Aptamer chemistry

Abstract

Aptamers are single-stranded DNA or RNA molecules capable of tightly binding to specific targets. These functional nucleic acids are obtained by an in vitro Darwinian evolution method coined SELEX (Systematic Evolution of Ligands by EXponential enrichment). Compared to their proteinaceous counterparts, aptamers offer a number of advantages including a low immunogenicity, a relative ease of large-scale synthesis at affordable costs with little or no batch-to-batch variation, physical stability, and facile chemical modification. These alluring properties have propelled aptamers into the forefront of numerous practical applications such as the development of therapeutic and diagnostic agents as well as the construction of biosensing platforms. However, commercial success of aptamers still proceeds at a weak pace. The main factors responsible for this delay are the susceptibility of aptamers to degradation by nucleases, their rapid renal filtration, suboptimal thermal stability, and the lack of functional group diversity. Here, we describe the different chemical methods available to mitigate these shortcomings. Particularly, we describe the chemical post-SELEX processing of aptamers to include functional groups as well as the inclusion of modified nucleoside triphosphates into the SELEX protocol. These methods will be illustrated with successful examples of chemically modified aptamers used as drug delivery systems, in therapeutic applications, and as biosensing devices.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…Nucleoside triphosphates can be modified at any position of the nucleosidic scaffold. The exact positioning of the modification is dictated by empirical rules as well as information gathered from structural investigations of polymerases ,. Essentially, modifications brought at the level of the C5 position of pyrimidines and N7 position of purines are particularly well tolerated by polymerases, as their Watson–Crick base‐pairing capacity is maintained and crucial interactions with the enzyme are not ablated or hindered .…”

“…The chemical diversity of modifications brought to the phosphate group is more modest than for the sugar and nucleobase and is merely restricted to phosphorothioates. Most DNA and RNA polymerases readily incorporate phosphorothioate‐modified triphosphates, albeit only the S P stereoisomers …”

“…In addition to metal‐mediated base pairs formed by canonical nucleobases, synthetic nucleotides that act as ligands can be incorporated into DNA to increase the diversity as well as the choice of the metal ion . Most reported systems rely on the introduction of artificial nucleosides by solid‐phase synthesis, which inherently imposes restrictions on the nature of the functional groups as well as the length of the sequences that can be crafted . Recently, the enzymatic incorporation of modified nucleotides has been proposed as a viable alternative for the construction of artificial metal–base pairs .…”

“…Solid‐phase synthesis is a highly convenient method for the introduction of chemical modifications into nucleic acids, especially if larger quantities are considered. However, this method is limited in terms of the length of the oligonucleotides (<100 nucleotides) and the nature of the functional groups (which need to be compatible with all the chemical steps involved in the process) . On the other hand, the enzymatic synthesis of modified nucleic acids only depends on the tolerance of natural or engineered polymerases at accepting dN*TPs as substrates .…”

Biologically Inspired, Cell‐Selective Release of Aptamer‐Trapped Growth Factors by Traction Forces

“…[45] Additional modifications, such as the use of phosphorothioate bases, flipped bases, locked nucleic acids (LNAs), Spiegelmers (mirror-image L-oligonucleotide aptamers), and g-quadruplex aptamers can additionally improve the resistance to both exo- and endonuclease degradation. [45–47] Coupled with the prediction that there is likely steric protection of the oligonucleotide from enzymatic attack due to the presence of a bound protein and close association with the matrix, the TrAP platform provides significant potential to allow persistence in tissue microenvironments actively undergoing repair.…”