Development of Phosphorothioate DNA and DNA Thioaptamers

Abstract

Nucleic acid aptamers are short RNA- or DNA-based affinity reagents typically selected from combinatorial libraries to bind to a specific target such as a protein, a small molecule, whole cells or even animals. Aptamers have utility in the development of diagnostic, imaging and therapeutic applications due to their size, physico-chemical nature and ease of synthesis and modification to suit the application. A variety of oligonucleotide modifications have been used to enhance the stability of aptamers from nuclease degradation in vivo. The non-bridging oxygen atoms of the phosphodiester backbones of RNA and DNA aptamers can be substituted with one or two sulfur atoms, resulting in thioaptamers with phosphorothioate or phosphorodithioate linkages, respectively. Such thioaptamers are known to have increased binding affinity towards their target, as well as enhanced resistance to nuclease degradation. In this review, we discuss the development of phosphorothioate chemistry and thioaptamers, with a brief review of selection methods.

Aptamer Therapeutics in Cancer: Current and Future

“…Methods for base substitutions including 2′-fluoro- [ 9 , 10 , 11 ], 2′-amino-, 2′-azido-, 2′-hydroxymethyl-, and 2′-methoxypyrimidines and 2′-methoxypurines have been established [ 12 , 13 , 14 , 15 ]. Phosphorothioate and phosphorodithioate substitutions are another option for the backbone modification [ 16 ]. Such chemical modifications of the DNA backbone provide resistance against nucleases, as was first shown by Eckstein’s group [ 17 ], and often increase binding affinity [ 16 ].…”

“…Phosphorothioate and phosphorodithioate substitutions are another option for the backbone modification [ 16 ]. Such chemical modifications of the DNA backbone provide resistance against nucleases, as was first shown by Eckstein’s group [ 17 ], and often increase binding affinity [ 16 ]. The introduction of functional groups in the aptamer backbone permits conjugation to other drugs, siRNA [ 18 , 19 ], and nanoparticles [ 20 , 21 , 22 ], further broadening their application as multivalent therapeutics [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ].…”

“…In addition to stabilization, thioation enhanced binding affinity as well, owing to decreased negative charge on DNA backbone [ 40 ]. Dithioaptamer developed by Gorenstein’s group [ 16 ] showed an increased binding affinity up to 28–600 fold compared to monothioaptamer [ 41 ]. “Mirror aptamers” (Spiegelmers) with oligonucleotide backbones composed of l -ribose (RNA Spiegelmers) or l -deoxy-ribose (DNA Spiegelmers) also lead to nuclease resistance.…”

Nucleic Acid Aptamers: Emerging Applications in Medical Imaging, Nanotechnology, Neurosciences, and Drug Delivery

“…The possibilities for the modification at the level of the phosphate unit are more limited than in the case of the sugar and nucleobases moieties and most efforts have focused on the α-phosphate, particularly α-phosphorothioates [ 242 , 279 , 292 , 293 , 294 ]. In this context, Yang et al have explored the alkylation of phosphorothioated thrombine-binding aptamers (TBA) with aim of improving the antitumor properties of the ligands by reducing the thrombin binding affinity [ 295 ].…”

Key Aspects of Nucleic Acid Library Design for in Vitro Selection

“…Therefore, the choice of library design has a great impact on the overall efficiency of the selection. When generating the initial library, a researcher should keep in mind the properties of the target (such as in capture SELEX for small molecules [ 32 ]) and the end use of an aptamer (whether nuclease resistance is necessary or not) [ 27 , 33 ]. The importance of covering a maximal sequence space (a multi-dimensional space of different sequences of a certain length), the necessity of introducing a particular sequence or structural element should also be taken into account.…”