Endonuclease IV discriminates mismatches next to the apurinic/apyrimidinic site in DNA strands: constructing DNA sensing platforms with extremely high selectivity

Download Endonuclease IV discriminates mismatches next to the apurinic/apyrimidinic site in DNA strands: constructing DNA sensing platforms with extremely high selectivity

Post on 28-Feb-2017

212 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

<ul><li><p>This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 2819--2821 2819</p><p>Cite this: Chem. Commun.,2013,49, 2819</p><p>Endonuclease IV discriminates mismatches next to theapurinic/apyrimidinic site in DNA strands: constructingDNA sensing platforms with extremely highselectivity</p><p>Xianjin Xiao, Yang Liu and Meiping Zhao*</p><p>A unique capability of Endonuclease IV in discrimination of mismatches</p><p>neighboring a natural abasic site in DNA strands has been demon-</p><p>strated, which enables genotyping of SNPs with high discrimination</p><p>factors and differentiation of as low as 0.10.01%of target DNA strands</p><p>from a large background of single-base different interfering strands.</p><p>Endonuclease IV (Endo IV) is an important DNA repair enzymein the base excision repair process.1 As a member of theapurinic/apyrimidinic (AP) endonuclease family,2 it recognizesthe abasic sites in DNA strands and cleaves the phosphodiesterbond 50 to the lesion, generating a hydroxyl at the 30 terminus.3</p><p>The catalytic mechanism of Endo IV has attracted extensivestudies, and a double-nucleotide flipping at the AP site and athree-metal-ion mechanism have been revealed for the damagebinding and incision.4 Meanwhile, the strong preference ofEndo IV to an AP-site within double-stranded DNA and itscapability to cleave the DNA strand containing the AP-site intotwo pieces have provided a useful tool for DNA biosensing.5</p><p>However, the effect of mismatches flanking the AP site on thecleavage activity of Endo IV has rarely been thoroughly inves-tigated. Takeuchi et al. studied the substrate specificity of EndoIV, but the substrates they used were modified oligonucleotidescontaining tetrahydrofuranyl abasic sites instead of naturalDNA abasic sites and they examined only three types ofmismatches.6 Kutyavin et al. designed an artificial lesion tomimic the abasic site, but the cleavage reaction by Endo IV wasmore like an exonuclease degradation process.7 In our previouswork,5b,c we synthesized uracil-containing DNA probes andobtained a natural abasic site by removing the uracil usingUracil-DNA glycosylase (UDG). Herein, we demonstrate theunique capability of Endo IV in discrimination of mismatchesneighboring the natural abasic site and describe its greatpotential in bioanalytical applications.</p><p>To investigate the effect of mismatches neighboring thenatural abasic site on the cleavage rate of Endo IV, we designed a21-nt dual labeled probe and tested four different target strands(see Fig. 1a). When Endo IV cleaves the AP-site, the two resultantfragments will dissociate from the target strand due to thermalinstability, tearing apart the fluorophorequencher pair and emittingfluorescent signals. In our previous work, we found that Endo IVexhibits strong activity in a wide temperature range, up to 60 1C.Based on the predicted melting temperatures of the probe and twofragments,8 we set the detection temperature at 42 1C to obtain aquick fluorescent signal upon cleaving. Interestingly, the resultsshowed that mismatches at the two different sides of the AP sitehad distinct influence on the cleavage rate of Endo IV. As shown inFig. 