Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

This protocol provides a powerful alternative for screening nuclease activity as biomarker of disease, with an easy-to-implement methodology even for researchers who are not as specialized in nucleic acid probes. The main advantage of this technique is the ability to select nucleic acid probes that can identify both known and unknown nuclease activities, taking advantage of the probe-nuclease dynamic interaction. Other advantages of this methodology are its flexibility, high reproducibility and ease of use.

The demonstration will be performed by Khadija, a Masters student, and Baris, a postdoc from our laboratory. When designing an oligonucleotide library, include at least one DNA and one RNA random sequence containing a combination of adenine, guanine, cytosine and thiamine or uracil. To prepare the oligonucleotide probes, spin down the lyophilized oligonucleotide probes and dilute each probe in Tris-EDTA buffer at a 500 picomolar per microliter concentration to prevent nuclease degradation.

For bacterial culture on solid medium, roll a single porous glass bead from cryogenic storage directly onto one culture dish containing TSA supplemented with defibrinated sheep blood to streak out individual bacterial colonies. Then place the plate at 37 degrees Celsius for 24 hours. For bacterial culture in liquid medium, transfer a single colony from a solid medium culture to 50 milliliters of TSB for incubation at 37 degrees Celsius for 24 hours at 200 rotations per minute.

The next day, dilute the culture at a one to 500 ratio in fresh TSB and incubate the bacteria for an additional 24 hours at 37 degrees Celsius and 200 rotations per minute in a shaking incubator. To set up a nuclease activity assay, first pre-warm a fluorometer to 37 degrees Celsius and carefully add 96 microliters of sterile TSB or supernatant from a Salmonella or E.Coli liquid medium culture to one 1.5 milliliter nuclease-free microcentrifuge tube per probe. Add four microliters of probe working solution to each tube and use a pipette to thoroughly mix each tube contents until homogenous solutions are achieved, taking care to avoid bubbles.

Next, carefully load 95 microliters of each solution close to the wall of individual wells of a black bottom, non-treated 96 well plate, taking care to avoid bubbles. When all of the solutions have been added, cover the plate and visually inspect the lid for pen markings or dust that may introduce measurement artifacts. To set up the software for nuclease activity measurement, open a suitable acquisition software program.

Select Read Now from the Task Manager window, and select New to create the kinetic measurement protocol. Click Set Temperature to select 37 degrees Celsius and confirm and save the settings by clicking OK.Click Start Kinetics. In the pop-up window, select two hours in the Run Time input box and two minutes in the interval input box before clicking OK to confirm and save the settings.

Click Read. In the pop-up window, select Fluorescence intensity as the Detection Method, Endpoint Kinetic as the Read Type and Filters as the Optics Type. Then click OK.In the pop-up window, select Green from the Filter Set and click OK.In the Procedure window, select Use lid and click Validate.

A pop-up window will appear confirming that the created protocol is valid. Under the Protocol menu, select Procedure. In the Procedure window, define the wells to be measured and enter the name of the experiment in the File name input box.

Then load the plate into the plate reader, taking care that the plate is in the right orientation and click the Read New button to begin the acquisition. For data analysis, open the data in the appropriate analysis software, and select one of the measured wells in the plate one window. Click Select Wells and include all of the measured wells in the Well Selection Dialog window before clicking OK.Then select Data in the plate one window to visualize the tabulated results and click QuickExport to export the data to a spreadsheet.

In a spreadsheet, label the data columns as appropriate for each sample and probe, and plot the relative fluorescence units versus time for the data to generate kinetic graphs. In this representative experiment, after the first round of screening, the Salmonella culture supernatants reported a clear preference for RNA probes over the DNA probes. Based on the identification of RNA as the preferred nucleic acid type by Salmonella nucleases, a new RNA-only library is designed to be used in the second round of screening.

In contrast, E.Coli in culture medium controls demonstrated a very limited ability to degrade RNA probes. After a second round of screening using chemically-modified nucleotides aimed at increasing the specificity of the RNA probes, RNA pyrimidine 2'O-methyl and RNA purine 2'O-methyl could be identified on the basis of their chemical modifications as exhibiting the best performing kinetic behavior when compared with the RNA pyrimidine 2'fluoro and RNA purine 2'fluoro respectively. These results suggest that Salmonella has an important RNAse activity with differential substrate chemistry preference that can be used for selecting probes capable of specifically recognizing this bacteria.

This procedure enables the selection of probes that are able to identify nuclease activities associated with disease conditions, such as cancer or bacterial infection, allowing the development of novel clinical diagnostic tools.