Spiegelmers Binding to Small Molecules

The first functional mirror-image oligonucleotides, spiegelmers, described in 1996 were generated to bind to small molecules, i.e. the amino acid arginine and the nucleoside adenosine [18, 19]. For in-vitro selection against adenosine, the target in the enantiomeric configuration was covalently coupled to a matrix. A standard

Spiegelmers

characterized and tested for binding to the mirror-image form of the naturally occurring target. The sequences of identified binders are then synthesized in the mirror-image form to create mirror-image oligonucleotides, the so called spiegelmers. Because of the rules of stereochemistry the spiegelmers bind to the naturally occurring form ofthe intended target molecule.

selected Binder (D-oMgonucleotide) Fig. 3.4.2. Schematic representation of spiegelmer technology. A library of 1015 different oligonucleotides is synthesized and incubated with the mirror-image (or enantiomeric) form of a naturally occurring target. After several cycles of selection and amplification the enriched library is cloned in an appropriate vector and sequenced. Individualized binding oligonucleotides are further

Spiegelmer (L-oligonucleotide)

characterized and tested for binding to the mirror-image form of the naturally occurring target. The sequences of identified binders are then synthesized in the mirror-image form to create mirror-image oligonucleotides, the so called spiegelmers. Because of the rules of stereochemistry the spiegelmers bind to the naturally occurring form ofthe intended target molecule.

library containing 60 random positions flanked by primer binding sites was synthesized. A typical scheme showing how to create an RNA library is depicted in Figure 3.4.3.

Isolation of L-adenosine binders was essentially as outlined in Figure 3.4.2 and described by Sassanfar and Szostak [20]. To improve stereospecificity counter-selection using the natural target D-adenosine was introduced in later stages of selection [21]. After ten cycles of in-vitro selection and subsequent cloning and sequencing, high-affinity binding motifs (see appendix) against adenosine were identified that had the expected reciprocal chiral specificity; Figure 3.4.4. Competition binding experiments using equilibrium dialysis and radioactively labeled Land D-adenosine were performed to estimate binding constants. Figure 3.4.4A shows the competition curves with the cognate and the non-cognate binding partners. The estimated dissociation constants for the binding complexes are, on average, 2.3 mM whereas the non-binding complexes result in significantly weaker binding of approx. 20 mM, if at all (Figure 3.4.4B). The mirror-inverted CD spectra of the adenosine binding aptamer (d-RNA) and spiegelmer (l-RNA) indicate that

254 I 3.4 Enantiomeric Nucleic Acids - Spiegelmers primer A

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