A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect
Identifieur interne : 000385 ( Pmc/Corpus ); précédent : 000384; suivant : 000386A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect
Auteurs : Po-Jung Jimmy Huang ; Mahsa Vazin ; Aneta Matuszek ; Juewen LiuSource :
- Nucleic Acids Research [ 0305-1048 ] ; 2014.
Abstract
Url:
DOI: 10.1093/nar/gku1296
PubMed: 25488814
PubMed Central: 4288186
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect</title><author><name sortKey="Huang, Po Jung Jimmy" sort="Huang, Po Jung Jimmy" uniqKey="Huang P" first="Po-Jung Jimmy" last="Huang">Po-Jung Jimmy Huang</name></author><author><name sortKey="Vazin, Mahsa" sort="Vazin, Mahsa" uniqKey="Vazin M" first="Mahsa" last="Vazin">Mahsa Vazin</name></author><author><name sortKey="Matuszek, Aneta" sort="Matuszek, Aneta" uniqKey="Matuszek " first=" Aneta" last="Matuszek"> Aneta Matuszek</name></author><author><name sortKey="Liu, Juewen" sort="Liu, Juewen" uniqKey="Liu J" first="Juewen" last="Liu">Juewen Liu</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25488814</idno><idno type="pmc">4288186</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288186</idno><idno type="RBID">PMC:4288186</idno><idno type="doi">10.1093/nar/gku1296</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000385</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000385</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect</title><author><name sortKey="Huang, Po Jung Jimmy" sort="Huang, Po Jung Jimmy" uniqKey="Huang P" first="Po-Jung Jimmy" last="Huang">Po-Jung Jimmy Huang</name></author><author><name sortKey="Vazin, Mahsa" sort="Vazin, Mahsa" uniqKey="Vazin M" first="Mahsa" last="Vazin">Mahsa Vazin</name></author><author><name sortKey="Matuszek, Aneta" sort="Matuszek, Aneta" uniqKey="Matuszek " first=" Aneta" last="Matuszek"> Aneta Matuszek</name></author><author><name sortKey="Liu, Juewen" sort="Liu, Juewen" uniqKey="Liu J" first="Juewen" last="Liu">Juewen Liu</name></author></analytic><series><title level="j">Nucleic Acids Research</title><idno type="ISSN">0305-1048</idno><idno type="eISSN">1362-4962</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>In vitro</italic> selection of RNA-cleaving DNAzymes was performed using three heavy lanthanide ions (Ln<sup>3+</sup>): Ho<sup>3+</sup>, Er<sup>3+</sup> and Tm<sup>3+</sup>. The resulting sequences were aligned together and about half of the library contained a new family of DNAzyme. These DNAzymes have a simple loop structure, and they are active only with the seven heavy Ln<sup>3+</sup>. Among the tested non-lanthanide ions, only Y<sup>3+</sup> induced cleavage and even Pb<sup>2+</sup> failed to cleave, suggesting a very high specificity. A representative DNAzyme, Tm7, has a sigmoidal metal binding curve with a Hill coefficient of 3, indicating that three metal ions are involved in the catalytic step. Its pH-rate profile has a slope of 1, suggesting a single deprotonation step is involved in the rate-limiting step. Tm7 has a cleavage rate of 1.6 min<sup>−1</sup> at pH 7.8 with 10 μM Er<sup>3+</sup>. Phosphorothioate substitution at the cleavage junction completely inhibits the activity, which cannot be rescued by Cd<sup>2+</sup> alone, or by a mixture of Er<sup>3+</sup> and Cd<sup>2+</sup>, suggesting that two interacting metal ions are involved in direct bonding to both non-bridging oxygen atoms. A new model involving three lanthanide ions is proposed based on this study. A biosensor is engineered using Tm7 to detect Dy<sup>3+</sup> down to 14 nM.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Komiyama, M" uniqKey="Komiyama M">M. Komiyama</name></author><author><name sortKey="Takeda, N" uniqKey="Takeda N">N. Takeda</name></author><author><name sortKey="Shigekawa, H" uniqKey="Shigekawa H">H. Shigekawa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kim, H K" uniqKey="Kim H">H.-K. Kim</name></author><author><name sortKey="Li, J" uniqKey="Li J">J. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Nucleic Acids Res</journal-id><journal-id journal-id-type="iso-abbrev">Nucleic Acids Res</journal-id><journal-id journal-id-type="hwp">nar</journal-id><journal-id journal-id-type="publisher-id">nar</journal-id><journal-title-group><journal-title>Nucleic Acids Research</journal-title></journal-title-group><issn pub-type="ppub">0305-1048</issn><issn pub-type="epub">1362-4962</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25488814</article-id><article-id pub-id-type="pmc">4288186</article-id><article-id pub-id-type="doi">10.1093/nar/gku1296</article-id><article-categories><subj-group subj-group-type="heading"><subject>Nucleic Acid Enzymes</subject></subj-group></article-categories><title-group><article-title>A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Po-Jung Jimmy</given-names></name></contrib><contrib contrib-type="author"><name><surname>Vazin</surname><given-names>Mahsa</given-names></name></contrib><contrib contrib-type="author"><name><surname>Matuszek</surname><given-names>Żaneta</given-names></name></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Juewen</given-names></name><xref ref-type="corresp" rid="COR1">*</xref></contrib><aff id="AFF1">Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Ontario, Canada N2L 3G1</aff></contrib-group><author-notes><corresp id="COR1"><label>*</label>To whom correspondence should be addressed. Tel: +1 519 888 4567 (Extn 38919); Fax: +1 519 746 0435; Email: <email>liujw@uwaterloo.ca</email></corresp></author-notes><pub-date pub-type="ppub"><day>09</day><month>1</month><year>2015</year></pub-date><pub-date pub-type="epub"><day>08</day><month>12</month><year>2014</year></pub-date><pub-date pub-type="pmc-release"><day>08</day><month>12</month><year>2014</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>43</volume><issue>1</issue><fpage>461</fpage><lpage>469</lpage><history><date date-type="accepted"><day>28</day><month>11</month><year>2014</year></date><date date-type="rev-recd"><day>26</day><month>11</month><year>2014</year></date><date date-type="received"><day>28</day><month>10</month><year>2014</year></date></history><permissions><copyright-statement>© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.