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

Forfatterne vil gerne anerkende Luiza I. Hernandez (Linköping Universitet) for hendes omhyggelige revision af manuskriptet og værdifuld rådgivning. Dette arbejde blev støttet af Knut og Alice Wallenberg Foundation og den svenske regering strategisk forskning område i materialevidenskab om avancerede funktionelle materialer på Linköping University (fakultet Grant SFO-mat-LiU No. 2009-00971).

1. oligonukleotid bibliotek design og forberedelse
<ol><li>Biblioteks design<ol>
<li>Baseret på følgende kriterier designe et bibliotek af standset fluorescerende oligonukleotid sonder mellem 8 og 12-Mer lang for at undgå sekundær struktur dannelse, med samme længde for alle sonder.<ol>
<li>Omfatte mindst ét DNA og en RNA-tilfældig sekvens, der indeholder en kombination af adenin (A), guanin (G), cytosin (C) og thymin (T)/uracil (U) (DNA og RNA-Proder i tabel 1). Bemærk: de DNA-og RNA-sekven...

<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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tudor+staphylococcal+nuclease+is+a+structure-specific+ribonuclease+that+degrades+RNA+at+unstructured+regions+during+microRNA+decay.">Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.</a> RNA. 24 (5), 739-748 (2018).</li><li>Wan, L., 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=MTDH-SND1+interaction+is+crucial+for+expansion+and+activity+of+tumor-initiating+cells+in+diverse+oncogene-+and+carcinogen-induced+mammary+tumors.">MTDH-SND1 interaction is crucial for expansion and activity of tumor-initiating cells in diverse oncogene- and carcinogen-induced mammary tumors.</a> Cancer Cell. 26 (1), 92-105 (2014).</li><li>Keyel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dnases+in+health+and+disease.">Dnases in health and disease.</a> Developmental Biology. 429 (1), 1-11 (2017).</li><li>Hernandez, F. J., 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=Noninvasive+imaging+of+Staphylococcus+aureus+infections+with+a+nuclease-activated+probe.">Noninvasive imaging of Staphylococcus aureus infections with a nuclease-activated probe.</a> Nature Medicine. 20 (3), 301-306 (2014).</li><li>Flenker, K. S., 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+Detection+of+Urinary+Tract+Infections+via+Bacterial+Nuclease+Activity.">Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.</a> Molecular Therapy. 25 (6), 1353-1362 (2017).</li><li>Kannan, N., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tipping+the+balance:+MTDH-SND1+curbs+oncogene-induced+apoptosis+and+promotes+tumorigenesis.">Tipping the balance: MTDH-SND1 curbs oncogene-induced apoptosis and promotes tumorigenesis.</a> Cell Stem Cell. 15 (2), 118-120 (2014).</li><li>Kemmer, T. P., Malfertheiner, P., Buchler, M., Kemmer, M. L., Ditschuneit, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+ribonuclease+activity+in+the+diagnosis+of+pancreatic+disease.">Serum ribonuclease activity in the diagnosis of pancreatic disease.</a> International Journal of Pancreatology. 8 (1), 23-33 (1991).</li><li>Fernandez-Salas, E., Peracaula, R., Frazier, M. L., de Llorens, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleases+expressed+by+human+pancreatic+adenocarcinoma+cell+lines.">Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.</a> European Journal of Biochemistry. 267 (5), 1484-1494 (2000).</li><li>Fujibayashi, K., 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=Serum+deoxyribonuclease+I+activity+can+be+a+useful+diagnostic+marker+for+the+early+diagnosis+of+unstable+angina+pectoris+or+non-ST-segment+elevation+myocardial+infarction.">Serum deoxyribonuclease I activity can be a useful diagnostic marker for the early diagnosis of unstable angina pectoris or non-ST-segment elevation myocardial infarction.</a> Journal of Cardiology. 59 (3), 258-265 (2012).</li><li>Kawai, Y., 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=Diagnostic+use+of+serum+deoxyribonuclease+I+activity+as+a+novel+early-phase+marker+in+acute+myocardial+infarction.">Diagnostic use of serum deoxyribonuclease I activity as a novel early-phase marker in acute myocardial infarction.</a> Circulation. 109 (20), 2398-2400 (2004).</li><li>Hernandez, L. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+plating+methods.">Aseptic laboratory techniques: plating methods.</a> Journal of Visualized Experiment. (63), e3064 (2012).</li><li>Worthington Biochemical Corporation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Clinical Enzyme Measurements. , (1972).</li><li>Burghardt, E. L., 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,+Culture-Free+Detection+of+Staphylococcus+aureus+Bacteremia.">Rapid, Culture-Free Detection of Staphylococcus aureus Bacteremia.</a> PLoS One. 11 (6), 0157234 (2016).</li><li>Bibette, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaining+confidence+in+high-throughput+screening.">Gaining confidence in high-throughput screening.</a> Proceedings of the National Academy of Science U. S. A. 109 (3), 649-650 (2012).</li><li>Hernandez, L. I., Machado, I., Schafer, T., 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=Aptamers+overview:+selection,+features+and+applications.">Aptamers overview: selection, features and applications.</a> Current Topics in Medicinal Chemistry. 15 (12), 1066-1081 (2015).</li><li>Pande, J., Szewczyk, M. M., Grover, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+display:+concept,+innovations,+applications+and+future.">Phage display: concept, innovations, applications and future.</a> Biotechnology Advances. 28 (6), 849-858 (2010).</li><li>Skerra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+binding+proteins:+anticalins+-+harnessing+the+structural+plasticity+of+the+lipocalin+ligand+pocket+to+engineer+novel+binding+activities.">Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities.</a> FEBS Journal. 275 (11), 2677-2683 (2008).</li><li>Ku, T. H., 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=Nucleic+Acid+Aptamers:+An+Emerging+Tool+for+Biotechnology+and+Biomedical+Sensing.">Nucleic Acid Aptamers: An Emerging Tool for Biotechnology and Biomedical Sensing.</a> Sensors (Basel). 15 (7), 16281-16313 (2015).</li><li>Jayasena, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers:+an+emerging+class+of+molecules+that+rival+antibodies+in+diagnostics.">Aptamers: an emerging class of molecules that rival antibodies in diagnostics.</a> Clinical Chemistry. 45 (9), 1628-1650 (1999).</li><li>Gray, B. P., Brown, K. 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Ændringer af nukleaseaktivitet har været forbundet med en bred vifte af sygdoms fænotyper, herunder forskellige typer af kræft og bakterielle infektioner. Disse ændringer foreslås at være det udløsende middel for en betingelse14, mens de i andre tilfælde er konsekvensen af en skadelig fysiologisk hændelse20 eller sygdomsfremkaldende agens16,26. Ikke overraskende, forsøg på at bruge nukleaser og nuclease ...

