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 sequences described in Table 1 have been useful as the initial substrates for classifying an unknown type of nuclease activity, either DNase or RNase. These two sequences are recommended as the starting point for any screening, as they have shown a wide capability to detect nuclease activity profiles in bacteria and tissue samples. If nuclease activity is not observed with these DNA and RNA sequences, the design of additional oligonucleotides is required.</li>
			<li>Include polynucleotide sequences consisting of a single type of nucleotide to determine sequence dependence for a given nuclease activity (DNA-Poly A, DNA-Poly T, DNA-Poly C, DNA-Poly G, RNA-Poly A, RNA-Poly U, RNA-Poly C and RNA-Poly G in Table 1).</li>
			<li>Using the same sequence of nucleotide residues as the initial DNA and RNA sequences, include sequences that contain nucleoside analogs harboring chemical modifications at the 2&#39;-position of the ribose sugar, such as 2&#39;-Fluoro and 2&#39;-O-Methyl, as a stringent step for increasing the selectivity of the nucleases.
			<ol>
				<li>Include fully modified sequences consisting entirely of 2&#39;-Fluoro nucleoside analogs or 2&#39;-O-Methyl nucleoside analogs (All 2&#39;-F and All 2&#39;-OMe, Table 1)</li>
				<li>Include chimeric sequences consisting of unmodified natural purines and 2&#39;-Fluoro pyrimidine nucleoside analogs (RNA Pyr-2&#39;F, Table 1).</li>
				<li>Include chimeric sequences consisting of unmodified natural purines and 2&#39;-O-Methyl pyrimidine nucleoside analogs (RNA Pyr-2&#39;OMe, Table 1).</li>
				<li>Include chimeric sequences consisting of unmodified natural pyrimidines and 2&#39;-Fluoro purine nucleoside analogs (RNA Pur-2&#39;F, Table 1).</li>
				<li>Include chimeric sequences consisting of unmodified natural pyrimidines and 2&#39;-O-Methyl purine nucleoside analogs (RNA Pur-2&#39;OMe, Table 1).</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Oligonucleotide probe preparation and storage 
	NOTE: Oligonucleotides are synthesized by the phosphoramidite method. After synthesis, the oligonucleotides are purified using high performance liquid chromatography (HPLC) and the mass is measured by mass spectrometry.
	<ol>
		<li>Spin down the lyophilized oligonucleotide probes using a centrifuge and dilute them in Tris-EDTA (TE) buffer to prevent nuclease degradation. Determine the dilution volume according to the yield of each probe to render a stock solution with a concentration of 500 pmol/&#956;L.</li>
		<li>Store lyophilized oligonucleotides at 4 &#176;C or room temperature for a short term. For long term storage (months to years), store lyophilized oligonucleotides at -20 &#176;C. Upon resuspension of lyophilized oligonucleotides in TE, store the stock solutions at -20 &#176;C or, preferably, at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Bacterial culture
<ol>
	<li>Take one porous glass bead from the cryogenic storage vial under sterile conditions.</li>
	<li>To isolate individual bacterial colonies (Salmonella and E. coli), use the quadrant method24 by streaking/rolling the bead directly onto a Petri dish containing Tryptic Soy Agar (TSA) medium supplemented with defibrinated sheep blood.</li>
	<li>Incubate the culture at 37 &#176;C for 24 h.</li>
</ol>
3. Supernatant preparation
<ol>
	<li>Transfer a single colony from solid media to 50 mL Tryptic Soy Broth (TSB) and incubate at 37 &#176;C for 24 h at 200 rpm.</li>
	<li>Dilute the culture (1:500) in TSB (sub-culture) and incubate at 37 &#176;C for 24 h at 200 rpm in a shaking incubator. 
	NOTE: It is expected that after 24 h of incubation the bacterial cultures are in stationary phase reaching values higher than 109 CFU/mL.</li>
	<li>After the incubation period, transfer the cultures to capped sterile tubes and centrifuge at 4,500 x g for 30 min.</li>
	<li>Collect the supernatant and use it immediately. Alternatively, store the supernatants at 4 &#176;C or -20 &#176;C.</li>
</ol>
4. Nuclease Activity Assay
<ol>
	<li>Preparation of working solutions
	<ol>
		<li>Prepare a 20 &#956;L working solution for each probe in 1.5 mL nuclease free microcentrifuge tubes by diluting (1:10 ratio) the stock solution (500 pmol/&#956;L) for a final working concentration of 50 pmol/ &#956;L. For that purpose, mix 18 &#956;L of Phosphate Buffer Saline (PBS) containing MgCl2 and CaCl2 with 2 &#956;L of probe stock solution. 
