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  • This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 2819--2821 2819

    Cite this: Chem. Commun.,2013,49, 2819

    Endonuclease IV discriminates mismatches next to theapurinic/apyrimidinic site in DNA strands: constructingDNA sensing platforms with extremely highselectivity

    Xianjin Xiao, Yang Liu and Meiping Zhao*

    A unique capability of Endonuclease IV in discrimination of mismatches

    neighboring a natural abasic site in DNA strands has been demon-

    strated, which enables genotyping of SNPs with high discrimination

    factors and differentiation of as low as 0.10.01%of target DNA strands

    from a large background of single-base different interfering strands.

    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

    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

    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.

    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

    mismatch (50,30 mismatch) almost completely inhibits the cleavage.We then examined the generality of the discrimination

    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

    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

    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

    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

    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.

    Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of

    Bioorganic Chemistry and Molecular Engineering, College of Chemistry and

    Molecular Engineering, Peking University, Beijing, 100871, China.

    E-mail: mpzhao@pku.edu.cn; Fax: +86-10-62751708 Electronic supplementary information (ESI) available: Experimental details andsupporting data. See DOI: 10.1039/c3cc40902c

    Received 2nd February 2013,Accepted 18th February 2013

    DOI: 10.1039/c3cc40902c






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  • 2820 Chem. Commun., 2013, 49, 2819--2821 This journal is c The Royal Society of Chemistry 2013

    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.

    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

    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.

    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.

    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

    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

    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.

    Another concept closely related to SNPs is the point muta-tions, which are often related to diseases, particularly cancer.9

    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


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