Nukleaser er en klasse af enzymer, der kan kløende fosfodiester-obligationerne, som danner rygraden i nukleinsyremolekyler. Den store mangfoldighed af nukleaser gør deres klassificering temmelig vanskelig. Der er dog nogle almindelige kriterier, der anvendes til at beskrive nukleaser, såsom substrat præference (deoxyribonukleinsyre (DNA) eller ribonukleinsyre (RNA)), spaltning site (endonukleaser eller exonucleaser), eller metal ion afhængighed, blandt andre1. Nukleaser er stærkt bevaret katalytiske enzymer, der har fundamentale roller i både prokaryotiske og eukaryotiske organismer og er blevet brugt, og fortsat anvendes, som genredigerings værktøjer2. De er også grundlæggende aktører inden for DNA-vedligeholdelse og-replikering, hvilket medvirker til at bevare genomstabiliteten og deltage i korrekturlæsning3. I bakterier, for eksempel, er nukleaser blevet identificeret som vigtige virulensfaktorer, i stand til at fremme bakteriel overlevelse ved at reducere effektiviteten af værtens immunsystem4,5,6,7 ,8. I pattedyr, er nukleaser blevet foreslået at være impliceret i apoptose9, mitokondrie Biogenese og vedligeholdelse10 og mægling af antibakterielle og antiviral medfødte immunrespons11. Ikke overraskende, nuclease aktivitet ændringer, uanset om forbedring eller mangel på, har været impliceret i en bred vifte af sygdomme hos mennesker. Disse sygdomme spænder fra en bred vifte af kræftformer12,13 til Hjertehypertrofi10 eller autoimmune sygdomme14. Derfor er nukleaser blevet interessante kandidater som biomarkører for en uensartet gruppe af menneskelige forhold. Faktisk har nukleaser allerede vist deres potentiale som vellykkede diagnostiske værktøjer til påvisning af infektioner forårsaget af specifikke bakterielle agenser, såsom Staphylococcus aureus eller Escherichia coli15, 16. i mange kræfttyper, udtryk for stafylokok nuclease domæne-holdige protein 1 (SND1) ribonuklease er tegn på dårlig prognose17. Hos patienter med kræft i bugspytkirtlen er forhøjet ribonuklease i (RNase i) serumniveauer blevet rapporteret18 og foreslået at være associeret med kræft celle fænotyper19. I iskæmiske hjertelidelser, såsom myokardieinfarkt eller ustabil angina pectoris, har deoxyribonuclease i (DNase i) serumniveauer vist sig at være en gyldig diagnostisk markør20,21.
Det har været en hypotese, at den globale plan for nukleaseaktivitet kan være forskellige i raske og sygdomstilstande. Faktisk har nylige rapporter brugt forskelle i nukleaseaktivitet til at skelne mellem sunde og kræft fænotyper22 eller til at identificere sygdomsfremkaldende bakterielle infektioner på en artsspecifik måde15,23. Disse fund har åbnet en ny Avenue for brugen af nukleaser som biomarkører af sygdom. Der er derfor en nødvendighed for at udvikle en omfattende screeningsmetode, der systematisk kan identificere sygdomsrelaterede forskelle i nukleaseaktiviteten, hvilket er af afgørende betydning for udviklingen af nye diagnostiske værktøjer.
Heri introducerer og beskriver vi en ny in vitro-screening tilgang (figur 1) for at identificere følsomme og specifikke sonder, der kan diskriminere mellem nukleaseaktiviteten i sund og usund, eller aktivitet, der er specifik for en type celle eller bakterier. Ved at udnytte nukleinsyrer modularitet designede vi et første bibliotek af dæmpede fluorescerende oligonukleotidsonder bestående af et omfattende sæt af forskellige sekvenser og kemi, som begge er vigtige parametre for Biblioteks design. Disse oligonukleotidsonder flankeres af en fluoroforet (fluorescein amidite, Fam) og en QUENCHER (tidevands QUENCHER 2, TQ2) ved henholdsvis 5 ' og 3 ' enderne (tabel 1). Ved at bruge denne fluorometriske analyse af energioverførsel (FRET) til måling af den enzymatiske nedbrydnings kinetik var vi i stand til at identificere kandidat sonder med potentiale til at diskriminere differentialmønstre af nukleaseaktivitet forbundet med raske eller sygdomstilstande. Vi designede en iterativ proces, hvor nye biblioteker oprettes baseret på de bedste kandidat sonder, som gør det muligt at identificere stadigt mere specifikke kandidat sonder i efterfølgende Screenings trin. Desuden udnytter denne fremgangsmåde den katalytiske karakter af nukleaser for at øge følsomheden. Dette opnås ved at drage fordel af den aktivatable karakter af reporter sonder og evne til nukleaser til kontinuerligt at behandle substrat molekyler, begge repræsenterer de vigtigste fordele i forhold til alternative antistof eller små molekyle-baserede screeningmetoder.
Denne tilgang tilbyder et yderst modulært, fleksibelt og let at gennemføre screeningsværktøj til identifikation af specifikke nukleinsyre sonder, der kan diskriminere mellem raske og sygdomstilstande, og en fremragende platform for udvikling af nye diagnostiske værktøjer, der kan tilpasses til fremtidige kliniske anvendelser. Som sådan blev denne metode anvendt til at identificere nukleaseaktiviteten afledt af salmonella typhimurium (i det følgende benævnt salmonella) til den specifikke identifikation af disse bakterier. I den følgende protokol rapporterer vi om en metode til at screene for bakteriel nukleaseaktivitet ved hjælp af kinetisk analyse.