1b, an A:A mismatch 30 to the AP site (30 mismatch) slightlyaccelerates the process, whereas a C:C mismatch 50 to the AP site(50 mismatch) causes an approximately 5-fold decrease in thecleavage rate. More importantly, simultaneous 50 mismatch and 30</p><p>mismatch (50,30 mismatch) almost completely inhibits the cleavage.We then examined the generality of the discrimination</p><p>capability of Endo IV. For the 30 mismatch, we tested all A:Xmismatches. Fig. S1a (ESI) shows that a 30 mismatch has slightinfluence on Endo IV, and the cleavage rate varies from 100%(30 A:C) to 130% (30 A:A). For the 50,30 mismatch, we fixed the 30</p><p>mismatch to be A:A, and then tested nine types of 50 mismatches:A:X, T:X and C:X. As shown in Fig. S1b (ESI), Endo IVs discrimina-tion toward different types of 50 mismatches in the presence of a 30</p><p>mismatch varies greatly. Defining the discrimination factor (DF) asthe ratio of the signal increase rate of a 30 mismatch to that of a 50,30</p><p>mismatch, we calculated the DFs of all the tested nine mismatches,which were found to be in the range from 3.11 (50 A:G and 30 A:A) to179 (50 C:C and 30 A:A). On the whole, the DFs of C:X are larger thanthose of A:X and T:X. As we know, a C:X mismatch is the mostinstable type of mismatch, so it seems that the thermal stability ofmismatches plays an important role in the discrimination process.We measured the melting temperatures of all the probetargetduplexes that contain the above-mentioned nine types of 50,30</p><p>mismatches. The results, along with the discrimination factors, arelisted in Table 1. Within each category of mismatch, the discrimina-tion factor increases as the melting temperature decreases.</p><p>Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of</p><p>Bioorganic Chemistry and Molecular Engineering, College of Chemistry and</p><p>Molecular Engineering, Peking University, Beijing, 100871, China.</p><p>E-mail: mpzhao@pku.edu.cn; Fax: +86-10-62751708 Electronic supplementary information (ESI) available: Experimental details andsupporting data. See DOI: 10.1039/c3cc40902c</p><p>Received 2nd February 2013,Accepted 18th February 2013</p><p>DOI: 10.1039/c3cc40902c</p><p>www.rsc.org/chemcomm</p><p>ChemComm</p><p>COMMUNICATION</p><p>Publ</p><p>ishe</p><p>d on</p><p> 19 </p><p>Febr</p><p>uary</p><p> 201</p><p>3. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Uni</p><p>vers</p><p>ity o</p><p>f C</p><p>alif</p><p>orni</p><p>a - </p><p>Irvi</p><p>ne o</p><p>n 26</p><p>/10/</p><p>2014</p><p> 11:</p><p>31:0</p><p>1. </p><p>View Article OnlineView Journal | View Issue</p><p>http://dx.doi.org/10.1039/c3cc40902chttp://pubs.rsc.org/en/journals/journal/CChttp://pubs.rsc.org/en/journals/journal/CC?issueid=CC049027</p></li><li><p>2820 Chem. Commun., 2013, 49, 2819--2821 This journal is c The Royal Society of Chemistry 2013</p><p>So we can conclude that the discrimination capability ofEndo IV toward a 50 mismatch in the presence of a 30 mismatchhas a positive relationship with the thermal instability of the50-mismatch-containing duplex.</p><p>According to the reported structure of the Endo IVDNAproduct complex, six nucleotides are involved in anchoring theflipped-out abasic nucleotide to the enzyme active site: the twobase pairs flanking the AP site and the two bases at positions +2and 2 from the AP site. Also, as mentioned above, Endo IV</p><p>cleaves the phosphodiester bond 50 to the AP site. So, thepresence of a mismatch 50 to the lesion may have changed theconformation of the phosphodiester bond in the catalytic center,rendering low efficiency of cleavage. Further investigation of theexact reason for this phenomenon is underway.</p><p>Actually, the above newly found property of Endo IV not onlymerits further study of the underlying mechanisms, but alsohas great potential in constructing DNA sensing platforms withextremely high selectivity. The first potential sensing systemis genotyping, because only one base is different between the30 mismatch target and the 50,30 mismatch target, just like thedifference between different single nucleotide polymorphisms(SNPs). It is easy to apply the unique discrimination property ofEndo IV to detect SNPs 50 to the AP site. All we need to do is todesign an AP site opposite the 30 position of the target SNPs andintroduce a mispaired base at the 30 side of the AP site. It isnoteworthy that the above design does not pose any require-ment on the sequence of the target since the probe can beflexibly designed according to the target strand, which ensuresthe methods generality.