</copyright-statement><copyright-year>2015</copyright-year><license license-type="creative-commons" xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="gku1296.pdf"></self-uri><abstract><p><italic>In vitro</italic> selection of RNA-cleaving DNAzymes was performed using three heavy lanthanide ions (Ln<sup>3+</sup>): Ho<sup>3+</sup>, Er<sup>3+</sup> and Tm<sup>3+</sup>. The resulting sequences were aligned together and about half of the library contained a new family of DNAzyme. These DNAzymes have a simple loop structure, and they are active only with the seven heavy Ln<sup>3+</sup>. Among the tested non-lanthanide ions, only Y<sup>3+</sup> induced cleavage and even Pb<sup>2+</sup> failed to cleave, suggesting a very high specificity. A representative DNAzyme, Tm7, has a sigmoidal metal binding curve with a Hill coefficient of 3, indicating that three metal ions are involved in the catalytic step. Its pH-rate profile has a slope of 1, suggesting a single deprotonation step is involved in the rate-limiting step. Tm7 has a cleavage rate of 1.6 min<sup>−1</sup> at pH 7.8 with 10 μM Er<sup>3+</sup>. Phosphorothioate substitution at the cleavage junction completely inhibits the activity, which cannot be rescued by Cd<sup>2+</sup> alone, or by a mixture of Er<sup>3+</sup> and Cd<sup>2+</sup>, suggesting that two interacting metal ions are involved in direct bonding to both non-bridging oxygen atoms. A new model involving three lanthanide ions is proposed based on this study. A biosensor is engineered using Tm7 to detect Dy<sup>3+</sup> down to 14 nM.</p></abstract><counts><page-count count="9"></page-count></counts><custom-meta-group><custom-meta><meta-name>cover-date</meta-name><meta-value>January 2015</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="SEC1"><title>INTRODUCTION</title><p>The interaction between lanthanide ions (Ln<sup>3+</sup>) and nucleic acids has been studied for a long time. At high lanthanide concentrations, both DNA and RNA can be cleaved (<xref rid="B1" ref-type="bibr">1</xref>). Therefore, Ln<sup>3+</sup> and their complexes have been developed as artificial nucleases and chemical probes for nucleic acids (<xref rid="B2" ref-type="bibr">2</xref>–<xref rid="B5" ref-type="bibr">5</xref>). Tb<sup>3+</sup> luminescence is significantly enhanced upon binding to DNA, and related biosensor and biochemical applications have been reported (<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>). Lanthanide ions are popular structural probes for ribozyme research as well, where they can sometimes replace the original metal ions and provide rich spectroscopic information (<xref rid="B8" ref-type="bibr">8</xref>). With strong hydrolytic properties, Ln<sup>3+</sup> was exploited in RNA-cleaving DNAzymes. For example, Ln<sup>3+</sup> can accelerate the leadzyme (a small Pb<sup>2+</sup>-dependent ribozyme) (<xref rid="B9" ref-type="bibr">9</xref>), but they inhibit the hammerhead ribozyme (<xref rid="B8" ref-type="bibr">8</xref>), and the 8–17 DNAzyme (<xref rid="B2" ref-type="bibr">2</xref>). Ln<sup>3+</sup> moderately activates GR5 (a Pb<sup>2+</sup>-dependent DNAzyme) (<xref rid="B10" ref-type="bibr">10</xref>). The application of Ln<sup>3+</sup> in other DNAzyme reactions was also reported (<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>).</p><p>We are interested in isolating new Ln<sup>3+</sup>-dependent DNAzymes. Using <italic>in vitro</italic> selection, we recently reported two new RNA-cleaving DNAzymes that are highly specific for Ln<sup>3+</sup>. The first one (named Ce13d) was selected in the presence of Ce<sup>3+</sup>/Ce<sup>4+</sup> (<xref rid="B13" ref-type="bibr">13</xref>), and the second one (named Lu12) was selected in the presence of Lu<sup>3+</sup> (<xref rid="B14" ref-type="bibr">14</xref>). Both are highly active with Ln<sup>3+</sup>, although Lu12 appears to be less active with the last few heavy ones. Using these two probes, we were able to discriminate only a few heavy Ln<sup>3+</sup>. Ideally, if more DNAzyme probes with distinct activity patterns across the Ln<sup>3+</sup> series can be obtained, it may enable a sensor array to discriminate between each of the 15 lanthanide ions (<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>). In our effort to isolate more Ln<sup>3+</sup>-dependent DNAzymes, we herein performed three new selections using Ho<sup>3+</sup>, Er<sup>3+</sup> and Tm<sup>3+</sup> respectively. For all known elements, holmium has the largest magnetic strength and is used for making magnets. It is also used in nuclear reactors due to its ability to strongly absorb neutrons. Erbium is mainly used for making lasers and thulium is used in X-ray devices as a portable source.