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	<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

Nukleaser er en klasse af enzymer, der nedbryder nukleinsyrer ved at katalysere hydrolyse af fosfodiester bindinger, der forbinder de ribose sukkerarter. Nukleaser viser en række vitale fysiologiske roller i prokaryotiske og eukaryotiske organismer, der spænder fra at opretholde genom stabilitet til at yde beskyttelse mod patogener. Ændret nukleaseaktivitet har været forbundet med flere patologiske tilstande, herunder bakterielle infektioner og kræft. Til dette formål har nukleaseaktiviteten vist et stort potentiale for at blive udnyttet som en specifik biomarkør. En robust og reproducerbar screeningsmetode, der er baseret på denne aktivitet, er dog fortsat meget ønskelig.
Heri introducerer vi en metode, der gør det muligt at screene for nukleaseaktivitet ved hjælp af nukleinsyre sonder som substrater med omfanget af differentiering mellem patologiske og sunde tilstande. Denne metode giver mulighed for at designe nye sonde biblioteker, med stigende specificitet, på en iterativ måde. Således er flere runder af screening er nødvendige for at raffinere sonder ' design med forbedrede funktioner, drage fordel af tilgængeligheden af kemisk modificerede nukleinsyre syrer. Den foreslåede teknologis betydelige potentiale ligger i dens fleksibilitet, høje reproducerbarhed og alsidighed til screening af nukleaseaktiviteten i forbindelse med sygdomstilstande. Det forventes, at denne teknologi vil gøre det muligt at udvikle lovende diagnostiske værktøjer med et stort potentiale i klinikken.

