Chemistry Reference
In-Depth Information
helix-turn-helix (HTH) motif where the two helices have been derived from the
DNA-binding engrailed homeodomain and the turn reproduces the sequence of the
calcium binding site of calmodulin. Addition of Ce(IV) or Eu(III) leads to the for-
mation of a peptide/lanthanide ion complex which retains the HTH tertiary struc-
ture. The Ce(IV) complex (and to a minor extent also the Eu(III) complex) promotes
the cleavage of plasmid and linear double-stranded DNA and the reactivity of the
complex is similar to that of the free ion. However, while the uncomplexed metal
ion cleaves randomly both the P-O3
′
and P - O5
′
bonds, the peptide complex is regi-
oselective for P-O3
bonds. Furthermore, modest sequence selectivity for T/C rich
sequences was also observed.
In a similar approach, Sugiura and Nomura have succeeded in the preparation
of zinc - fi nger peptides with hydrolytic ability toward phosphate esters, by mutating
some amino acid residues coordinated to the zinc ion.
46a
This mutant is based on
the sequence of the second fi nger in the three-tandem zinc fi nger protein Sp1, a
DNA binding protein that binds specifi cally to GC boxes. Tandem alignment of
three zinc fi nger mutants in a way similar to natural proteins resulted in effi cient
and site selective cleavage of plasmid DNA pUC19GC, and of a 37 bp DNA duplex,
both containing a GC box. In another example, two zinc-fi nger peptides were con-
nected to a peptide linker able to bind lanthanide ions.
46b
In the presence of Ce(IV)
ions, the system cleaves linear dsDNA with high sequence selectivity.
′
13.6 Conjugation with Sequence - Selective Elements
A more exciting and fascinating application of the DNA hydrolytic catalysts is the
obtainment of artifi cial restriction enzymes with higher or different sequence spe-
cifi city than natural systems. However, such a goal requires highly specifi c sequence
recognition while most of the systems so far described produce random cleavage of
DNA or show only modest sequence selectivity.
A much higher selectivity is required and, in principle, this can be obtained by
metal complexes conjugated with DNA oligonucleotides (for antisense recognition
or triple helix formation) or PNA fragments. At the present time, only a very few
examples of such systems are known. Komiyama and coworkers
47
fi rst reported, in
1994, a conjugate in which an iminodiacetate metal-binding group was appended
to a 19-mer DNA oligonucleotide. In the presence of Ce(IV) ions, the DNA-
iminodiacetate conjugate effi ciently cleaved single-stranded 40-residue DNA at the
linkage between residues 30 and 31, according to the predicted selectivity, with scis-
sion of the P-O5
bond. Noticeably, the cleavage of the single-stranded DNA by this
conjugate is much more effi cient than hydrolysis of the dinucleotide TpT by Ce(IV)-
iminodiacetate, suggesting that the DNA moiety also has a role in delivering the
metal ion close to cleavable phosphate groups.
Krämer and coworkers described more recently a family of PNA-metal chelat-
ing group conjugates whose Zr(IV) complexes selectively cleave single-stranded
DNA oligonucleotides.
48
The cleavage is less effi cient than in the case of Komiya-
′