		NOTE: Be aware that in the working solution, the oligonucleotides are more vulnerable to nuclease degradation, therefore, it is recommended to prepare the working solutions just before setting up the reaction.</li>
		<li>Avoid direct light contact during the preparation and keep in the dark (wrapped in foil), when dealing with fluorophores.</li>
	</ol>
	</li>
	<li>Reaction set up
	<ol>
		<li>Pre-warm the fluorometer (e.g. Cytation 1) to 37 &#176;C.</li>
		<li>Prepare a set (one tube/probe) of 1.5 mL nuclease free microcentrifuge tubes for each sample (TSB, E. coli and Salmonella) and label accordingly.</li>
		<li>Carefully add 96 &#956;L of TSB sterile culture media, Salmonella supernatant or E. coli supernatant (from the step 3.4.). Subsequently, add 4 &#956;L/tube of probe working solution accordingly. Perform this step at room temperature. 
		NOTE: 10 probes were used for the first round of the screening and 6 probes for the second round.</li>
		<li>Mix thoroughly by pipetting up and down to obtain a homogenous solution. Avoid introducing air bubbles into the samples while mixing.</li>
		<li>Load 95 &#956;L of each solution (probe + supernatant or culture media) into a separate well of a black bottom, non-treated 96 well plate. Minimize the formation of bubbles in the wells upon loading by dispensing carefully with the tip close to the wall of the well.</li>
		<li>Cover the plate with its lid. Visually inspect the lid and check for pen markings or dust particle accumulation that may introduce measurement artifacts. Replace the lid for a new one if that is the case.</li>
	</ol>
	</li>
	<li>Measurement set up
	<ol>
		<li>Open the acquisition software (Gen5 3.05) by clicking the software shortcut icon (Figure S1A).</li>
		<li>Select Read Now from the task manager window and choose New&#8230; to create the kinetic measurement protocol (Figure S1B).</li>
		<li>Click on Set Temperature in the dialog window titled Procedure and select 37 &#730;C. Confirm and save the settings by pressing OK (Figure S1C).</li>
		<li>Click on Start Kinetics in the dialog window titled Procedure. Then in the pop-up dialog window, titled Kinetic Step, select 2 h in the Run Time input box and 2 min in the Interval input box. Confirm and save the settings by pressing OK (Figure S1D).</li>
		<li>Click on Read in the dialog window titled Procedure. Then in the pop-up dialog window, titled Read Method, select Fluorescence Intensity as a detection method, Endpoint/Kinetic as a read type and Filters as optics type. Confirm and save the settings by pressing OK (Figure S1E).</li>
		<li>In the pop-up dialog window titled Read Step (Kinetic), select Green from the Filter Set. Confirm and save the settings by pressing OK (Figure S2A).</li>
		<li>In the dialog window titled Procedure, select use lid and click on validate to ensure that the created protocol is valid. This is confirmed by a pop-up dialog window (Figure S2B).</li>
		<li>Select protocol in the menu bar and choose procedure (Figure S2C).</li>
		<li>In the dialog window titled Procedure, define the wells to be measured (Figure S2D).</li>
		<li>Enter the name of the experiment in the file name input box (Figure S2E).</li>
		<li>Load the plate with its lid into the plate reader. Ensure the plate is in the right orientation.</li>
		<li>Start the acquisition by clicking read new button in the toolbar (Figure S2F).</li>
	</ol>
	</li>
	<li>Data Analysis
	<ol>
		<li>Click on one of the measured wells in the dialog window titled Plate 1 (Figure S3A).</li>
		<li>Click select wells and include all the measured wells in the well selection dialog window. Confirm and save the settings by pressing OK. (Figure S3B).</li>
		<li>Select data in the dialog window titled Plate 1 to visualize the tabulated results (Figure S3C).</li>
		<li>Export the data to a spread sheet by selecting quick export from the context menu (Figure S3C).</li>
		<li>In the spread sheet, label the data columns accordingly for each sample and probe.</li>
		<li>Generate kinetic graphs, using line with markers style, by plotting relative fluorescence units (RFU) versus time (x-axis: timeline of the reaction and y-axis: RFUs). 
		NOTE: The description of generating a graph is applicable when using spreadsheet programs (e.g., Excel). However, any other programs can be used to generate graphs from the raw data obtained, by plotting RFU versus time.</li>
