Selection of RNA Aptamers

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 

In almost every published SELEX protocol, the experiment starts with a pool of chemically synthesized ssDNA. The DNA usually consists of a central randomized region of 20-60 nucleotides that is flanked by two constant regions that are necessary for primer binding. 

The randomized region is obtained by using a mixture of all four bases in each synthesis step. In most cases, the four building blocks are mixed with balanced stoichiometry (IUB mix code N ). However, pools may also be synthesized for the selection of aptamers that contain only three different bases in the randomized region [12] or unequal frequencies of the four bases [13]. 

Such ssDNA pools are available from various commercial suppliers of oligonucleotides. The main tasks in designing starting pools concern the definition of primer 

Amplification of the DNA template is achieved by PCR (for details, see [14]). The size and amount of the PCR product should be routinely checked by agarose gel electrophoresis. An insufficient amount of product may be improved by increasing the number of PCR cycles. However, care should be taken not to overdo the cycling, because this leads to a loss of dsDNA after several PCR cycles the amount of free primers decreases dramatically so that only a fraction of the DNA template strands anneal with a primer and yield new dsDNA. The high complexity of the pool causes that ssDNA is not able to rehybridize with the complementary strand. 

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Ulrich H, Magdesian MH, Alves MJ et al (2002) In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion. J Biol Chem 277 20756-20762... 

Barfod A, Persson T, Lindh J (2009) In vitro selection of RNA aptamers against a conserved region of the Plasmodium falciparum erythrocyte membrane protein. Parasitol Res 105 1557-1566... 

Klug, S.J., Huttenhofer, A. and Famulok, M. (1999) In vitro selection of RNA aptamers that bind special elongation factor Sel, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl... 

Misono, T.S. and Kumar, P.K.R. 2005. Selection of RNA aptamers against human influenza virus hemagglutinin using surface plasmon resonance. Anal Biochem 342 312-317. 

We have succeeded in the selection of RNA aptamers carrying multiple biotin groups in their side chains (70). Cytidine triphosphate (CTP) carrying the biotinyl group at the A -position was used for in vitro selection. A pool of... 

Ohuchi, S. P., Ohtsu, T., Nakamura, Y. (2006). Selection of RNA aptamers against recombinant transforming growth factor-beta type III receptor displayed on cell surface. Biochimie 88, 897-904. 

Figure 14.1 General scheme for the in vitro selection of RNA aptamers...

Hall, B. et al. In vitro selection of RNA aptamers to a protein target by filter immobilization. Current Protocols in Molecular Biology Chapter 24, Unit, 2009. 

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]. 

Figure 3.20 TAR RNA DCC SELEX system, employing 2 -ammo-2-deoxyuri-dine (U-NH ) capable of reversible imine formation with the appended aldehydes Rb, Rc, and Re. Selected appended RNA aptamers and their corresponding dissociation constants are shown at the bottom.

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... 

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.

Hlwang B, Lee SW, Improvement of RNA aptamer activity against myasthenic autoantibodies by extended sequence selection, Biochem. Biophys. Res. Commun., 290 656-662, 2002. 

Burgstaller P, Famulok M. Isolation of RNA aptamers for bi- 47. ological cofactors by in-vitro selection. Angew. Chem. Int. Ed. 

Konig J, Julius C, Baumann S, Homann M, Goringer HU, Feldbmgge M. Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers. RNA 2007 13 614-622. 

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],... 

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