**4. Challenges for aptamer technology**

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.

Therapeutic aptamers selected against intracellular or nuclear proteins represent bigger challenges as they need to go across physiological barriers (i.e. cell membrane) before they reach their targets. DNA and RNA aptamers are characterized by rapid renal clearance leading to short half-lives in the bloodstream. To address this issue, aptamers can be conjugated to synthetic polymers such as polyethylene glycol (PEG) to increase their *in vivo* half-life and pharmacodynamics [96]. Additionally, PEG-conjugated aptamers show higher cellular uptake than the unconjugated form [96]. Alternatively, delivery systems such as viral and nonviral vectors may have improved aptamer cell uptake and nuclear distribution [97]. Vectors or aptamers alone can be delivered either *ex vivo* or *in vivo. In vivo* approaches include intravenous injection or local implantation and *ex vivo* refer to the removal of cells followed by *in vitro* genetic manipulation and the reintroduction of modified cells. These therapies are still under evaluation and further studies are necessary to demonstrate their clinical safety. Aptamers against extracellular or surface viral targets have obvious advantages over aptamers targeting viral proteins intracellularly expressed, as they can reach exposed areas of infection, such as the respiratory tract or reproductive organs. This availability makes it possible to develop new antiviral drugs administrated by noninvasive methods, such as aerosols in case of respiratory tract infection or topical creams/lotions in case of reproductive organ infections. Many aptamers are undergoing clinical trials, some of them administrated by noninvasive methods but, so far, no antiviral aptamer has been approved for human use [18, 98].
