Nuclease-resistant aptamers

The insufficient stability of RNA, mentioned above, limits the use of RNA aptamers. However, some established techniques yield nuclease-resistant aptamers. One alternative is called the Spiegelmer approach [29], another method involves incorporation of 2 -methoxy purine nucleotides [30]. 

Aptamers have not shown toxicity in rodents. Furthermore, aptamers have not been immunogenic in mice, even after repeated injections of nuclease-resistant aptamers formulated so as to provide very long lifetimes in the vasculature. Aptamers have been injected with adjuvants and foreign basic proteins, as suggested by the literature for the preparation of antibodies against DNA, and no IgG s have been raised. More work will be required in the clinical trials of aptamers, but so far the data are promising. 

The cloned sequences often (but not always) have obvious secondary structures in common, leading to visual truncations by which the fixed sequences and other unnecessary sequences from the random regions are eliminated from the aptamer, often resulting in slightly increased affinity over the starting full-length aptamer. When visual truncation is not possible, experimental truncation is done instead. After truncation the aptamer may be further altered by substituting nuclease-resistant purines for the normal... 

An alternative to the mirror image-approach is the direct selection of an aptamer from libraries of chemically modified RNAs. Many modifications in the ribose moiety of nucleic acids have been shown to dramatically increase their nuclease resistance. Modifications have to be chosen so as to be compatible with nucleic acid replicating enzymes such as reverse transcriptase, or DNA- and RNA-polymerases. The modifications most commonly used are those in which the 2 -OH group of pyrimidines is substituted by a 2 -fluoro-, or a 2 -amino group (1) [64,65],... 

One of the aptamers significantly reduced intradermal VEGF-induced vascular permeability in vivo. Thus, these nuclease-resistant molecules may be useful for the development of novel pharmaceutical lead compounds for epithelial hyperproliferative diseases. 

New strategies for post-SELEX optimization continue to be developed. Schmidt et al. (79) have demonstrated that LNA residues are substituted into a tenascin-C aptamer to increase the thermal stability and nuclease resistance of the aptamer. In common with other aptamer substitution strategies, some substitutions have led to a loss of target binding. 

Aptamer is a globular-shaped oligonucleotide that is identified by the systematic evolution of ligands by exponential enrichment (SELEX) process. It has the similar composition as natural nucleic acid, but the nucleotides of aptamer have 2 -modified sugars to enhance the nuclease resistance [122]. Since its discovery in 1990, aptamer has become a valuable research tool. Aptamers can specifically bind to target protein with high affinity, therefore it can be utilized in the targeted delivery of siRNA [37,123]. For example, prostate-specific membrane... 

White, R.R. et al. Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2. Proceedings of the National Academy of Sciences of the United States of America 100,5028-5033,2003. 

DNA aptamers are suitable for designing rensable aptasensors, whereas RNA aptamers allow single shot measnrements since they are more susceptible to nucleases action (Sassolas et al., 2009 Strehlitz et al., 2008). Some chemical modifications can enhance nuclease resistance (ie, biostability in serum) and increase the half-life of RNA aptamers. These chemical modifications can be introduced in the oligonucleotides libraries and either dnring or after the SELEX cycle (Kuwahara and Sugimoto, 2010 Stoltenburg et al., 2007). 

The RNA-based aptamer consists of 28 nucleontides of predefined sequence, chemically modified in order to render it resistant to nuclease degradation. Two 20 kDa PEG molecules are also covalently attached at one end of the nucleotide. The overall product molecular mass is 50 kDa. Final product is presented as a sterile solution containing sodium chloride and sodium phosphate as excipients. 

FIGURE 21.5 Chemical structures of modified nucleotides used in the SELEX procedure. Aptamers containing 2 F- and 2 NH2-modified nucleotides are resistant against degradation by nucleases and therefore suitable for in vitro and in vivo applications. Aptamers containing modifications (I or Br) at the C-5 position of pyrimidine can be photo-cross-linked to their protein targets. Arrows indicate the respective chemical modifications. 

Importantly, these mirror-image aptamers exhibited complete resistance against nuclease degradation, similar to the small-molecule-binding mirror-image aptamers previously selected by another group [15, 26]. When tested in a bioassay, the anti-vasopressin L-ssDNA-aptamer inhibited cAMP release mediated by vasopressin, but cAMP release induced by oxytocin (a related peptide hormone) was not affected. 

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