</p><p>From these selections, we identified a new class of DNAzymes with many interesting properties. For example, they work only with the seven heavy Ln<sup>3+</sup>. Different from all the previously reported examples (lanthanide or other metal dependent), this is the first RNA-cleaving DNAzyme showing metal cooperativity, indicating that multiple metal ions are involved for catalysis. Finally, phosphorothioate modification has resulted in complete inactivation of the DNAzyme, such that it cannot be rescued by adding thiophilic metals. With these observations, a trinuclear Ln<sup>3+</sup> mechanism is proposed.</p></sec><sec sec-type="materials|methods" id="SEC2"><title>MATERIALS AND METHODS</title><sec id="SEC2-1"><title>Chemicals</title><p>The <italic>in vitro</italic> selection related DNA samples and the beacon DNA were purchased from Integrated DNA Technologies (IDT, Coralville, IA). See Supplementary Table S1 for their names, sequences and modifications. For characterization, the enzyme strands were from Eurofins (Huntsville, AL). The Ln<sup>3+</sup> other metal salts were from Sigma–Aldrich at the highest possible purity. Tris(hydroxymethyl)aminomethane (Tris), 2-(<italic>N</italic>-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, sodium chloride and ammonium acetate were from Mandel Scientific Inc. (Guelph, Ontario, Canada). SsoFast EvaGreen supermix was from Bio-Rad for real-time polymerase chain reaction (PCR). T4-DNA ligase, deoxynucleotide (dNTP) mix, Taq DNA polymerase with ThermoPol buffer and low molecular weight DNA ladder were from New England Biolabs.</p></sec><sec id="SEC2-2"><title><italic>In vitro</italic> selection</title><p>The method of <italic>in vitro</italic> selection is similar to the one we reported previously (<xref rid="B14" ref-type="bibr">14</xref>). In brief, the initial library was obtained by ligating Lib-FAM-N<sub>35</sub> and Lib-rA. For each subsequent round, the library was produced by PCR. For each cleavage step, the DNA library was incubated with fresh Ln<sup>3+</sup> solutions. For all the selections, the metal incubation time was maintained at 60 min. For the first four rounds, 50 μM Ln<sup>3+</sup> was used while for the last three rounds, 10 μM Ln<sup>3+</sup> was used. After incubation, the solution was mixed with 8 M urea and purified by 10% dPAGE (denaturing polyacrylamide gel electrophoresis). The position corresponding to the cleaved product was excised from the gel, the DNA was extracted by crushing and soaking the gel and was further desalted with a Sep-Pak C18 column (Waters). After drying in an Eppendorf Vacufuge at 30°C overnight, the dried DNA was re-suspended in 70 μl of 5 mM HEPES buffer (pH 7.5). The round 6 libraries for all the three selections were cloned and sequenced.</p></sec><sec id="SEC2-3"><title>PCR</title><p>During the <italic>in vitro</italic> selection experiment, three PCR reactions were carried out for each round. After the cleavage step, real-time PCR was carried out to quantify the cleaved DNA extracted from the gel. Each 20 μl reaction mixture contained 1 μl purified DNA template, 400 nM primer (P1 and P2) and 10 μl of SsoFast EvaGreen Supermix (Bio-Rad). The thermocycling steps were 95°C for 30 s, 95°C for 5 s followed by 55°C for 5 s. To amplify the library, in PCR1, a 50 μl reaction mixture contained the following: 1 μl DNA template, 200 nM of each of P1 and P2, 200 μM dNTP mixture, 1× Taq buffer and 1.25 units of Taq DNA polymerase. The reaction was carried out for 15–20 cycles (94°C for 5 min; 94°C for 30 s, 55°C for 30 s and 72°C for 30 s). A gel/PCR DNA fragment extraction kit (IBI Scientific) was used to purify the PCR1 product. The purified product was used as the template for PCR2. One-tenth of the purified PCR1 product was further amplified for 12 cycles using P3 and P4 as the primers. A 200 μl PCR reaction mixture contains 4 μl diluted template, 250 nM each of P3 and P4, 200 μM dNTP mixture, 1× Taq buffer and 5 units of Taq DNA polymerase. The final PCR2 product was again purified by 10% dPAGE. The single-stranded FAM-labeled DNA was excised from the gel, ethanol precipitated and used as the library for the subsequent round of selection.</p></sec><sec id="SEC2-4"><title>Activity assay</title><p>Gel-based activity assays were performed with a final concentration of 0.7 μM of the FAM-labeled substrate strand and 1.1 μM of the enzyme. The DNAzyme complexes were prepared by annealing them in buffer C (50 mM MES, pH 6.0, 25 mM NaCl) and a final concentration of 10 μM Ln<sup>3+</sup> was added to initiate the cleavage reaction. The products were separated on a denaturing polyacrylamide gel and analyzed using a Bio-Rad ChemiDoc MP imaging system. For pH-dependent activity assay, the 2-(N-morpholino)ethanesulfonic acid (MES) and 3-(N-morpholino)propanesulfonic acid (MOPS) buffers (50 mM with 25 mM NaCl) were used.</p></sec><sec id="SEC2-5"><title>Sensing</title><p>The sensing kinetics studies were carried out using a microplate reader (SpectraMax M3). The sensor complex was formed by annealing the FAM-labeled substrate and the quencher-labeled enzyme (1:1.5 ratio) in buffer C. In each well, 100 μl of the complex containing 50 nM FAM-labeled substrate was diluted in 10 mM HEPES (pH 7.5). One microliter of metal ion was added after 5 min of background reading and the signaling kinetics was monitored. Most assays were run in triplicates and error bars represent one times of standard deviation.