Abstract

Helicases are enzymes that couple nucleoside triphosphate turnover
(“NTPase”) with the separation of double-stranded oligonucleotides (“helicase”
activity).  Due to helicases’ central
role in protein synthesis, as well as other cellular functions, many cancers
are highly dependent on the activity of one or more, making helicases
candidates as drug targets for the treatment of various cancers.  Traditionally helicase activity has monitored
using radiochemistry; however more recently fluorescent dye-labelled
oligonucleotides have become the mainstay to overcome the practical limitations
of using radionuclides.  To this end, fluorescent
dye-labelled oligonucleotides were synthesised as single-stranded
oligonucleotides; which were subsequently used to produce the double-stranded
substrates.  In order to ensure reagent quality
and therefore assay quality, we utilised several biophysical analytical
techniques to monitor and quantify the annealing reactions.  The results clearly indicated that such analytical
techniques are essential to ensuring precise and accurate assay reagent quality.