Aptamers

Recently, intramers, aptamers that can be expressed inside cells and retain their function [71], were developed. Such intramers might also be generated against biomolecules that are part of a signal transduction pathway. In the future they might be used to elucidate signal transduction cascades triggered by a receptor. 

The search for molecules for the production of protein biochips led us to aptamers. Aptamers are DNA or RNA oligonucleotides with a lengtii of 15 to 60 nucleotides that bind specifically (for example) to protein. The binding affinities lie between Kd 1 pM to Kd 1 fiM. In vitro, aptamers can be produced easily, inexpensively, and in large amoimts. If you incorporate modified nucleotides, they become resistant against nucleases. Because DNA and RNA do not have souls as complicated as proteins, it is also not a problem to incorporate reporter molecules at any location. 

Aptamers can apparently be produced against anything—against ions such as as well 

Low epitope variety, low affinity, and a specificity that is not always encouraging— these are the main problems with the application of aptamers. Does the application of more flexible RNA molecules and special nucleotides help solve these difficulties This question will stiU burn through some doctoral candidates. 

Free signal aptamer (the fluorescence is suppressed by the spatial closeness of quencher and fluorophor) 

Bound signal aptamer (quencher and fluorophor are spatially separated and fluorophor can be stimulated) 

Two modified deoxynucleoside triphosphates, an adenosine derivative modi- 

A DNA aptamer with iV-glycosylase activity has been isolated by in vitro selection. The catalytic rate enhancement is of the order of 10, and the aptamer catalyses the AT-glycosidic cleavage of a specific dG residue. It is dependent on divalent metal cations, and has optimal activity at pH 5. DNA aptamers that catalyse the capping of DNA with AMP to the 5 -end of the DNA, thus creating a 5, 5 -pyrophosphate linkage, have been evolved. The aptamers require Cu and have a catalytic efficiency of 10 M min using either ATP or dATP. 

Kore et have used mutagenic PCR with the triphosphates dPTP (155) and 8-oxo-dGTP to isolate purine-specific hammerhead ribozymes. After five rounds of selection, new ribozymes were isolated with up to 90 times higher in trans cleavage than the starting ribozyme. 

An RNA aptamer has been selected to activate the carboxylic acid of amino acids that mimics the formation of a mixed phosphate anhydride synthesis of aminoacyl tRNA synthetases. The optimal aptamer requires only Ca + for the reaction, and operates at low pH with Xm 50 mM and /ccat 1.1 min for the activation of leucine. This lends support to the concept of translation in an RNA-based world. An RNA-cleaving DNAzyme has been used to target a chemokine receptor required by HIV-1 for entry into susceptible cells. The DNAzyme was found to be very efficient, and specifically interfered with the fusion of cells that harboured the T-lymphocytotropic HIV-1 envelope. 

A series of allosteric hammerhead ribozymes that are activated by theophylline have been evolved by in vitro selection. The ribozymes show a 3000-fold 

These are artificial/natural oligonucleotides (DNA or RNA), in which the principle of the biological lock-and-key recognition is preserved (Fig. 2.5a). 

There is a limited number of studies on aptasensors (aptamer sensors), particularly for the detection of small organic molecules. An electrochemical aptasensor was developed by Kim et al. for the detection of tetracycline using an ssDNA aptamer that selectively binds to tetracycline as the recognition element. The aptamer was highly selective for tetracycline and distinguishes minor structural 

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By similar logic, protein affinity Hbraries have been constmcted to identify protein—protein combining sites, as in antibody—antigen interaction (19) and recombinant Hbraries have been made which produce a repertoire of antibodies in E. coli (20). In another case, a potential DNA-based therapeutic strategy has been studied (21). DNAs from a partially randomized Hbrary were selected to bind thrombin in vitro. Oligonucleotides, termed aptamers that bound thrombin shared a conserved sequence 14—17 nucleotides long. 

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

VEGF inhibitors Humanized neutralizing antibodies, antisense oligonucleotides, siRNA, aptamers... 

Oligonucleic acid or peptide molecules that bind a specific target molecule. Nucleic acid aptamer species can be engineered through treated rounds of in vitro selection to bind to various molecular targets such as... 

Pegaptanib (Macugen) Aptamer VEGF-165 Inhibition of ligand binding AMD... 

Bunka DH, Stockley PG (2006) Aptamers come of age - at last, Nat Rev 4 588-596 Damm EM, Pelkmans L (2006) Systems biology of virus entry in mammalian cells. Cell Microbiol 8 1219-1227... 

Townsend L, Devivar R, Turk S, Nassiri M, Drach J (1995) Design, synthesis, and antiviral activity of certain 2,5,6-trihalo-l-(beta-d-ribofuranosyl)benzimidazoles. J Med Chem 38 4098 105 Turlure F, Devroe E, Silver PA, Engelman A (2004) Human cell proteins and human immunodeficiency virus DNA integration. Front Biosd 9 3187-3208 Umehara T, Fukuda K, Nishikawa F, Kohara M, Hasegawa T, NisUkawa S (2005) Rational design of dual-functional aptamers that inhibit the protease and helicase activities of HCV NS3. J Biochem 137 339-347... 

Kleimnan et al. 2008). In addition, synthetic siRNAs are also subject to degradation in vivo by nuclease activity. Besides side effects and instability, the efficient and specific delivery of the RNAi indncers to the target cell still requires optimization. Here we snmmarize the cnrrent statns of nncleic acid-based antiviral therapentics. The focns will be on antiviral strategies nsing antisense and RNAi technology. Additionally, antiviral ribozymes and aptamers will be discussed briefly, with a focus on recent studies. Gene therapy approaches and delivery systems are the subject of Chapter 11 of this book. 

Matsugami A, Kobayashi S, Ouhashi K, Uesugi S, Yamamoto R, Taira K, Nishikawa S, Kumar PK, Katahira M (2003) Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer. Structure 11 533-545... 

Bridonneau. R, Bunch, S., Tengler, R., Hill, K., Carter, J., Pieken, W., Tinner-meier, D., Lehrman, R., and Drolet, D. W., Purification of a highly modified RNA-aptamer. Effect of complete denaturation during chromatography on product recovery and specific activity, /. Chromatogr. B, 726, 237, 1999. 

Bracht F., Schror K. Isolation and identification of aptamers from defibrotide that act as thrombin antagonists in vitro. Biochem Biophys Res Commun 1994 200,933-7. 

Van den Bossche, J. et al. (2010) Efficient receptor-independent intracellular translocation of aptamers mediated by conjugation to carbon nanotubes. Chemical Communications, 7379-7381. Mu, Q.X., Broughton, D.L. and Yan, B. 

Holeman LA, Robinson SL, Szostak JW, Wilson C (1998) Isolation and characterization of fluorophore-binding RNA aptamers. Fold Des 3 423 -31... 

Gouda, H. Kuntz, I.D. Case, D.A. Kollman, P.A., Free energy calculations for theophylline binding to an RNA aptamer Comparison of MM-PBSA and thermodynamic integration methods, Biopolymers 2003, 68,16-34. 

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