Aspects of Chirality

The essential step during in-vitro selection is amplification of the rare species represented in the combinatorial oligonucleotide library that bind to the target molecule. Amplification of nucleic acids can be accomplished very easily by employing enzymes - DNA polymerases (Box 16) - in a process called PCR. On the other hand, the degradation of aptamers occurs as a result of abundant nucleases which attack specific sites within the naturally configured nucleic acids.

Most enzymes are proteins composed of chiral building blocks, the L-amino acids (with the exception of achiral glycine). Because of the chirality of its building blocks the enzymes themselves are inherently chiral. The word chiral is derived from the Greek word "chtir'', meaning hand. The phenomenon of chirality as an inherent property of biological products was first described by Louis Pasteur [14]. A satisfying definition was given by Lord Kelvin in 1904, in his Baltimore Lectures on molecular dynamics and the wave theory of light: ''I call any geometrical figure, or group of points, chiral, and say it has chirality, if its image in a plane mirror, ideally realized, cannot be brought to coincide with itself.''

The basic concepts of stereochemistry were independently developed by van't Hoff and Le Bel in 1874. Based on their findings Emil Fischer suggested that biological macromolecules are composed of chiral l amino acids and d sugars and in 1894 proposed his famous ''key and lock'' hypothesis that two chiral molecules interact through shape complementarity with each other and are therefore stereo-specific [15].

Thus, enantiomeric nucleic acids which are composed of mirror image nucleo-

tides should be resistant against nucleolytic degradation by naturally occurring enzymes. In mirror-image nucleic acids all nucleotides are converted to the synthetic, enantiomeric form which means inversion of each chiral center (1', 2', 3', 4' in RNA and 1', 3', 4' in DNA). In the two-dimensional representation of mirror-image nucleic acids only the nucleobase of each nucleotide is switched from the right to the left hand side.

Unfortunately, enantiomeric or mirror-image nucleic acids cannot be used directly in the SELEX process because of the lack of (mirror-image) enzymes which would be needed to amplify them.

Most interesting targets for aptamers are peptides or proteins which are chiral targets; both interacting partners are of the same natural chirality. Following the principles of stereochemistry, the mirror-image configurations of identified ap-tamers should interact with the corresponding enantiomers of the targets (first line of Figure 3.4.1). In this circumstance both partners in the complex have the un-

aptamer • target

Fig. 3.4.1. Biological proteins or peptides are composed of naturally occurring i 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 enantio-target * aptamer

Fig. 3.4.1. Biological proteins or peptides are composed of naturally occurring i 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

enantio-target . enantio-aptamer

enantio-aptamer * enantio-target (Spiegelmer)

form a complex having the same characteristics. If an aptamer recognizes a synthetic mirror-image protein, the same aptamer sequence synthesized in the synthetic mirror-image form should consequently bind to the mirror form of the mirror-image protein, i.e. the naturally occurring protein.

enantio-aptamer * enantio-target (Spiegelmer)

form a complex having the same characteristics. If an aptamer recognizes a synthetic mirror-image protein, the same aptamer sequence synthesized in the synthetic mirror-image form should consequently bind to the mirror form of the mirror-image protein, i.e. the naturally occurring protein.

natural chirality. By having two different chiralities in two substance classes - proteins and nucleic acids - in principle two additional pairings or complexes are possible, Figure 3.4.1, bottom line - an aptamer that would bind with high affinity and specificity to the synthetic mirror image form of a target molecule should code the sequence information for a shape, a mirror-image oligonucleotide, that would bind to the naturally occurring form of that target molecule. If these chiral principles are coupled to the powerful screening technology of SELEX, oligonucleotide ligands should be generated that would have the desired binding properties for a given target and at the same time these ligands should be unsusceptible to nucle-olytic degradation by naturally occurring enzymes.

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