Figur 1 viser arbejdsstrømmen for denne metode, som er opdelt i to screening runder. I den første runde af screening, vi brugte 5 DNA-sonder (DNA, DNA poly A, DNA poly T, DNA poly C og DNA poly G) og også 5 RNA prop'er (RNA, RNA poly A, RNA poly U RNA poly C og RNA poly G). De rå data for denne screening runde kan findes i supplerende tabel 1. I anden runde blev kemisk modificerede sonder syntetiseret ved at udskifte RNA-sekvensen med kemisk modificerede Nukleosider (all...

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

Ændret nukleaseaktivitet har været forbundet med forskellige menneskelige forhold, underliggende dets potentiale som en biomarkør. Den modulære og nem at implementere screeningmetode præsenteret i dette papir giver mulighed for udvælgelse af specifikke nukleinsyre sonder til at udnytte nuclease aktivitet som en biomarkør af sygdom.

Kinetisk screening af Nukleaseaktivitet ved hjælp af nukleinsyre sonder

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

Denne protokol giver et stærkt alternativ til screening nukleos aktivitet som biomarkør for sygdom, med en let at gennemføre metode, selv for forskere, der ikke er så specialiseret i nukleinsyre sonder. Den største fordel ved denne teknik er evnen til at vælge nukleinsyre sonder, der kan identificere både kendte og ukendte nuklease aktiviteter, drage fordel af sonden-nuklease dynamisk interaktion. Andre fordele ved denne metode er dens fleksibilitet, høje reproducerbarhed og brugervenlighed.Demonstrationen vil blive udført af Khadija, en Masters studerende, og Baris, en postdoc fra vores laboratorium. Når du designer et oligonucleotidbibliotek, skal der omfattes mindst én DNA- og én RNA-tilfældig sekvens, der indeholder en kombination af adenin, guanine, cytosin og thiamin eller uracil. For at forberede oligonucleotid sonder, spin ned lyophilized oligonucleotid sonder og fortynde hver sonde i Tris-EDTA buffer på en 500 picomolar per mikroliter koncentration for at forhindre nuklease nedbrydning.For bakteriekultur på fast medium rulle en enkelt porøs glasperle fra kryogen opbevaring direkte på en kulturskål, der indeholder TSA suppleret med defibrineret fåreblod for at stribe individuelle bakteriekolonier. Derefter placere pladen på 37 grader Celsius i 24 timer. For bakteriekultur i flydende medium, overføre en enkelt koloni fra en fast medium kultur til 50 milliliter TSB til inkubation ved 37 grader Celsius i 24 timer ved 200 rotationer i minuttet.Den næste dag, fortyndes kulturen på en til 500 forholdet i frisk TSB og inkubere bakterierne i yderligere 24 timer ved 37 grader Celsius og 200 rotationer i minuttet i en rystende inkubator. For at oprette en nuklease aktivitet assay, først pre-varme et fluorometer til 37 grader Celsius og omhyggeligt tilføje 96 mikroliter af steril TSB eller supernatant fra en Salmonella eller E.Coli flydende medium kultur til en 1,5 milliliter nuklease-fri mikrocentrifuge rør pr sonde. Tilføj fire mikroliter af sonde arbejdsopløsning til hvert rør og bruge en pipette til grundigt at blande hvert rør indhold, indtil homogene løsninger er opnået, idet du skal passe på at undgå bobler.Dernæst forsigtigt indlæse 95 mikroliter af hver opløsning tæt på væggen af individuelle brønde af en sort bund, ikke-behandlet 96 godt plade, idet du skal passe på at undgå bobler. Når alle opløsningerne er tilsat, dækkes pladen, og låget inspiceres visuelt for penmarkeringer eller støv, der kan indføre måleartefakter. Hvis du vil konfigurere softwaren til nukleaseaktivitetsmåling, skal du åbne et passende anskaffelsesprogram.Vælg Læs nu i vinduet Jobliste, og vælg Ny for at oprette den kinetiske måleprotokol. Klik på Angiv temperatur for at vælge 37 grader Celsius og bekræft og gem indstillingerne ved at klikke på OK. Klik på Start kinetik. I pop op-vinduet skal du vælge to timer i inputfeltet Køretid og to minutter i feltet intervalinput, før du klikker på OK for at bekræfte og gemme indstillingerne.Klik på Læs. I pop op-vinduet skal du vælge Fluorescensintensitet som registreringsmetode, slutpunkt kinetisk som læsetype og filtre som optiktype. Klik