Aptamers isolation

A large collection of aptamers (probably more than 200 at this time) aimed at protein targets has been made and studied. The affinities have been measured (usually in solution) as monovalent binding interactions between a unique aptamer sequence and the protein the measured binding constants (kjs) are shown in Figure 20.3 for the first 100 or so aptamers isolated at NeXstar Pharmaceuticals and at the University of Colorado in Boulder. 

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. 

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

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]. 

A new dimension in the development of nucleic acid based catalysts was introduced by Breaker and Joyce in 1994 when they isolated the first deoxyribozyme [111]. It is not unexpected that DNA is also able to catalyze chemical reactions because it was shown previously that ssDNA aptamers which bind to a variety of ligands can be isolated by in vitro selection [141]. In the meantime, several deoxyribozymes have been described which expand the range of chemical transformations accelerated by nucleic acid catalysts even further and raising question whether even catalytic DNA might have played some role in the pre-biotic evolution of hfe on earth [69-71]. 

Isolate individual white colonies, each containing one aptamer sequence. 

Usually, a minimum of five repetitions of steps 2-8 is required to yield an RNA pool that predominantly consists of the best target-binding RNA molecules. Individual aptamers are then isolated from this pool by cloning, and identified by sequencing. 

Figure 7.2 shows the individual steps of the isolation of RNA aptamers. The steps of one selection cycle are highlighted in gray. Usually, it takes 2 or 3 days to complete one cycle. Robots, however, can perform such an experiment in just a few hours... 

After 5 or, up to > 15 rounds of selection, single aptamers can be isolated and identified by cloning and sequencing. The characterization of aptamers can be... 

This mixture was heated for 1 min to 95 °C and was then immediately incubated on ice. Then 10 pL 5x RT buffer, 2pL DTT (100 mM), 4pL dNTP-mix (4mM of each clNTP), and 1 j lL Super Script1 II reverse transcriptase (200 U pL 1) was added. Reverse transcription was performed for lh at 42 °C. This mixture was subsequently used in the PCR (see above). After round 11, several aptamers were isolated and identified by cloning and sequencing [28]. 

Davis, J.H. and Szostak, J.W. (2002) Isolation of high-affinity GTP aptamers from partially structured RNA libraries, Proc. Natl. Acad. Sci. USA 99, 11616-11621. 

Figure 8. A sketch of the SELEX technique used to select for molecules with optimal binding constants to predefined target molecules. The SELEX procedure selects for molecules with sufficiently high binding constants, so called aptamers, in two steps. Target molecules are attached to a chromatographic column which allows for selective retention of sufficiently strong binders. A different solvent is applied to release the binders and canalize them to the next selection round. Commonly some tens of selection cycles (Figure 7) are sufficient to isolate optimal binding RNA molecules.

Because it is necessary to employ enzymes during in-vitro selection, natural nucleotides must be used in the process to isolate aptamers. Consequently, the re-... 

Fig. 3.4.1. Biological proteins or peptides are composed of naturally occurring L amino acids whereas all naturally occurring nucleic acids are composed of D sugars. If an isolated aptamer interacts with a protein target by building a stable complex, both structures composed of the synthetic mirror image form (enantio-target and enantio-aptamer) should...

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