiveness could be integrated into aptamer designs
Regulatory feedback loops	Currently limited to design logic	Present in many cellular pathways	Aptamers could be designed to modulate reader/writer proteins
Application overlap	Synthetic biology, diagnostics, therapeutics	Functional genomics, disease mechanisms	Cross-disciplinary innovation in RNA-based systems

Table 1. Potential intersections between RNA aptamer function and epitranscriptomic mechanisms.

Feature	RNA Aptamers	Epitranscriptomics	Possible Intersection
Functional dependency	Structure-dependent binding	Structure-dependent regulation	RNA modifications may alter aptamer folding or stability
Selection/design methods	SELEX-based in vitro evolution	Genetic, enzymatic, or chemical modification	Modified RNA libraries could be used for aptamer selection
Key chemical elements	Unmodified or chemically stabilized nucleotides	m⁶A, Ψ, m⁵C, etc.	Aptamers could be sensitive to or recognize these modifications
Role in gene regulation	Target inhibition; molecular recognition	mRNA splicing, stability, translation	Combined tools may control gene expression in a condition-specific manner
Therapeutic use	Targeted drug delivery; inhibition of proteins	Cancer, neurological diseases, immune regulation	Aptamer-based delivery of epitranscriptome-modifying agents
Response to cellular environment	May be degraded or misfolded	Responds to stress, development, signaling	Aptamer activity may vary depending on local modification state
Potential for biosensing	High specificity, real-time detection	Biomarker potential (modification levels)	Aptamers as sensors for detecting RNA modification patterns
Compatibility with in vivo systems	Requires stabilization strategies	Naturally occurring mechanisms	Modified-state-responsiveness could be integrated into aptamer designs
Regulatory feedback loops	Currently limited to design logic	Present in many cellular pathways	Aptamers could be designed to modulate reader/writer proteins
Application overlap	Synthetic biology, diagnostics, therapeutics	Functional genomics, disease mechanisms	Cross-disciplinary innovation in RNA-based systems

Box 1. Key concepts and definitions 

RNA Aptamers: Short, single-stranded RNA molecules selected for their ability to bind specific targets with high affinity; their function depends on precise 3D folding.

SELEX (Systematic Evolution of Ligands by EXponential enrichment): An in vitro method for selecting aptamers from large RNA libraries through iterative binding and amplification cycles.

Epitranscriptomics: The study of reversible chemical modifications on RNA that influence structure, stability, localization, and translation without altering sequence.

m⁶A (N⁶-methyladenosine): A common RNA modification that modulates transcript fate by destabilizing local structures and attracting specific reader proteins.

Pseudouridine (Ψ): An isomer of uridine that enhances RNA backbone rigidity and base pairing; commonly found in non-coding and coding RNAs.

RNA Modifications: Covalent chemical changes to RNA bases; installed by writers, interpreted by readers, and removed by erasers in a dynamic manner.

Writers, Readers, Erasers: Enzymes that install (writers), recognize (readers), or remove (erasers) RNA modifications to regulate RNA behavior.

RNA Secondary Structure: Local base pairing patterns (e.g., stem-loops, bulges) that influence RNA stability and interaction potential.

Aptamer Folding: The process by which aptamers adopt specific 3D shapes essential for target binding; sensitive to sequence and chemical context.

Structural Plasticity: The ability of RNA to adopt multiple conformations depending on cellular or chemical cues; often altered by modifications.

RNA Biosensors: Aptamer-based tools designed to detect specific molecules, ions, or RNA modifications in vitro or in cells.

RNA-based Therapeutics: Strategies that use RNA molecules (e.g., aptamers, siRNAs) to treat disease by modulating molecular pathways.

Modification-sensitive Aptamers: Hypothetical or designed aptamers whose structure or function is altered by the presence of RNA modifications.

RNA Modification Mapping: Techniques used to identify and locate chemical modifications across the transcriptome; often requiring enrichment or sequencing.

Direct RNA Sequencing: A method that reads RNA molecules without reverse transcription, allowing detection of certain base modifications in situ.

Targeted RNA Delivery: The use of aptamers to guide therapeutic cargos to specific cells or tissues based on target recognition.

Chemical Probing (e.g., SHAPE): Methods to study RNA structure by modifying reactive groups; used to infer folding and flexibility.

Aptamer-Protein Interactions: Non-covalent bindings between aptamers and proteins; used for inhibition, sensing, or targeted modulation.

Context-aware RNA Design: The development of RNA molecules that function reliably within the biochemical environment of living cells, including modification states.