Aptamers to Nucleotides NucleosidesNucleobases

The first aptamer to a nucleotide was isolated in 1993 by Sassanfar and Szostak (1993). The selection of this RNA aptamer to adenosine was performed with an ATP-agarose column; bound RNA was eluted with free ATP. A 40-nucleotide truncated aptamer bound ATP with a Kd of 0.7 mmol/L. Elution experiments with ATP analogs revealed that the base and the sugar of the ATP were recognized by the aptamer while the 5' phosphates did not take part in binding.

To determine whether a DNA aptamer can also bind adenosine/ATP, Huizenga and Szostak (1995) selected DNA aptamers for ATP. One characterized aptamer bound adenosine with a Kd of 6 mmol/L. Just like the RNA aptamer to ATP, this DNA bound ATP via the base and the sugar and the 5' phosphate had no in-

Fig. 4.1 Xanthine and guanine. The RNA aptamer selected for xanthine also binds guanine, while adenine, cytosine, and uracil are not bound.

Xanthine Guanine

Xanthine Guanine

Fig. 4.1 Xanthine and guanine. The RNA aptamer selected for xanthine also binds guanine, while adenine, cytosine, and uracil are not bound.

fluence on binding. Furthermore, the sizes of the RNA and DNA aptamers to ATP were similar (40 versus 42, respectively), so one could draw the conclusion that aptamers (of similar complexity) for one target molecule can be isolated from both RNA and DNA pools. However, the selected RNA and DNA aptamers had significantly different sequences and fold into different structures because the DNA lacks the 2'-hydroxyl group of the RNA. This was demonstrated when the DNA version of the RNA aptamer for ATP and the RNA version of the DNA aptamer for ATP were tested for binding, respectively, and neither recognized ATP. A difference between the RNA and DNA aptamers was detected by nuclear magnetic resonance (NMR) studies of the aptamers, revealing that the DNA aptamer binds two ATP molecules, while the RNA aptamer binds just one (Lin and Patel, 1997).

Kiga et al. (1998) isolated an RNA aptamer binding xanthine and guanine (Fig. 4.1). The aptamers were selected via affinity chromatography with xanthine agarose; bound RNA was eluted with free xanthine. A representative RNA aptamer was truncated to 32 nucleotides and turned out to bind xanthine with a Kd of 3.3 mmol/L. Guanine was bound by the aptamer as well, while adenenine, cyto-sine, and uracil were not bound.

RNA aptamers that exhibited a new mode of purine recognition were published by Meli et al. (2002). The aptamer contained two relatively independent secondary structure elements, which formed a bipartite RNA structure that bound adenine with a Kd of 10 mmol/L. In this aptamer the imidazole moiety of adenine is not trapped in the binding side, as it is in other adenine/ATP-binding aptamers. Hence this aptamer could be supposed to be a cofactor-binding site in a complex ribozyme.

In 2004 RNA aptamers that recognized the triphosphate of ATP were selected in the laboratory of Jack W. Szostak (Sazani et al., 2004). By employing a restrictive selection protocol containing AMP-agarose for counterselection and washing the selection column (ATP-agarose) with free AMP prior to elution of ATP-bound RNA, they could isolate aptamers that strongly interacted with the p and g phosphate. The Kd-value of a 57-nucleotide minimized aptamer was 11 mmol/L for ATP and 1700 mmol/L for AMP. In contrast to other ATP aptamers, this aptamer showed binding to GTP, UTP, and CTP, also. The aptamer was not isolated in an earlier selection for ATP aptamers, although it had an affinity comparable to other ATP aptamers. That might be due to its more complex structure. Therefore, this triphosphate-binding aptamer could first be isolated after counterselection for the simpler ATP-binding molecules.

Koizumi and Breaker (2000) published the selection of RNA aptamers for cAMP. For this selection cAMP was attached at the C8 position of agarose via a nine-atom spacer. Elution of bound RNA was achieved by washing the column with buffer containing cAMP. After four rounds of selection the enriched pool contained two classes of aptamers. Class II aptamers exhibited Kd-values of about 10 mmol/L for cAMP. These aptamers did not bind to 5' or 3' phosphory-lated adenosine like ATP, 5'-AMP, or 3'-AMP. However, adenine and adenosine were bound with the same affinity as cAMP. Hence it is assumed that the apta-mer recognizes the target via the adenosine and the (groups on the) ribose can influence the specificity via steric interactions. Remarkable is the fact that the ap-tamers seemed to interact with the part of the target molecule that faced the matrix, and did not strongly interact with the part that had greatest accessibility.

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