</p></sec></sec><sec id="SEC3"><title>RESULTS AND DISCUSSION</title><sec id="SEC3-1"><title><italic>In vitro</italic> selection</title><p>Free Ln<sup>3+</sup> and some of their complexes can cleave RNA at high metal concentrations (<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B17" ref-type="bibr">17</xref>). We hope to use DNA as a scaffold to more efficiently use Ln<sup>3+</sup> ions so that the same reaction takes place at much lower Ln<sup>3+</sup> concentrations and at a designated position. <italic>In vitro</italic> DNAzyme selection provides a powerful tool to achieve this goal (<xref rid="B18" ref-type="bibr">18</xref>–<xref rid="B23" ref-type="bibr">23</xref>), and a number of metal-dependent DNAzymes have been isolated. In this work, selections were separately carried out using three different Ln<sup>3+</sup> to increase sequence diversity. The design of our library is shown in Figure <xref ref-type="fig" rid="F1">1A</xref>. It contains a FAM (carboxyfluorescein) label at the 5′-end, so that the cleavage reaction can be quantified. The cleavage junction is the rAG (indicated by the arrowhead), where the rA (ribo-adenosine) is the only RNA linkage in this whole sequence. Since RNA is about one million fold less stable compared to DNA, cleavage is most likely to take place at this junction (<xref rid="B24" ref-type="bibr">24</xref>). The cleavage site is positioned in proximity to the randomized N35 loop (35 random nucleotides) and is flanked by two Watson–Crick base pairing regions. If any sequence in this library can cleave the RNA linkage in the presence of an added Ln<sup>3+</sup>, a shorter DNA is generated (from 102 nucleotides to 74, Figure <xref ref-type="fig" rid="F1">1B</xref>). This shortened piece is isolated after gel electrophoresis and amplified by two PCR steps to seed the next round of selection.</p><fig id="F1" position="float"><label>Figure 1.</label><caption><p>(<bold>A</bold>) The DNA library sequence. The cleavage site is indicated by the arrowhead. (<bold>B</bold>) A simplified scheme of the <italic>in vitro</italic> selection process. Three independent selections with the different Ln<sup>3+</sup> were carried out. PAGE means gel electrophoresis and two PCR steps were used in step 3 to amplify and re-generate the library. (<bold>C</bold>) Selection progress and the round 6 library was sequenced. Ln<sup>3+</sup> concentration was 50 μM for rounds 1–4 and 10 μM for rounds 5–7. Incubation time was maintained at 60 min. (<bold>D</bold>) Sequence alignment in the enzyme loop region. (<bold>E</bold>) The secondary structure of the trans-cleaving version of the Tm7 DNAzyme.</p></caption><graphic xlink:href="gku1296fig1"></graphic></fig><p>The selection progress is presented in Figure <xref ref-type="fig" rid="F1">1C</xref>. All the three selections experienced a steady improvement in cleavage yield until round 6. At this moment, 15–38% cleavage was achieved. Since the activity of the round 7 libraries dropped, the round 6 libraries were sequenced. We obtained a total of 60 sequences from the three selections and they were aligned altogether for a systematic comparison (Supplementary Table S2). The DNAzymes are named by the Ln<sup>3+</sup> used in selection followed by the clone number for sequencing. Most of them can fold into a simple loop structure and the folding of Tm7 is shown in Supplementary Figure S1 as an example (predicted by Mfold) (<xref rid="B25" ref-type="bibr">25</xref>). Based on this, a trans-cleaving version was designed (Figure <xref ref-type="fig" rid="F1">1E</xref>).</p><p>Assuming such a simple loop structure flanked by two base paired regions, we aligned the sequences in the loop region in Figure <xref ref-type="fig" rid="F1">1D</xref>. However, it is difficult to identify a long-stretch of conserved nucleotides. This situation is quite different from our previously reported two selections (<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>). In those two cases, the conserved nucleotides were easily identified. In Figure <xref ref-type="fig" rid="F1">1D</xref>, the sequences are aligned based on an <italic>AT</italic> dinucleotide (marked in red) and a thymine in blue. About half of them end with <italic>TCTT</italic> (also marked in blue), and many contain a stretch of adenines in the middle prior to the conserved <italic>AT</italic> dinucleotide. The sequences in Figure <xref ref-type="fig" rid="F1">1D</xref> represent about 50% of the library. For the rest, 16 of them (∼27%) are the Lu12 type of DNAzyme we previously reported (<xref rid="B14" ref-type="bibr">14</xref>). It is not surprising that Lu12 was selected again since it is active with all these three lanthanide ions.</p></sec><sec id="SEC3-2"><title>Lanthanide selectivity</title><p>Since the sequences of this new DNAzyme family are quite diverse, it is difficult to rationally predict the most optimal sequence. Therefore, we picked seven of them for further assay (those marked in green in Figure <xref ref-type="fig" rid="F1">1D</xref>). Each was designed in a trans-cleaving format, all sharing the same substrate as shown in Figure <xref ref-type="fig" rid="F1">1E</xref>. The DNAzyme secondary structures are shown in Figure <xref ref-type="fig" rid="F2">2A</xref> (substrate binding arms are denoted by the bars). All these DNAzymes contain a stretch of four unpaired nucleotides in the substrate strand at the 5′ side of the cleavage junction.</p><fig id="F2" position="float"><label>Figure 2.</label><caption><p>(<bold>A</bold>) Secondary structures of six DNAzymes in this study. Gel images showing cleavage activity of (<bold>B</bold>) Tm7 and (<bold>C</bold>) Ho11 with different Ln<sup>3+</sup> ions (10 μM) after 1 h reaction. (<bold>D</bold>) Quantification of the cleavage results of the six DNAzymes by Ln<sup>3+</sup>.