Biology Reference
In-Depth Information
2.2. Protein/nucleic acid interactions
Several technologies have been developed to evaluate protein/nucleic acid
interactions.
32
As for nucleotides, new chemistry has been proposed for
labeling of nucleic acids, and a large panel of dyes have been attached to
DNA or RNA molecules (Fluorescein, Cyanine, Alexa, ATTO, etc.).
One of the major breakthroughs in the design of tools for measuring
protein/nucleic acid interactions is the development of chemistry for accu-
rate high-throughput DNA sequencing by a synthetic approach. DNA
sequencing by synthesis is based on the extension of a primer hybridized
to its target sequence by DNA polymerase with a reversible fluorescent
chain terminator 2
0
-deoxynucleotide.
33,34
Fluorescently labeled reversible
chain terminator nucleotides are stable during the polymerase-mediated
extension step, and their structure (geometry and size) and the location of
the dye within the 2
0
-deoxynucleotide moiety do not prevent their
recognition by standard DNA polymerases.
35
Oligonucleotide can be
easily labeled at the 5
0
-position through the introduction of a primary
amine (NH
2
) at the 5
0
-position to functionalize the corresponding
terminus of the nucleotide for conjugation with an activated
N-Hydroxysuccinimide (NHS) ester or isothiocyanate fluorescent label.
Several hydrophobic spacers have been proposed of 3, 6, or 12 methylene
(CH
2
) groups between the terminal phosphate and the amino part.
Amino-modified nucleotides have been used to incorporate a dye in post-
coupling reactions on the base, with the main advantage of providing
labeling anywhere in the oligonucleotide sequence without altering the
5
0
-position for elongation. Finally, modifications on the deoxyribose have
also been proposed to add more than one dye anywhere in the sequence
or on either terminus after post-coupling reactions between the amine
group and an activated label. In most cases, the dye is linked to the
deoxyribose via a six-carbon-atom spacer, which reduces steric hindrance
(
Fig. 4.8
). As most of the dyes and probes are largely hydrophobic, the risk
of nonspecific association with the protein is present and it is essential to
confirm interactions with displacement experiments using unlabeled
oligonucleotides and other approaches to confirm the affinity values. As for
fluorescently labeled nucleotides, it remains essential to take into account
the impact of the probe on the interaction and binding parameters.
Although RNA and DNA polymerases are known to share the same
general catalytic mechanism, they are all unique in their structural dynamics
and constitute a major challenge for the enzymologist.
36
RNA and DNA
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