The above-described SELEX experiments led to the identification of high affinity RNA ligands that target the trypanosomal surface and thus suggest several pharmacological approaches. In order to optimize the selected RNAs for their pharmaceutical use, the original molecules have to be converted from in vitro binding reagents to therapeutic compounds, which can be tested for their pharmacological effects in vivo. Within this context an important consideration is the cost of synthesis. Since aptamer-derived pharmaceuticals are chemically synthesized and therefore fairly expensive, the size of the aptamer is a critical feature and has to be reduced to its minimal active core. This can be experimentally addressed in so-called boundary experiments (Fitzwater and Polisky 1996). As already mentioned above, the minimal binding domain of aptamer 2-16 was determined as a 64-nt sequence (Homann et al. 2001), while the core structure of the VSG-specific aptamer was 44 nt long (Lorger et al. 2003).
Another critical characteristic of a pharmacologically active compound is its stability in biological fluids, especially in blood. The stability of nucleic acids is limited by their sensitivity towards nucleases, which are highly abundant in almost all in vivo environments. The in vitro half-life of a typical DNA oligonucleotide in human serum is 30-60 min, while that of a typical RNA molecule is in the range of only a few seconds. However, the stability of RNAs can be significantly increased by the incorporation of 2/-fluoro-, 2'-amino-or 2/-0-methyl-substituted ribonucleotides (Eaton and Pieken 1995). This is due to the fact that the naturally occurring cleavage of the phosphodiester backbone relies on the availability of a 2/-hydroxyl group. The 2/-OH enables the nucleophilic attack that leads to the formation of a 2/,3/-cyclic phosphodiester hydrolysis product, which further reacts to the corresponding 3/-nucleoside monophosphate.
A simple way of creating serum-stable aptamers is to introduce the modified nucleotides into the starting pool at the onset of the selection. This was done in the case of the VSG-specific aptamers by using 2/-F-modified pyrimidine nucleotides and led to RNA molecules with serum stabilities greater than 24 h (Lorger et al. 2003). However, aptamers can also be modified after the selection process, provided that the structure and function ofthe molecules remains unaffected by the modifications. This is not necessarily the case, given the critical role that 2/-hydroxyl groups can play in the structural organization of RNA. Aptamer 2-16 was originally selected as an unmodified RNA. The introduction of 2/ amino and/or 2/ fluoro modifications required a detailed analysis of the structural and functional consequences. C2/-modifications are known to affect the sugar puckering of nucleic acids. The preferred conformation of RNA helices is the A-form, which is characterized by a C3/-endo ribose conformation. Heteroatom modifications like 2/-amino-groups favour C2/-endo conformations, which is one of the major determinants of the "DNA-like" B-helix. In contrast, oligonucleotides substituted with 2/-0-methyl- or 2/-fluoro-groups shift the equilibrium towards the "RNA-like" C3/-endo form of the sugar.
Unmodified aptamer 2-16 RNA was characterized with a half-life of < 5 s in human serum. The co-transcriptional incorporation of2/-fluoro- and/or 2/-amino-substituted pyrimidine nucleotides increased the stability of the RNA up to a half-life of several days (unpublished data). A cell-binding analysis demonstrated, that 2/-amino-modifications led to a complete loss of the ap-tamer/parasite interaction, while 2/-fluoro-substitutions retained the binding capacity of the modified RNA. This observation is consistent with the above-described assumption that 2/-fluoro-substitutions are the preferred candidates for maintaining the structural integrity and thus function of modified ap-tamers. The binding affinity of 2/-fluoro-modified 2-16 RNA to parasite cells was marginally enhanced (Kd of 70 nM versus 60 nM for unmodified 2-16 RNA), suggesting a contribution of the 2/-F group in the interaction. In addition, 2/-F-modified 2-16 RNA bound to the flagellar pocket indistinguishable from unmodified aptamer preparations, and the molecules were internalized and transported to the lysosome via the same endosomal routing pathway. This was further confirmed in a structural analysis by chemical and enzyme probing experiments. The data confirmed that the overall structure of aptamer 2-16 was by and large unaltered by the 2'-substitutions, and as a consequence the RNA molecules had retained their functionality.
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