Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

So my research group were really interested in enzymes involved in DNA replication and repair. In particular, we're studying enzymes from extremophilic microorganisms and also from pathogens, because we want to understand how these enzymes let those organisms survive environmental challenges. We're interested both in the biological function of the enzymes and also in their biotechnological applications.

So we work a lot with DNA and RNA ligases, and we've recently established the role of a minimal type of DNA ligase, which seems to be trafficked to the outside of the bacterial cell. And we've determined that this plays a role in biofilm formation in the pathogen neisseria gonorrhoeae. We've also, together with our collaborators from ArcticZymes characterized a novel RNA to DNA ligase, which is able to attach DNA adapters to either end of a piece of RNA.

So our collaborators at ArcticZymes, who've also contributed to this protocol, are characterizing different DNA and RNA metabolizing enzymes, which have potential applications in molecular biology. And so they're using this type of protocol as a first step for understanding the spectrum of activities these enzymes have and benchmarking them relative to what's already available on the market. The protocol offers an easier and safer alternative for SA nucleic acid metabolizing enzymes, compared to the use of radio-labeled substrates.

The fluorescently labeled DNA or RNA substrates make our approach more efficient, as we can mix and match the oligonucleotides to make a variety of substrates. So, at the moment, we're really focused on understanding the biological function of some novel nucleases that we've discovered in an Antarctic environmental genome. These nucleases have homologs in other extremophilic organisms, and so we're trying to understand what role they play in allowing these microbes to survive.

We're also really interested in the activity of some of our novel enzymes on non-natural nucleic acid analogs and their potential uses as part of a molecular biology toolkit with non-natural DNA and RNA substrates To begin, centrifuge the lyophilized oligonucleotide containing tube at full speed in a benchtop centrifuge for two to five minutes. Resuspend the oligonucleotides in TE buffer to prepare a master stock of 100 micromolar and gently vortex the tube. Dilute an aliquot of master stock with TE buffer to prepare a 10 micromolar stock and prepare the reaction master mix.

After dispensing the mix to the required tubes, anneal the oligonucleotides in a thermocycler at 95 degrees Celsius for five minutes. Let the tube cool to room temperature for 30 minutes to one hour. Then add nucleotide co-factors and other heat sensitive buffer components to the master mix.

If required, store the mix at minus 20 Degrees Celsius for future use. Combine 22.5 microliters of the substrate master mix with 2.5 microliters of the DNA ligase in a PCR tube. Immediately place the reaction tubes in a PCR machine at 25 degrees Celsius for 30 minutes.

To quench the reaction, add five microliters of loading dye and incubate at 95 degrees Celsius for five minutes. To begin, prepare a stock of 20%acrylamide, seven molar urea, and one XTBE solution. For one gel, combine 10 milliliters of acrylamide and urea solution with 100 microliters of 10%ammonium persulfate, and three microliters of tetramethylethylenediamine, and cast it in a gel caster.

After the gel solidifies, pre-run the gel in one XTBE buffer for 30 minutes at 10 milliamperes per gel with external heating. Using a pasture pipette flush one XTBE into the gel wells to remove access urea. Load 10 microliters of each reaction and run for one to 1.5 hours at 45 to 55 degrees Celsius.

Visualize the gel with the correct settings for the chosen fluorophore. Finally, quantify the band intensity of the product and the substrate using Image J and calculate the percentage of the product, using the formula. DNA Ligase activity led to an increase in oligonucleotide size from 20 nucleotides to 40 nucleotides observed on a urea page gel.

The bacterial DNA ligase ligated both nick and mismatched DNA substrates and can utilize both magnesium and manganese for ligation activity with a preference for magnesium.