</p><p>For a genotyping assay, a large discrimination factorbetween the perfect match duplex and the single-base mis-match duplex is very important. The inherent discriminationcapability of Endo IV provides a discrimination factor rangingfrom 3.11 to 179 for different base pairs, which is alreadyfeasible for genotyping. We conceived that elevating thetemperature may further increase the discrimination factorsby reinforcement of the instability of mismatched duplexes. Wemeasured the melting temperatures of 30 mismatch duplexesand 50,30 mismatch duplexes. Then, we set the temperature atan optimized value between the melting temperature of the30 mismatch duplex and that of the 50,30 mismatch duplex. Asshown in Table 1, column 4, the DFs of the 30 mismatch/50,30</p><p>mismatch for the tested six types of 50,3 0 mismatches increasedfrom 99 to 860. We attribute this substantial increase to thesynergetic amplification effect of the discriminating capabilityof Endo IV and the difference in thermal stability of the 30</p><p>mismatch and 50,30 mismatch probetarget duplex strands.Though in total there are 12 types of mismatches in nature,all of them can be covered by the six types shown in column 4 inTable 1 because genomic DNA or PCR products are all doublestrands and both can be used for performing the detection.These results indicate that Endo IV is able to genotype all kindsof mismatches with great selectivity.</p><p>Another concept closely related to SNPs is the point muta-tions, which are often related to diseases, particularly cancer.9</p><p>Thus, detection of low-abundance mutations has long been ahot issue in clinical diagnosis and the vital point is that thedetection method must have the ability to identify low levels oftarget strands in a large background of interfering sequenceswith only a single-base difference from the targets. In ourprevious work, by taking advantage of the difference in thermalstability between the perfect-match duplex and the single-basemismatch duplex, we established an Endo IV-based selectivesignal amplification method which allows detection of targetstrands at abundance down to 1%.5b Herein, we further inte-grate the newly disclosed discrimination property of Endo IV</p><p>Fig. 1 (a) Schematic illustration of the effects of different mismatches next tothe AP site on the cleavage rate of Endo IV. The blank frame in the dual labeledAP-probe (50-FAM-TATCTGCAC&amp;AGATGCACCTT-30-BHQ1) indicates the AP site.FAM and BHQ1 are carboxyfluorescein and Black Hole Quencher, respectively.50 mismatch represents a mismatch positioned 50 to the AP site. 50 ,30 mismatchindicates that in addition to the mismatch at the 50 position, a second mismatch islocated 30 to the AP site. (b) Fluorescence intensity responses of solutions thatcomprise of 0.1 units of Endo IV, 5 pmol of AP-probes and 5 pmol of the testedtarget strand.</p><p>Table 1 The melting temperatures (Tm) and corresponding discriminationfactors (DF) of duplexes containing different types of 50 ,30 mismatches</p><p>Type of mismatch Tm (1C) DF DF (optimized temperature)</p><p>50C:C &amp; 30A:A 46.9 179 860 (52.5 1C)50C:T &amp; 30A:A 47.5 18.9 498 (52.5 1C)50C:A &amp; 30A:A 48.0 11.6 244 (52.5 1C)50T:C &amp; 30A:A 50.7 11.1 169 (53.5 1C)50T:T &amp; 30A:A 51.0 6.97 144 (53.5 1C)50T:G &amp; 30A:A 51.7 4.44 50A:C &amp; 30A:A 51.5 6.59 99 (53.0 1C)50A:A &amp; 30A:A 52.3 6.19 50A:G &amp; 30A:A 53.0 3.11 </p><p>Communication ChemComm</p><p>Publ</p><p>ishe</p><p>d on</p><p> 19 </p><p>Febr</p><p>uary</p><p> 201</p><p>3. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Uni</p><p>vers</p><p>ity o</p><p>f C</p><p>alif</p><p>orni</p><p>a - </p><p>Irvi</p><p>ne o</p><p>n 26</p><p>/10/</p><p>2014</p><p> 11:</p><p>31:0</p><p>1. </p><p>View Article Online</p><p>http://dx.doi.org/10.1039/c3cc40902c</p></li><li><p>This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 2819--2821 2821</p><p>with the previous approach to construct a more powerfulselective signal amplification system (Fig. 2a). We determinedthe detection limits of the six types of mismatches shown inTable 1, column 4, which were observed to be 0.1% for A:C, T:C,T:T, 0.02% for C:T, C:A, and 0.01% for C:C, as shown in Fig. 2b andFig. S2S6 in the ESI. The detection limits are very impressive foronly one-step reaction. Coupling with prior selective PCR suchas Co-amplification at Lower Denaturation temperature PCR(COLD-PCR)10 or Allele Specific PCR (AS-PCR),11 the detectionlimits can be further improved.</p><p>All of the above experiments were carried out using single-stranded DNA. However, conventional PCR products are doublestranded. Several methods have been developed to generatesingle-stranded PCR products.12 Higuchi and Ochman used a50-phosphorylated forward primer and a regular reverse primer toobtain double-stranded DNA with a 50-phosphate terminal on onestrand.12b By addition of l exonuclease, the 50 phosphorylatedstrands are degraded, resulting in single-stranded DNA products.We tested the adaptability of our system to post-PCR detection. Thedetailed experimental procedures and results are all shown inFig. S7 (ESI). Clearly, our system can be readily applied to thepost-PCR reaction solution after degradation of the PCR productswith l exonuclease and enzyme inactivation. The discriminationfactor between the 30 A:A strand and the 50 C:C &amp; 30 A:A strandachieved was 725 for the PCR products, which is comparable to theDF value (860) obtained by direct detection (see Table 1). The aboveresults proved that by incorporating the newly found property of</p><p>Endo IV, the signal amplification system is able to differentiate thetarget DNA strand at very low abundance from single-base differentbackground sequences and can work on double-stranded DNA.These distinct advantages hold great potential for further applica-tions in detection of low-abundance mutations. A limitation of thepresent assay is the relatively high cost of the dual labeled probe,which may be further modified to a simpler and less expensivelabel-free detection system in the future.7b</p><p>To sum up, we report for the first time the unique capabilityof Endo IV in discrimination of mismatches neighboring anatural abasic site in DNA strands. Furthermore, we found thatthe intrinsic property of the enzyme in differentiation of a 30</p><p>mismatch from a 50,30 mismatch had a positive relationshipwith the 50 mismatch-induced thermal instability of the probetarget duplex. These new findings are not only important forrelated biological studies, but also have significant potential inconstructing DNA sensing platforms with extremely high selec-tivity. Genotyping of SNPs with high discrimination factors inthe range from 99 to 860 and differentiation of 0.10.01% oftarget DNA strands from a large background of single-basedifferent interfering strands have been demonstrated. Thereaction system can be easily adapted to post-PCR detection.</p><p>The authors acknowledge the NSF of China (Grant No.21175007 and 91132717) for financial support.</p><p>Notes and references1 (a) T. Lindahl, P. Karran and R. D. Wood, Curr. Opin. Genet. Dev.,</p><p>1997, 7, 158169; (b) C. D. Mol, D. J. Hosfield and J. A. Tainer,Mutat.Res., 2000, 460, 211229.</p><p>2 (a) L. Aravind, D. R. Walker and E. V. Koonin, Nucleic Acids Res.,1999, 27, 12231242; (b) J. L. Huffman, O. Sundheim andJ. A. Tainer, Mutat. Res., 2005, 577, 5576.</p><p>3 J. D. Levin, A. W. Johnson and B. Demple, J. Biol. Chem., 1988, 263,80668071.</p><p>4 (a) D. Ramotar, Biochem. Cell Biol., 1997, 75, 327336; (b) D. J. Hosfield,Y. Guan, B. J. Haas, R. P. Cunningham and J. A. Tainer, Cell, 1999, 98,397408; (c) I. Ivanov, J. A. Tainer and J. A. McCammon, Proc. Natl.Acad. Sci. U. S. A., 2007, 104, 14651470; (d) E. D. Garcin, D. J. Hosfield,S. A. Desai, B. J. Haas, M. Bjoras, R. P. Cunningham and J. A. Tainer,Nat. Struct. Mol. Biol., 2008, 15, 515522.</p><p>5 (a) M. Pflaum, O. Will and B. Ep...</p></li></ul>

Recommended

View more >