Functional aptamers

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... 

Fig. 1. In vitro selection scheme, showing an outline of the steps involved in the in vitro selection of functional aptamers. Sequences from an RNA pool created by combined chemical and enzymatic synthesis are partitioned according to their abilities to perform a desired task. Unfit RNAs are discarded, and the RNAs that are fit to do the task are reverse transcribed, PCR amplified, and regenerated through in vitro transcription. Multiple cycles of selection and amplification should result in the selective enrichment ofthe fittest species.

Functional Aptamers for Proteins and their Application in Biotechnology, Molecular Medicine and Diagnostics... 

Functionally, aptamers can be imagined as nncleic acid versions of antibodies. Their properties are comparable. 

Zhou, J., Li, H., Li, S., Zaia, J., and Rossi, J.J. Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy. Molecular Therapy 16,1481-1489,2008. 

Some strategies with this aim have been reported using, for example, a dual-functional aptamer (Min et al., 2010 Xie and Walton, 2010), an aptamer-cDNA that hybridizes with two aptamers (Zhao et al., 2012), or nanomaterials (Bai et al., 2012 Qian et al., 2010). 

There is a useful public database of functionally active aptamers aptamer. icmb. utexas. edu. 

Maehashi et al. (2007) used pyrene adsorption to make carbon nanotubes labeled with DNA aptamers and incorporated them into a field effect transistor constructed to produce a label-free biosensor. The biosensor could measure the concentration of IgE in samples down to 250 pM, as the antibody molecules bound to the aptamers on the nanotubes. Felekis and Tagmatarchis (2005) used a positively charged pyrene compound to prepare water-soluble SWNTs and then electrostatically adsorb porphyrin rings to study electron transfer interactions. Pyrene derivatives also have been used successfully to add a chromophore to carbon nanotubes using covalent coupling to an oxidized SWNT (Alvaro et al., 2004). In this case, the pyrene ring structure was not used to adsorb directly to the nanotube surface, but a side-chain functional group was used to link it covalently to modified SWNTs. 

Capture array involves the immobilization of non-protein molecules onto the surface which can interact with proteins in the solute phase. Generally, capture molecules may be broad capture agents based on chromatography type surface chemistries such as ion exchange, hydrophobic and metal affinity functionality, or they may be highly specific such as molecular imprinted polymers or oligonucleotide aptamers. 

Hoppe-Seyler, F., Crnkovic-Mertens, I., Tomai, E., and Butz, K. 2004. Peptide aptamers specific inhibitors of protein function. Current Molecular Medicine 4(5), 529-583. 

While indirect selections work quite well for antibodies they have been less successful in the case of catalytic nucleic acids. There are only three examples which prove that it is possible in principle to obtain a ribo- or deoxyribozyme by selecting an aptamer that binds to a TSA A rotamase ribozyme [7], a ribozyme capable of catalyzing the metallation of a porphyrin derivative [92], and one catalytic DNA of the same function [93]. Another study reported the selection of a population of RNA-aptamers which bind to a TSA for a Diels-Alder reaction but the subsequent screen for catalytic activity was negative for all individual RNAs tested [94]. The attempt to isolate a transesterase ribozyme using the indirect approach also failed [95]. 

The fact that, in both selection experiments, new solutions regarding the structure of the functional molecules have been adopted demonstrates that the best sequence for binding is not necessarily the best sequence for performing catalysis. It seems likely that many of the sequence solutions could also have been selected from completely randomized pools. This notion is confirmed by the aforementioned study by Hager and Szostak [82], in which the mutated ATP-aptamer motif was also included in the starting library but where the resulting ribozyme had no relationship to the parent ATP-binding motif. 

Prerequisites to this approach are methods of incorporating non-natural moieties at predetermined positions in the biopolymer. This volume is meant to serve as a source in this respect describing the state of the art of some major lines of attack with this goal in mind. As a modern alternative, the creation of novel catalysts by directed evolution of nucleic acid aptamers is included. In this case, too, it is of prime importance to learn about the structural details which cooperate to bring about the catalytic function underlying the selection process. 

Screening for biological activity The biological activity of the selected RNA aptamers are determined in vitro, as to whether they inhibit the nAChR function as cocaine does or whether they compete with cocaine but do not have any biological activity by themselves and, therefore, protect the receptor against inhibition by cocaine (25). 

Repeat the experiment with 100 pM carbamoylcholine in the presence of increasing concentrations of the RNA aptamer to be tested, in order to determine if the RNA aptamer inhibits receptor function. If an RNA aptamer does not inhibit receptor function, experiments are performed in the presence of a constant concentration of carbamoylcholine and cocaine with increasing concentrations of the RNA aptamer to determine whether the aptamer alleviates inhibition (25). 

A more recent application of redox labeled ODNs is redox-active aptamers that exploit molecular recognition between the aptamer and a target analyte. Briefly, aptamers are functional nucleic acids that selectively bind to a variety of targets. Due to a well-defined three-dimensional structure, aptamers can achieve selectivity comparable to that of antibodies but are readily accessible taking advantage of well-known nucleic acid chemistry, polymeric chain reaction and contemporary separation methods, followed by aptamer selection from random pools of nucleic acids (DNA or RNA) by in vitro selection process called systematic evolution of ligands by... 

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