</p></caption><graphic xlink:href="gku1296fig2"></graphic></fig><p>We next measured the enzyme activity as a function of Ln<sup>3+</sup> atomic number after 1 h reaction. With a FAM label in the substrate, the cleavage reaction was conveniently followed using gel electrophoresis. Figure <xref ref-type="fig" rid="F2">2B</xref> and <xref ref-type="fig" rid="F2">C</xref> show the trend for the Tm7 and Ho11 DNAzymes, respectively. Interestingly, hardly any cleavage was observed with the seven lighter ions (Pm<sup>3+</sup> was not tested since it is radioactive) and only moderate cleavage was observed with Tb<sup>3+</sup>. Efficient cleavage occurred from Dy<sup>3+</sup> to Tm<sup>3+</sup>, and then the cleaved product decreased rapidly with Yb<sup>3+</sup> and Lu<sup>3+</sup>. This is a general trend for all the six tested DNAzymes and their activities are quantified in Figure <xref ref-type="fig" rid="F2">2D</xref>. Based on this study, we reason that these DNAzymes belong to the same family. From the sequence diversity in Figure <xref ref-type="fig" rid="F1">1D</xref>, they seem to be quite tolerant to mutations and insertions.</p><p>Tm7, Ho11 and Ho15 are the three fastest DNAzymes among the six. They all have a stretch of pyrimidines followed by a few purines in the loop (this is also true for Tm8, see Supplementary Figure S2). The other three DNAzymes are slower and their base contents in the loop region are more mixed up, especially on the pyrimidine side. It is also interesting to note that for the three most active sequences, only Tm7 has a single guanine in the loop, while Ho11 and Ho15 do not contain any guanines. Among the four nucleobases, guanine is the most efficient ligand for Ln<sup>3+</sup> binding (<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B26" ref-type="bibr">26</xref>–<xref rid="B28" ref-type="bibr">28</xref>). The other two Ln<sup>3+</sup>-dependent DNAzymes are richer in guanine in their conserved sequences (<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>). This might be a reason for the completely different activity trends for these DNAzymes across the Ln<sup>3+</sup> series.</p><p>The ionic radii of Ln<sup>3+</sup> decrease gradually and steadily from 1.17 to 1.0 Å, while the p<italic>K</italic><sub>a</sub> values of their bound water decrease from 9.3 to 8.2 from La<sup>3+</sup> to Lu<sup>3+</sup>. Given the abrupt change of the cleavage activity from Gd<sup>3+</sup> to Tb<sup>3+</sup>, it is unlikely for either the size or p<italic>K</italic><sub>a</sub> to be the main factor. However, the ‘gadolinium break’ in Figure <xref ref-type="fig" rid="F2">2D</xref> may be attributed to the change of coordination number. As the lanthanide atomic number increases, the coordinated water decreases from 9 to 8 due to lanthanide contraction and steric effects (<xref rid="B29" ref-type="bibr">29</xref>). This transition takes place around Gd<sup>3+</sup> for water ligand. It might be that the low coordination number allows better metal binding and catalysis in this DNAzyme.</p><p>Gadolinium break was reported previously with cleavage of a dinucleotide (5 mM lanthanide) (<xref rid="B17" ref-type="bibr">17</xref>), and with the GR5 DNAzyme (60 μM Ln<sup>3+</sup>) (<xref rid="B10" ref-type="bibr">10</xref>). In those two examples, however, Lu<sup>3+</sup> was among the most active ions, but our Tm7 has almost no activity with Lu<sup>3+</sup>. Therefore, the mechanism is still different for Tm7. Since Tm7 has the highest efficiency among all the tested sequences and it has a small enzyme loop containing only 11 nucleotides, it was chosen for subsequent studies.</p></sec><sec id="SEC3-3"><title>Biochemical characterization of Tm7</title><p>To further understand this new DNAzyme, we first tested metal selectivity for non-lanthanide ions. A gel image of Tm7 reacting with 10 μM other metal ions for 1 h is shown in the inset of Figure <xref ref-type="fig" rid="F3">3A</xref>. In addition to Er<sup>3+</sup> (used as a representative Ln<sup>3+</sup>), only Y<sup>3+</sup> produced cleavage, while all the other metals were inactive. The property of Y<sup>3+</sup> is between Ho<sup>3+</sup> and Er<sup>3+</sup>, which explains its activity. The same experiment was repeated with 100 μM metals (the red bars in Figure <xref ref-type="fig" rid="F3">3A</xref>), and still only Y<sup>3+</sup> showed moderate cleavage. At 100 μM, even Er<sup>3+</sup> inhibited the activity and we did not test even higher metal concentrations. It is important to note that Pb<sup>2+</sup> was not active at all. Almost all the previously reported DNAzymes are active with Pb<sup>2+</sup> (<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B30" ref-type="bibr">30</xref>,<xref rid="B31" ref-type="bibr">31</xref>). This is a rare example where Pb<sup>2+</sup> failed to show activity. Such high selectivity makes Tm7 another useful probe for Ln<sup>3+</sup>, especially for the heavy ones.</p><fig id="F3" position="float"><label>Figure 3.</label><caption><p>(<bold>A</bold>) Fraction of substrate cleavage by the Tm7 DNAzyme using 10 or 100 μM metal ions. Inset: gel image of cleavage in the presence of 10 μM metal ions. The lanes correspond to the metal ions in the <italic>x</italic>-axis. Only Au<sup>3+</sup> produced streaking and Er<sup>3+</sup> and Y<sup>3+</sup> produced cleavage. (<bold>B</bold>) Kinetics of Tm7 cleavage at a few Er<sup>3+</sup> concentrations. (<bold>C</bold>) Cleavage rate as a function of Er<sup>3+</sup> concentration. Inset: the same data plotted using the log scale.