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In the early 1980s, RNA molecules that can catalyze enzymatic
reactions were discovered and named ribozymes. This discovery was
followed by demonstrations in the 1990s that DNA also can act as
enzymes, termed deoxyribozymes or DNAzymes.
With only four nucleotides as building blocks, versus 20 in proteins,
nucleic acid enzymes may need to recruit cofactors (helper molecules)
to perform some functions. Metal ions are a natural choice, and indeed
most nucleic acid enzymes require metal ions for function under
physiological conditions (and therefore are called metalloenzymes).
Metalloenzymes use various modes for functions for which
metal-dependent conformational change (induced fit) is required in
some cases but not in others (lock and key). In contrast, most
ribozymes require conformation change that almost always precedes the
enzyme reactions.
Using an extremely sensitive measurement technique called
single-molecule fluorescence resonance energy transfer, Lu, physics
professor Taekjip Ha and their research team studied the
metal-dependent conformational change and cleavage activity of a
particular lead-sensitive DNAzyme.
In single-molecule fluorescence resonance energy transfer, researchers
add two dye molecules � one green and one red � to the molecule they
want to study. Then they excite the green dye with a laser. Some of
the energy moves from the green dye to the red dye, depending upon the
distance between them.
�The changing ratio of the two intensities indicates the relative
movement of the two dyes,� said Ha, who also is an affiliate of the
university�s Institute for Genomic Biology and of the Howard Hughes
Medical Institute. �By monitoring the brightness of the two dyes, we
can measure conformational changes with nanometer precision.�
The researchers found that, in the presence of zinc or magnesium, a
conformational change took place in the DNAzyme, followed by a
cleavage reaction (behavior similar to many proteins and ribozymes).
In the presence of lead, however, the cleavage reaction occurred
without a preceding conformational change.
�This presents very strong evidence that the lead-specific enzyme uses
the lock and key reaction mechanism,� Lu said. �This DNAzyme appears
to be prearranged to accept lead for the activity.�
In previous work, Lu and his research group fashioned highly sensitive
and selective fluorescent, colorimetric and magnetic resonance imaging
sensors from the lead-specific DNAzyme used in this study. They have
also constructed simple, disposable sensors using a different,
uranium-specific DNAzyme.
�We think the answer to faster, more sensitive sensors lies with the
lock and key mechanism,� Lu said. �Our next step is to look for other
metals that use the lock and key mechanism with other specific
DNAzymes. In addition, we want to investigate the structural details
at the metal-binding sites and see how they change during catalysis.�
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