cleave different target sequences providing that Watson–Crick recognition between the enzyme and sub-
strate is maintained. Recently a ‘10 –23’ DNAzyme capable of achieving cleavage rates of up to 10 min^1
under certain metal ion, concentration and pH conditions has been identified, while a related DNAzyme
also reported by Joyce^53 , the ‘8–17’ motif, has been exploited as a biosensor for Pb^2 ions.
5.7.3.4 Modified Nucleotides Used in SELEX. The diversity of functional groups available for
binding and catalysis offered by the four natural nucleotides is limited. Furthermore, the pKavalues of the
nucleobases are not ideal to permit electrostatic transition-state stabilisation or general acid–base mech-
anisms at appropriate neutral pH. Consequently, both sugar and base modified nucleotides have been exploited
in SELEX, but such modified nucleotides must be substrates for T7 RNA polymerase or a suitable DNA
polymerase to replace their natural analogues during replication or transcription of the template DNA or
RNA pool respectively.
Analogues such as 5-(1-pentynyl)-2-deoxyuridine (Figure 5.29, structure c) have been used in the
selection of aptamers designed to bind thrombin. These aptamers display similar binding to thrombin as
the unmodified 15-mer aptamers, but have different structures and do not function if the analogue is
replaced by 2-deoxyuridine or thymidine. The potential use of aptamers as therapeutics has led to attempts
to increase their stability towards degradation by nuclease enzymes.Thus 2-fluoro- and 2-amino modi-
fied ribonucleotides have been employed in SELEX.^54 These analogues impart a considerably increased
stability to RNA in respect of both chemical and enzymatic hydrolysis. Complete resistance to nucleases
can be achieved if the aptamers are comprised of mirror image or L-RNA (spiegelmers).^61
A recent variation suitable for the isolation of very high affinity nucleic acid ligands is photo-SELEX.^62
Here a 5-bromo-UTP, -2-fluoro-UTP or -dUTP is employed as one of the nucleotide units and the affinity
chromatography step is followed by a brief UV laser irradiation step at 308 nm, during which the single-
stranded nucleic acid ligand is cross-linked to a proximal electron-rich amino acid in a protein target. The
complexes are purified by SDS–PAGE and following proteolysis with proteinase K, the aptamer functions as
202 Chapter 5
Figure 5.29 A selection of modified nucleoside triphosphates that have been used in the selection of nucleic acid
aptamers and catalysts