Phosphate Substitutions

Both phosphorothioate and phosphorodithioate modifications have been explored in the context of aptamers. These substitutions introduce sulfur(s) in place of non-bridging phosphodiester oxygen(s) and thereby confer nuclease resistance, li-pophilicity, and polarizability to oligonucleotides that contain them. While phos-phorothioate oligonucleotides can be generated by either transcription or solidphase chemical synthesis, phosphorodithioate NTPs are not utilized by any poly-merases tested to date and thus phosphorodithioate-containing libraries have been prepared by solid-phase chemical synthesis. King et al. (2002) used phos-phorothioate SELEX to discover phosphorothioate-containing aptamers ("thioap-tamers") to NF-IL6, NFkB, p65, and p50 proteins. They successfully isolated an aptamer with 800pmol/L affinity binding to p50, and using an electrophoretic gel-shift approach, attributed enhanced affinities and specificities for this aptamer to the presence of phosphorothioate modifications. Tam et al. (1999) discovered an 18-mer fully-phosphorothioate G-rich aptamer that inhibits the expression of CD28. By reducing the number of phosphorothioate linkages the authors showed that only some of these residues are required for nuclease resistance, and that the functional properties of these phosphorothioate linkages are responsible for enhancing the bioactivity of this aptamer.

Extending upon the work of King and co-workers, Yang et al. (2002, 2003) have developed in vitro screening methods for generating phosphorodithioate-contain-ing aptamers and have described the application of these methods to isolate anti-NFkB aptamers. The inability to directly transcribe sequence pools containing this modification forced these authors to develop a split synthesis method in which each solid-phase support bead contained a single phosphorothioate-containing sequence. Direct, post-screening sequencing of beads with the highest affinity binding made it possible to identify functional aptamers. However, relatively limited data are currently available concerning the characteristics of molecules discovered using this approach.

A handful of additional types of phosphate modifications may ultimately be shown to have utility in the context of aptamers. Methylphosphonate linkages have also been considered for oligonucleotides administered for imaging and therapeutic purposes (Younes et al., 2002) and in order to confer nuclease resistance (Agrawal et al., 1997). P-borano linkages can be incorporated into transcripts using T7 RNA polymerase and 5'-(a-P-borano) GTP or 5'-(a-P-borano) UTP, and these modifications have been incorporated into aptamers to ATP using SELEX (Lato et al., 2002). These authors suggested that because 10B can capture thermal neutrons and thereby become radioactive, 10B-containing apta-mers could find utility as activatable radiotherapeutics that are specifically delivered to tumor cells.

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