</p></caption><graphic xlink:href="gku1296fig3"></graphic></fig><p>Next, the cleavage kinetics was measured at various Er<sup>3+</sup> concentrations (Figure <xref ref-type="fig" rid="F3">3B</xref>). With 0.7 μM DNAzyme, we did not observe any cleavage over 4 h in the presence of 0.5 μM Er<sup>3+</sup>. On the other hand, the rate increased by 10-fold from 1 to 2 μM Er<sup>3+</sup>. Since the rate does not scale linearly with the Er<sup>3+</sup> concentration, it indicates that multiple metal binding is required for activity. Figure <xref ref-type="fig" rid="F3">3C</xref> plots the cleavage rate as a function of Er<sup>3+</sup> concentration and a sigmoidal curve was obtained with a Hill coefficient of 3.0. In the inset, the double log plot is presented, where the initial slope is 2.7, suggesting that three Er<sup>3+</sup> ions are involved in catalysis. The highest rate was ∼0.06 min<sup>−1</sup> in the presence of 20 μM Er<sup>3+</sup> at pH 6.0, which is similar to our selection condition. This is however slower than the rate of the Lu12 DNAzyme. The survival of this class of slower DNAzyme is attributed to that a long incubation time was used. They are also highly tolerant to mutations with good sequence diversity. At the moment of sequencing, after six rounds of selection we already have obtained 16 Lu12 DNAzymes out of 60 (27%). If we continue the selection with shorter time, the library is likely to be dominated by the Lu12 type of DNAzymes. Most of the sequences still have very low activity with Lu<sup>3+</sup>, and this might be the reason that we did not see any Tm7 sequences in our previous Lu<sup>3+</sup>-dependent selection (<xref rid="B14" ref-type="bibr">14</xref>). At higher pH, the rate of Tm7 increases significantly (see below) and can reach higher than 1.6 min<sup>−1</sup>.</p><p>To the best of our knowledge, this is the first time that metal cooperativity is reported for RNA-cleaving DNAzymes. All the previous reported enzymes employ only a single metal ion (<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B31" ref-type="bibr">31</xref>–<xref rid="B34" ref-type="bibr">34</xref>). Most small ribozymes use only one metal as well (<xref rid="B35" ref-type="bibr">35</xref>,<xref rid="B36" ref-type="bibr">36</xref>), although multiple metals are involved in large ribozymes performing more complex reactions such as RNA splicing (<xref rid="B37" ref-type="bibr">37</xref>,<xref rid="B38" ref-type="bibr">38</xref>). There is one example that Ln<sup>3+</sup> (especially Nd<sup>3+</sup>) can accelerate the Pb<sup>2+</sup>-dependent activity of the leadzyme, where a two-metal mechanism was proposed. However, the best rate was only ∼0.01 min<sup>−1</sup> when both metals were used (<xref rid="B39" ref-type="bibr">39</xref>). In addition, those two metals do not show much cooperativity.</p><p>Using multiple Ln<sup>3+</sup> ions for RNA cleavage was reported with a high concentration (e.g. mM) of free ions (no DNAzymes) (<xref rid="B1" ref-type="bibr">1</xref>). It is proposed that one Ln<sup>3+</sup> directly bonds with the leaving phosphate to decrease its negative charge density at the transition state. A bridging water (bridging two Ln<sup>3+</sup> ions) may deprotonate and act as a general base to activate the 2′-OH of the ribose to make it a better nucleophile.</p></sec><sec id="SEC3-4"><title>The effect of pH</title><p>To gain further insights into Tm7, a pH-dependent study was performed, and the logarithm of cleavage rate was plotted (Figure <xref ref-type="fig" rid="F4">4A</xref>). While the selection was carried out at pH 6.0, the rate became progressively faster at higher pH (<xref rid="B31" ref-type="bibr">31</xref>,<xref rid="B34" ref-type="bibr">34</xref>,<xref rid="B40" ref-type="bibr">40</xref>). At pH higher than 7.0, the rate increase slowed down. By fitting the data in the pH range from 6.0 to 7.0, a slope of 0.97 was obtained (Figure <xref ref-type="fig" rid="F4">4B</xref>), suggesting only a single deprotonation step is involved in the rate-limiting step of the cleavage reaction. This is typical for RNA-cleaving DNAzymes and this deprotonation is often linked to the 2′-OH group on the ribose.</p><fig id="F4" position="float"><label>Figure 4.</label><caption><p>The pH-rate profile of the Tm7 DNAzyme over (<bold>A</bold>) a wide pH range and (<bold>B</bold>) the initial linear range. A slope of 0.97 is obtained in (B), indicating a single deprotonation step.</p></caption><graphic xlink:href="gku1296fig4"></graphic></fig></sec><sec id="SEC3-5"><title>Phosphorothioate modification</title><p>Since lanthanide ions are hard Lewis acids that prefer oxygen-based ligands, we further probed metal binding by introducing a phosphorothioate (PS) modification at the cleavage junction, where one of the non-bridging oxygen atoms is replaced by a sulfur (Figure <xref ref-type="fig" rid="F5">5A</xref>) (<xref rid="B41" ref-type="bibr">41</xref>,<xref rid="B42" ref-type="bibr">42</xref>). Depending on the position of replacement, two diastereomers are possible. With the PS-modified substrate (a mixture of the two isomers), Tm7 barely showed cleavage with any metal, including thiophilic Cd<sup>2+</sup> (Figure <xref ref-type="fig" rid="F5">5B</xref>), which is typically used to rescue the activity of PS-modified enzymes. Only Er<sup>3+</sup> showed a trace amount of cleavage after 1 h. We performed a quantitative kinetic measurement (Figure <xref ref-type="fig" rid="F5">5D</xref>) and the initial kinetics is very similar to that of the PO substrate (within 10% difference) and no further cleavage occurred beyond ∼5%. Therefore, this small fraction of cleavage is attributed to that the PS substrate is only ∼95% pure and the remaining 5% are PO, explaining the observed Er<sup>3+</sup>-dependent initial cleavage kinetics.</p><fig id="F5" position="float"><label>Figure 5.