Name: kinetic screening of nuclease activity using nucleic acid probes
Uploaded: 2019-11-01T12:30:00
Description: Watch this Scientific Journal Video about Kinetic Screening of Nuclease Activity using Nucleic Acid Probes at JoVE.com

The authors would like to acknowledge Luiza I. Hernandez (Link&#246;ping University) for her careful revision of the manuscript and valuable advice. This work was supported by The Knut and Alice Wallenberg Foundation and The Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Link&#246;ping University (Faculty Grant SFO-Mat-LiU No. 2009-00971).

1. Oligonucleotide library design and preparation
<ol>
	<li>Library design
	<ol>
		<li>Based on the following criteria design a library of quenched fluorescent oligonucleotide probes between 8 and 12-mer long to avoid secondary structure formation, with equal length for all probes.
		<ol>
			<li>Include at least one DNA and one RNA random sequence containing a combination of adenine (A), guanine (G), cytosine (C) and thymine (T)/uracil (U) (DNA and RNA probes in Table 1). 
			NOTE: The DNA and RNA...

<ol>
	<li>Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleases:+diversity+of+structure,+function+and+mechanism.">Nucleases: diversity of structure, function and mechanism.</a> Quarterly Reviews of Biophysics. 44 (1), 1-93 (2011).</li><li>Adli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR+tool+kit+for+genome+editing+and+beyond.">The CRISPR tool kit for genome editing and beyond.</a> Nature Communication. 9 (1), 1911 (2018).</li><li>Mason, P. A., Cox, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+DNA+exonucleases+in+protecting+genome+stability+and+their+impact+on+ageing.">The role of DNA exonucleases in protecting genome stability and their impact on ageing.</a> Age (Dordr). 34 (6), 1317-1340 (2012).</li><li>Berends, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclease+expression+by+Staphylococcus+aureus+facilitates+escape+from+neutrophil+extracellular+traps.">Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps.</a> Journal of Innate Immunity. 2 (6), 576-586 (2010).</li><li>Kiedrowski, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+Nuc2+is+a+functional,+surface-attached+extracellular+nuclease.">Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.</a> PLoS One. 9 (4), 95574 (2014).</li><li>Morita, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall-anchored+nuclease+of+Streptococcus+sanguinis+contributes+to+escape+from+neutrophil+extracellular+trap-mediated+bacteriocidal+activity.">Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.</a> PLoS One. 9 (8), 103125 (2014).</li><li>Hasegawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+virulence-associated+and+cell-wall-located+DNase+of+Streptococcus+pyogenes.">Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes.</a> Microbiology. 156, 184-190 (2010).</li><li>Moon, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+catalytic+and+substrate+binding+mechanisms+of+the+strategic+EndA+nuclease+from+Streptococcus+pneumoniae.">Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae.</a> Nucleic Acids Research. 39 (7), 2943-2953 (2011).</li><li>Li, L. Y., Luo, X., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endonuclease+G+is+an+apoptotic+DNase+when+released+from+mitochondria.">Endonuclease G is an apoptotic DNase when released from mitochondria.</a> Nature. 412 (6842), 95-99 (2001).</li><li>McDermott-Roe, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endonuclease+G+is+a+novel+determinant+of+cardiac+hypertrophy+and+mitochondrial+function.">Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.</a> Nature. 478 (7367), 114-118 (2011).</li><li>Wang, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+link+between+adipogenesis+and+innate+immunity:+RNase-L+promotes+3T3-L1+adipogenesis+by+destabilizing+Pref-1+mRNA.">A link between adipogenesis and innate immunity: RNase-L promotes 3T3-L1 adipogenesis by destabilizing Pref-1 mRNA.</a> Cell Death &amp; Disease. 7 (11), 2458 (2016).</li><li>Li, C. L., Yang, W. Z., Shi, Z., Yuan, H. 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I., Ozalp, V. C., Hernandez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclease+activity+as+a+specific+biomarker+for+breast+cancer.">Nuclease activity as a specific biomarker for breast cancer.</a> Chemical Communication (Cambridge). 52 (83), 12346-12349 (2016).</li><li>Machado, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+specific+detection+of+Salmonella+infections+using+chemically+modified+nucleic+acid+probes.">Rapid and specific detection of Salmonella infections using chemically modified nucleic acid probes.</a> Analytica Chimica Acta. 1054, 157-166 (2019).</li><li>Sanders, E. 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Alterations of nuclease activity have been associated with a wide variety of disease phenotypes, including different types of cancer and bacterial infections. These alterations are proposed to be the causative agent of a condition14, while in other cases they are the consequence of a detrimental physiological event20 or pathogenic agent16,26. Not surprisingly, attempts to use nucleases and nuclease activity as a dia...

