Download Deoksyribozymes active in the presence of cadmium ions and at low

Deoksyribozymes active in the presence of cadmium ions and at low pH
conditions obtained by selection in vitro
Aleksandra Kasprowicz
This dissertation is devoted to DNAzymes, catalytic DNA molecules, which –
similarly to protein-based enzymes – have the ability to catalyze many biochemical reactions.
So far, no DNAzymes have been found in nature. Those which are known by now have been
obtained using in vitro selection. DNAzymes are applied in medicine as potential therapeutics
as well as in biotechnology as specific molecular tools. Nowadays, they are most commonly
applied in qualitative and quantitative analysis. Since most DNAzymes require cofactors in
the form of divalent metal ions for their catalytic activity, they have been successfully used
for construction of biosensors which are capable of metal ions detection.
The aim of this thesis was to obtain DNAzymes which are catalytically active in the
presence of cadmium ions. Cadmium is considered to be one of the main chemical pollutants
in the environment. It is extremely toxic to living organisms, even at very low concentrations.
Due to its high toxicity cadmium poses a serious threat to the health of humans and animals.
Search for DNAzymes which are able to catalyze cleavage of the RNA phosphodiester bond
in the presence of Cd2+ ions was performed by in vitro selection using the oligonucleotides
combinatorial library with a 23-nucleotide random region.
Unexpectedly, the first selection yielded DNAzymes active in a low pH environment
with the optimum activity at pH 4.0–4.5. In vitro selection experiments are always associated
with a risk of not obtaining variants with the expected characteristics or obtaining variants
with a broader spectrum of activity than we originally assumed. So far, a majority of literature
reports refer to variants of catalytic DNA molecules which require cofactors in the form of
divalent metal ions and a physiological pH environment. In view of the interesting features of
the selected pH-dependent DNAzymes, a series of experiments were performed to better
characterize their catalytic properties. Interestingly, the selected DNAzymes require neither
Cd2+ ions nor other metal ions for their activity. The results of spectroscopic studies using CD
and UV-vis spectroscopy suggest that in an environment of pH 4.0–4.5 there occur
protonation of functional groups of nucleotides involved in the forming of the DNAzymes
catalytic centre and the folding of molecules into an appropriate secondary structure. It is
highly probable that nitrogen bases of the sugar-phosphate backbone of deoxyribozymes
which undergo protonation do not only play a structural role but also directly participate in the
catalysis. Based on the results of the secondary structures probing of pH-dependent
DNAzymes by nuclease S1 and dimethyl sulfate, essential catalytic elements were defined.
Additionally, it was shown that the 5'- and 3'-terminal regions of these molecules have no
effect on their catalytic properties. Catalytically active pH-dependent trans-acting variants
were also designed.
As a result of the second experiment of in vitro selection, DNAzymes active in the
presence Cd2+ ions were obtained. However, the obtained variants showed broader specificity
and were active also with other metal ions. They exhibited the highest catalytic activity in the
presence of Cd2+ ions and lower in the presence of Co2+, Mn2+ and Zn2+ions. This suggests
that forming of an ion-DNAzyme complex does not require a highly specific coordination
mode of metal ions. Based on the results of the chemical and enzymatic mapping of the
secondary structure of one of the selected DNAzymes and a comparison of the sequence of all
the obtained variants, it was found that they belong to the 8-17 type DNAzymes. In order to
maintain high catalytic activity, the obtained DNAzymes required a temperature of 25°C,
which may be needed to ensure the proper folding of the molecules into a catalytically active
structure. Studies using directed mutagenesis have shown that the presence of a purine residue
in position 12 of the catalytic core is essential for the catalytic activity. On the other hand, the
nucleotide composition of the hairpin stem which is present in the catalytic region can affect
the catalytic activity level of the DNAzymes as well as their metal ions preferences. An
attempt was made at obtaining Cd2+-dependent trans-acting DNAzymes, however none of the
six engineered variants showed catalytic activity.