</label><caption><p>(<bold>A</bold>) Structures of the normal phosphate linkage (PO), and the two diastereomers of the PS modification. (<bold>B</bold>) Cleavage of the Tm7 DNAzyme with the PS-modified substrate (racemic mixture) in the presence of various divalent metal ions and Er<sup>3+</sup>. (<bold>C</bold>) Cleavage of Tm7 with the PS substrate using a mixture of Er<sup>3+</sup> and Cd<sup>2+</sup>. (<bold>D</bold>) Kinetics over 24 h for Tm7 cleaving the PO (black dots), and PS (red triangles) substrate in the presence of 10 μM Er<sup>3+</sup>. The green squares are the Tm7/PS complex incubated without Er<sup>3+</sup>. Inset is the re-plot of the PS sample magnifying the initial kinetics.</p></caption><graphic xlink:href="gku1296fig5"></graphic></fig><p>All the previously studied ribozymes and DNAzymes (e.g. the hammerhead ribozyme (<xref rid="B43" ref-type="bibr">43</xref>), HDV ribozyme (<xref rid="B44" ref-type="bibr">44</xref>), 10–23 DNAzyme (<xref rid="B45" ref-type="bibr">45</xref>), and RNase P (<xref rid="B46" ref-type="bibr">46</xref>)) use the pro-<italic>R</italic><sub>p</sub> oxygen for metal binding (mostly Mg<sup>2+</sup>). Replacing this oxygen by sulfur usually completely inhibits the Mg<sup>2+</sup>-dependent activity (by over 100-fold), but this inhibition can be rescued by using thiophilic metals such as Mn<sup>2+</sup> or Cd<sup>2+</sup>. The activity of the other PS isomer (<italic>S</italic><sub>p</sub>) is only slightly decreased (e.g. ∼5-fold). This is because Mg<sup>2+</sup> only coordinates to one of the non-bridging oxygen atoms (pro-<italic>R</italic><sub>p</sub>), and thus the pro-<italic>S</italic><sub>p</sub> substitution does not have much influence on activity. For the Tm7 experiment, we used a racemic mixture of <italic>R</italic><sub>p</sub> and <italic>S</italic><sub>p</sub> (no separation performed). Since no activity was observed with either Er<sup>3+</sup> or Cd<sup>2+</sup>, neither <italic>R</italic><sub>p</sub> nor <italic>S</italic><sub>p</sub> is active. In other words, the metal(s) must interact with both phosphate oxygen atoms through inner sphere coordination. This is different from all the previously reported nucleic acid enzymes, including another Ln<sup>3+</sup>-dependent DNAzyme, Ce13d, whose activity can be rescued by Cd<sup>2+</sup> (<xref rid="B47" ref-type="bibr">47</xref>).</p><p>If both non-bridging oxygen atoms are important, we reason that a mixture of Er<sup>3+</sup> and Cd<sup>2+</sup> might rescue the activity if these two metals do not need to interact with each other. By fixing the total concentration at 20 μM, their ratio was varied (Figure <xref ref-type="fig" rid="F5">5C</xref>). Still only ∼5% cleavage was observed when the Er<sup>3+</sup> concentration was more than 6.7 μM. This is again cleavage of the impure PO substrate. This implies that the metals must act synergistically instead of independently. If simply one metal binds to one oxygen, the use of this mixture should have boosted the activity with the PS substrate. The independent action of metal ions is best illustrated in the leadzyme, where a Ln<sup>3+</sup> is proposed to bind to the phosphate oxygen and the Pb<sup>2+</sup> is used to deprotonate the 2′-OH group (<xref rid="B39" ref-type="bibr">39</xref>). To the best of our knowledge, this is the first RNA-cleaving nucleic acid enzyme that is completely inactivated with the PS modification under all the metal conditions, which further confirms its new mechanism of catalysis.</p></sec><sec id="SEC3-6"><title>Cleavage mechanism</title><p>Before proposing mechanism, we performed mass spectrometry studies on the substrate cleavage product (Supplementary Figure S3). The 5′-fragment contains a cyclic phosphate, which is typical for most ribozymes and DNAzymes. We also tested the cleavage of a substrate with seven consecutive RNA bases (Supplementary Figure S4). However, Tm7 is inactive for this substrate. Most DNAzymes selected with a chimeric substrate are not active with full RNA. However, they are still called RNA-cleaving DNAzymes because of the same reaction mechanism.</p><p>Dinuclear Ln<sup>3+</sup> complexes have been proposed to cleave RNA (<xref rid="B1" ref-type="bibr">1</xref>), which however cannot explain our results. Based on all the above studies, we propose a mechanism involving a trinuclear Ln<sup>3+</sup> center (Figure <xref ref-type="fig" rid="F6">6</xref>). From the PS studies, both non-bridging oxygen atoms are in direct inner sphere coordination with Ln<sup>3+</sup> (Er<sup>3+</sup> used as an example here). These two can contribute to the electrostatic interactions to stabilize the transition state of the nucleophilic attack by the 2′-OH. Our pH studies indicate that only a single deprotonation reaction is involved, and this is likely to be related to the 2′-OH. Since the Ln<sup>3+</sup> concentration-dependent studies indicate a total of three Ln<sup>3+</sup> ions are involved, the third ion is thus proposed to interact with the 2′-OH. The three ions could be linked together with hydroxyl bridges. Since heavy Ln<sup>3+</sup> ions have a lower p<italic>K</italic><sub>a</sub> value for the bound water, it is easier for them to form polynuclear hydrolyzed products, which could also explain the tendency of heavy Ln<sup>3+</sup> being much more effective. The role of the DNAzyme loop is likely to stabilize this trinuclear complex.</p><fig id="F6" position="float"><label>Figure 6.</label><caption><p>Proposed mechanism of the lanthanide-induced RNA cleavage for the Tm7 DNAzyme. The red arrow indicates nucleophilic attack of the phosphorus center. The bridging oxygen atoms linking the Er<sup>3+</sup> ions are originated from deprotonated water.