Nucleases are a class of enzymes capable of cleaving the phosphodiester bonds that form the backbone structure of nucleic acid molecules. The vast diversity of nucleases makes their classification rather difficult. However, there are some common criteria used to describe nucleases, such as substrate preference (deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), cleavage site (endonucleases or exonucleases), or metal ion dependency, among others1. Nucleases are highly conserved catalytic enzymes that have fundamental roles in both, prokaryotic and eukaryotic organisms and have been used, and continue to be used, as gene editing tools2. They are also fundamental actors in DNA maintenance and replication, helping to keep genome stability and participating in proof-reading processes3. In bacteria, for example, nucleases have been identified as important virulence factors, able to promote bacterial survival by reducing the efficacy of the host&#39;s immune system4,5,6,7,8. In mammals, nucleases have been suggested to be implicated in apoptosis9, mitochondrial biogenesis and maintenance10 and mediation of antibacterial and antiviral innate immune responses11. Not surprisingly, nuclease activity alterations, whether enhancement or lack of, have been implicated in a wide array of human diseases. These diseases range from a wide variety of cancers12,13 to cardiac hypertrophy10 or autoimmune diseases14. Therefore, nucleases have become interesting candidates as biomarkers for a heterogeneous group of human conditions. In fact, nucleases have already shown their potential as successful diagnostic tools for the detection of infections caused by specific bacterial agents, such as Staphylococcus aureus or Escherichia coli15,16. In many cancer types, expression of staphylococcal nuclease domain-containing protein 1 (SND1) ribonuclease is indicative of poor prognosis17. In pancreatic cancer patients, elevated ribonuclease I (RNase I) serum levels have been reported18 and proposed to be associated with cancerous cell phenotypes19. In ischemic heart conditions, such as myocardial infarction or unstable angina pectoris, deoxyribonuclease I (DNase I) serum levels have been shown to be a valid diagnostic marker20,21.
It has been hypothesized that the global blueprint of nuclease activity may be different in healthy and disease states. In fact, recent reports have used differences in nuclease activity to distinguish between healthy and cancerous phenotypes22 or to identify pathogenic bacterial infections in a species-specific manner15,23. These findings have opened a new avenue for the use of nucleases as biomarkers of disease. Therefore, there exists a necessity for the development of a comprehensive screening method able to systematically identify disease associated differences in nuclease activity, which may be of key importance in the development of new diagnostic tools.
Herein, we introduce and describe a new in vitro screening approach (Figure 1) to identify sensitive and specific probes capable of discriminating between nuclease activity in healthy and unhealthy, or activity specific to a type of cell or bacteria. Taking advantage of the modularity of nucleic acids, we designed an initial library of quenched fluorescent oligonucleotide probes consisting of a comprehensive set of different sequences and chemistries, both being important parameters for library design. These oligonucleotide probes are flanked by a fluorophore (fluorescein amidite, FAM) and a quencher (tide quencher 2, TQ2) at the 5&#39; and 3&#39; ends respectively (Table 1). By using this fluorescent resonance energy transfer (FRET) based fluorometric assay to measure the kinetics of enzymatic degradation, we were able to identify candidate probes with the potential to discriminate differential patterns of nuclease activity associated with healthy or disease states. We designed an iterative process, in which new libraries are created based on the best candidate probes, that allows the identification of ever more specific candidate probes in subsequent screening steps. Moreover, this approach takes advantage of the catalytic nature of nucleases to increase sensitivity. This is achieved by taking advantage of the activatable nature of the reporter probes and the ability of nucleases to continually process substrate molecules, both representing key advantages over alternative antibody or small molecule-based screening methods.
This approach offers a highly modular, flexible and easy to implement screening tool for the identification of specific nucleic acid probes capable of discriminating between healthy and disease states, and an excellent platform for the development of new diagnostic tools that can be adapted for future clinical applications. As such, this approach was used to identify the nuclease activity derived from Salmonella Typhimurium (herein referred to as Salmonella) for the specific identification of this bacteria. In the following protocol, we report on a method to screen for bacterial nuclease activity using kinetic analysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:759px;" width="759">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Black bottom, non-treated 96 well plate</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:128px;">10000631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Cytation1</td>
			<td style="width:199px;">BioTek</td>
			<td style="width:128px;">CYT1FAV</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Eppendorf tubes</td>
			<td>Thermofisher</td>
			<td style="width:128px;">11926955</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Escherichia coli</td>
			<td style="width:199px;">ATCC</td>
			<td style="width:128px;">25922</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Microbank cryogenic storage vial containing beads</td>
			<td style="width:199px;">Pro-Lab Diagnostics</td>
			<td style="width:128px;">22-286-155</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Nucleic acid probes</td>
			<td style="width:199px;">Biomers.net</td>
			<td style="width:128px;">#</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Phosphate Buffer Saline containing MgCl2 and CaCl2</td>
			<td style="width:199px;">Gibco&#8482;</td>
			<td style="width:128px;">14040117</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Salmonella enterica subs. Enterica</td>
			<td style="width:199px;">ATCC</td>
			<td style="width:128px;">14028</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Tris-EDTA</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:128px;">10647633</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Tryptone Soya Agar with defibrinated sheep blood</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:128px;">10362223</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:344px;">Tryptic Soy Broth</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:128px;">22092</td>
			<td></td>
		</tr>
	</tbody>
</table>

kinetic screening of nuclease activity using nucleic acid probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Figure 1 shows the work flow of this methodology, which is divided into two screening rounds. In the first round of screening, we used 5 DNA probes (DNA, DNA Poly A, DNA Poly T, DNA Poly C and DNA Poly G) and also 5 RNA probes (RNA, RNA Poly A, RNA Poly U RNA Poly C and RNA Poly G). The raw data of this screening round can be found in Supplementary Table 1. In the second round, chemically modified probes were synthesized by replacing the RNA sequence with chemically modified...

Watch this Scientific Journal Video about Kinetic Screening of Nuclease Activity using Nucleic Acid Probes at JoVE.com

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

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.

Research

JoVE Journal

Biochemistry

A Polyaniline-based Sensor of Nucleic Acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...