derefter på OK. Vælg Grøn i filtersættet i pop op-vinduet, og klik på OK. Vælg Brug låg i vinduet Procedure, og klik på Valider.Der vises et pop op-vindue, der bekræfter, at den oprettede protokol er gyldig. Vælg Procedure under menuen Protokol. Definer de brønde, der skal måles, i vinduet Procedure, og angiv navnet på eksperimentet i feltet Inputboks til filnavn.Læg derefter pladen ind i pladelæseren, idet du skal passe på, at pladen er i den rigtige retning, og klik på knappen Læs ny for at starte anskaffelsen. Til dataanalyse skal du åbne dataene i den relevante analysesoftware og vælge en af de målte brønde i pladen ét vindue. Klik på Vælg brønde, og medtag alle de målte brønde i dialogboksen Godt markering, før du klikker på OK. Vælg derefter Data i pladen ét vindue for at visualisere de tabelbelagte resultater, og klik på QuickExport for at eksportere dataene til et regneark.I et regneark skal du mærke datakolonnerne efter behov for hver prøve og sonde og afbilde de relative fluorescensenheder i forhold til den tid, dataene skal generere kinetiske grafer. I dette repræsentative eksperiment, efter den første runde af screening, rapporterede Salmonella kultur supernatants en klar præference for RNA sonder over DNA-sonder. Baseret på identifikation af RNA som den foretrukne nukleinsyre type af Salmonella nukleaser, en ny RNA-only bibliotek er designet til at blive brugt i anden runde af screening.I modsætning hertil viste E.Coli i kulturmediumkontrol en meget begrænset evne til at nedbryde RNA-sonder. Efter en anden screeningsrunde med kemisk modificerede nukleotider, der havde til formål at øge RNA-sondernes specificitet, kunne RNA pyrimidin 2'O-methyl og RNA purin 2'O-methyl identificeres på grundlag af deres kemiske modifikationer som de mest effektive kinetiske adfærd sammenlignet med henholdsvis RNA pyrimidin 2'fluor og RNA purin 2'fluoro. Disse resultater tyder på, at Salmonella har en vigtig RNAse aktivitet med differentiale substrat kemi præference, der kan bruges til at vælge sonder i stand til specifikt at anerkende denne bakterie.Denne procedure gør det muligt at vælge sonder, der er i stand til at identificere nuklease aktiviteter forbundet med sygdomssygdomme, såsom kræft eller bakteriel infektion, så udviklingen af nye kliniske diagnostiske værktøjer.

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

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 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.

En Polyanilin-baserede sensor af nukleinsyrer

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.

Forberedelse og<em&gt; In vivo</em&gt; Anvendelse af en aktivitetsbaseret Probe til<em&gt; N</em&gt; -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 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.

Anaerob Protein oprensning og kinetisk analyse via ilt elektrode til at studere DesB Dioxygenase aktivitet og hæmning

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.

Udtryk og rensning af nuklease-fri 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, ...

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.

Skøn over indholdet af plantebiomasse lignin ved hjælp af thioglykolsyre (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 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.

Præcis og præcis enkeltmolekyle FRET-målinger ved hjælp af 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 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.

Bestemmelse af den termodynamiske og kinetiske forening af en DNA-aptamer og tetracyclin ved hjælp af isotermisk titreringskalorimetri

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 ...

Identifikation af G-quadruplexer i en DNA-skabelon ved hjælp af kemisk kortlægning

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.

Forfatter Spotlight: Karakterisering af DNA G-Quadruplex ved Bis-3-chloropiperidinbaseret kemisk kortlægning 

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.

In vitro Kemisk kortlægning af G-quadruplex DNA-strukturer af bis-3-chloropiperidiner

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