</p></caption><graphic xlink:href="gku1296fig6"></graphic></fig></sec><sec id="SEC3-7"><title>Sensing heavy lanthanide ions</title><p>Apart from proposing a novel reaction mechanism, this study has contributed a new probe for Ln<sup>3+</sup> detection. Lanthanides are known as industrial vitamins since they are often doped at small quantities but are indispensable for modern technological applications. Rational design of small molecule ligands that selectively bind individual Ln<sup>3+</sup> is quite difficult since these 15 elements have the same charge, similar sizes and comparable chemical properties (<xref rid="B48" ref-type="bibr">48</xref>). We are interested in developing DNA-based biosensors for Ln<sup>3+</sup>. DNA is a good candidate for Ln<sup>3+</sup> ligand since the phosphate backbone provides high binding affinity through hard acid/base interactions and the nitrogen containing nucleobases may offer specificity to discriminate between different Ln<sup>3+</sup> ions (<xref rid="B49" ref-type="bibr">49</xref>).</p><p>To test the application of this DNAzyme for Ln<sup>3+</sup> detection, we aim to engineer this DNAzyme into a biosensor. Although many sensor design methods are available (<xref rid="B50" ref-type="bibr">50</xref>,<xref rid="B51" ref-type="bibr">51</xref>), a simple catalytic beacon design was used (<xref rid="B52" ref-type="bibr">52</xref>). The 3′-end of the substrate labeled was labeled with a FAM and the 5′-end of the enzyme labeled with a quencher (Supplementary Figure S5). In the initial state, the beacon is dark. Fluorescence is unmasked upon cleavage and release of the cleaved fragment. Fluorescence enhancement was observed with Dy<sup>3+</sup> and Y<sup>3+</sup>, but the non-rare earth metals failed to produce signal (Figure <xref ref-type="fig" rid="F7">7A</xref>). Among the lanthanide ions, only those seven heavy ones produce signal (Figure <xref ref-type="fig" rid="F7">7B</xref>). This is also consistent with the gel-based assays, indicating that the signal generation is indeed due to cleavage. Interestingly, the fastest signaling was observed with Dy<sup>3+</sup>. This might be attributed to the use of higher pH (7.5) in sensing as compared to pH 6.0 in the gel-based assay. We next performed a metal concentration dependency study using Dy<sup>3+</sup> as the target metal ion (Figure <xref ref-type="fig" rid="F7">7C</xref>). Interestingly, barely any cleavage was observed with 5 nM Dy<sup>3+</sup> and significant improvement was achieved with 10 and 20 nM Dy<sup>3+</sup>. This trend also confirms metal cooperativity. The Dy<sup>3+</sup>-concentration dependent rate of fluorescence increase is shown in Figure <xref ref-type="fig" rid="F7">7D</xref> and the detection limit is 14 nM Dy<sup>3+</sup> based on the signal greater than three times of background variation.</p><fig id="F7" position="float"><label>Figure 7.</label><caption><p>(<bold>A</bold>) Sensor response to 0.5 μM of divalent and trivalent metal ions. The list of the other metal ions tested can be found in Figure <xref ref-type="fig" rid="F2">2B</xref>. (<bold>B</bold>) Sensor response to 0.5 μM of various Ln<sup>3+</sup>. (<bold>C</bold>) Sensor signaling kinetics in the presence of various concentrations of Dy<sup>3+</sup>. DNAzyme sensor concentration = 50 nM. (<bold>D</bold>) Quantification of Dy<sup>3+</sup> based on the initial rate of sensor fluorescence enhancement. Inset: the initial linear response at low Dy<sup>3+</sup> concentrations.</p></caption><graphic xlink:href="gku1296fig7"></graphic></fig></sec></sec><sec sec-type="conclusions" id="SEC4"><title>CONCLUSIONS</title><p>In summary, we carried out three independent <italic>in vitro</italic> selection experiments using Ho<sup>3+</sup>, Er<sup>3+</sup> and Tm<sup>3+</sup> as metal cofactors. Out of the 60 obtained sequences, half of them belong to a new family of DNAzyme, which are active to the seven heavy Ln<sup>3+</sup> ions only. Importantly, this new DNAzyme shows metal cooperativity as indicated by metal concentration dependency and phosphorothioate replacement studies. Combined with the pH-rate profile, we propose a new mechanism involving a trinuclear Ln<sup>3+</sup> complex. Two Ln<sup>3+</sup> ions interact with the non-bridging oxygen atoms and the third one interacts with the 2′-OH. This is the first RNA-cleaving DNAzyme showing such a metal binding property. Finally, we engineered a catalytic DNA beacon that can detect the heavy Ln<sup>3+</sup> such as Dy<sup>3+</sup> down to 14 nM.</p></sec><sec sec-type="supplementary-material" id="SEC5"><title>SUPPLEMENTARY DATA</title><p><ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gku1296/-/DC1">Supplementary Data</ext-link> are available at NAR Online.</p><supplementary-material id="PMC_1" content-type="local-data"><caption><title>SUPPLEMENTARY DATA</title></caption><media mimetype="text" mime-subtype="html" xlink:href="supp_43_1_461__index.html"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="supp_gku1296_nar-03117-f-2014-File009.pdf"></media></supplementary-material></sec></body><back><ack><p>We thank Dr Richard Smith and the Mass Spectrometry Facility at the University of Waterloo for assistance.</p></ack><sec id="SEC6"><title>FUNDING</title><p>Canadian Institutes of Health Research (CIHR); Ontario Ministry of Research & Innovation; Natural Sciences and Engineering Research Council (NSERC) of Canada (Discovery Grant and Strategic Project Grant). 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