**5. Conclusion**

internal loops and bulges generally present different conformations in solution and adopt defined secondary and tertiary structures on ligand–aptamer complex [92]. This effect was observed on aptamer Sc5-c3 selected against HPV-16 VLPs. Sc5-c3 showed a hairpin structure with an internal loop, where the main loop (ML) presented two different structures in the absence of a target (Table 2). Sc5-c3 transition structure was demonstrated by ribonuclease mapping. Further experiments using Sc5-c3 mutants generated both stable stem and stable loop conformations, demonstrating that the loop structure binds better to the VLPs [58]. Thus, as observed in several aptamers, the binding region remains as a flexible single strand as bulges or loops stabilize conformation arrangements in the presence of a target, producing a very

Although bulges and loops are quite common target-binding motifs in aptamer RNAs, they are not the only structures present in aptamer–target complexes. Pseudoknots and G-quad‐ ruplexes have also been reported as functional components of aptamers [93]. For example, some of the aptamers isolated against HIV integrase (93 del and 112 del) presented a G-rich nucleic acid sequence that was stabilized in the presence of K+ as G-tetrad, increasing their inhibitory effect [25]. Later reports showed that 93 del adopts an unusually stable dimeric

The binding properties of an aptamer are dictated by its sequence and subsequent folding into secondary and tertiary structures. Recently, functional RNA structures were classified as critical, connecting, neutral and forbidden structures regarding their particular roles within a structure [95]. This classification is also applicable to nucleic acid aptamers and is an important

According to their molecular characteristics, RNA or DNA aptamers have some limitations in their use in animal models and humans. They have limited stability in biological fluids and are readily degraded by nucleases, unmodified aptamers in the bloodstream possess a halflife time of less than two minutes. However, many post-SELEX modifications have been developed to avoid nuclease attack and improve stability in biological fluids. Some modifica‐ tion examples include nucleotide substitutions by 2′-modified variants such as 2′-fluoro (2′- F), 2′-amino (2′-NH2) or 2′-O-alkyl. Because the most abundant nucleases in biological fluids are specific to pyrimidines, substitutions in pyrimidine positions appear to be sufficient to prevent degradation. Another method to stabilize RNA aptamers is the substitution of Dribose by L-ribose. As a first step, the aptamers bind the mirror image of the target molecule to obtain a D-aptamer, then the selected aptamer sequence is synthesized in L-conformation. As a result of molecular symmetry, the L-ribose–containing aptamer can bind to the target molecule avoiding degradation by D-ribose–specific nucleases. Moreover, to efficiently overcome binding issues produced by the introduction of modified nucleotides on the aptamer sequence, the SELEX procedure can be carried out in the presence of modified libraries.

clue to design novel and functional variants for viral detection or therapy.

**4. Challenges for aptamer technology**

specific binding.

quadruplex structure [94].

110 Nucleic Acids - From Basic Aspects to Laboratory Tools

In the last few years, aptamers have become successful tools for specific viral diagnosis and genotyping, resulting in the development of many methods based on aptamer–target detection with very high sensibility and accuracy. On the other hand, aptamer's role as an antiviral drug has demonstrated the inhibition of viral infection through *in vitro* assays and *in vivo* experi‐ ments using cell lines or animal models. Nevertheless, most aptamers failed to produce results in clinical trials mostly due to nuclease-associated degradation. Therefore, further develop‐ ment of aptamer's stability in biofluids and improved pharmacodynamics and delivery methods are required to overcome clinical issues that would allow its successful therapeutic application.