		<li>Calculate the rate of the enzymatic reaction for each measured interval according to the following formula25: rate= <img alt="Equation 1" src="/files/ftp_upload/60005/60005eq1.jpg" />, where If is RFU at the maximal interval time point and Ii is RFU at the minimal interval time point, and Tf and Ti are the maximal and minimal interval time points, respectively.</li>
		<li>Select the rate with the highest value (Rmax) for each curve. If there is more than one Rmax per sample, select the one that occurs at the earliest measurement time point.</li>
		<li>Calculate the ratio between Rmax and the average value of the time interval at which Rmax occurs: rate coefficient = <img alt="Equation 2" src="/files/ftp_upload/60005/60005eq2.jpg" /></li>
		<li>Calculate the fold difference (FD) between the rate coefficient of Salmonella and E. coli for each probe.</li>
		<li>Consider a fold difference value higher than 3 as significant.</li>
		<li>Consider the significant probes with the highest fold difference values (FD) as candidate probes and as the basis for the library design in the next screening round.</li>
	</ol>
	</li>
</ol>
5. Screening Rounds&#8217; Selection Criteria (Figure 1)
<ol>
	<li>First Round of Screening
	<ol>
		<li>Perform the nuclease activity assay (as described in section 4) using DNA, RNA and polynucleotide probes.</li>
		<li>Evaluate the preference for DNA chemistry or RNA chemistry by comparing the number and performance (FD value) of DNA and RNA candidate probes.</li>
		<li>Select the type of nucleic acid chemistry rendering the greatest number of candidate probes, as illustrated in Figure 1.</li>
	</ol>
	</li>
	<li>Second Round of Screening
	<ol>
		<li>Perform the nuclease activity assay (as described in section 4) using fully modified probes and chimeric probes designed based on the nucleic acid substrate selected in the first screening round.</li>
		<li>Evaluate the preference towards sequences containing 2&#39;-Fluoro and 2&#39;-O-Methyl nucleoside analogs by comparing the results obtained for 2&#39;-Fluoro and 2&#39;-O-Methyl modified candidate probes.</li>
		<li>Select the type of nucleoside analog modification rendering the greatest number of candidate probes, as illustrated in Figure 1.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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">
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		<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. ...

kinetic-screening-of-nuclease-activity-using-nucleic-acid-probes

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JoVE Journal

Biochemistry

8.2K Views.  Linköping University. 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.

Video: Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

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 rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

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

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 probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

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

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

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 purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

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

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, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

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

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 and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

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 measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

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 application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

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 ...

Identifying G-Quadruplexes in a DNA Template Using Chemical Mapping

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

Author Spotlight: Characterizing DNA G-Quadruplex by Bis-3-Chloropiperidine Based Chemical Mapping 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

Author Spotlight: Advancements in DNA Nanosensors &#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Assays for DNA Enzymes Using Synthetic Oligonucleotides

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

Methods Article

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes