Sensitive Detection of DNA Lesions by Bulge Enhanced Highly

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Sensitive Detection of DNA Lesions by Bulge Enhanced Highly Specific COLD-PCR Yu Feng, Shuang Cai, Guoliang Xiong, Guanfei Zhang, Shi-Hui Wang, Xin Su, and Changyuan Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01599 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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Analytical Chemistry

Sensitive Detection of DNA Lesions by Bulge Enhanced Highly Specific COLD-PCR Yu Feng,† Shuang Cai,† Guoliang Xiong,‡ Guanfei Zhang,† Shihui Wang,† Xin Su*,† and Changyuan Yu*,† †

College of Life Science and Technology, Beijing University of Chemical Technology, Beijing

100029, China. ‡

Department of Nephrology, Shenzhen Affiliated Hospital, Guangzhou University of Chinese

Medicine, Guangdong, Shenzhen 518033, China. *

Corresponding author

Email: [email protected], [email protected] Tel: +86-10-64421335 Fax: +86-10-64416248

1

ACS Paragon Plus Environment


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Abstract Mutagenic modifications of nucleotides or DNA lesions that result from environmental stress have proven associated with a variety of diseases, particularly cancer. The method for accurately detecting the lesions is therefore of great importance for biomedical research and toxicity study. We develop a sensitive and low-cost bulge enhanced COLD-PCR method for detecting DNA lesions (uracil and 8-oxoguanine) by combining in vitro base excision repair (BER) pathway and COLD-PCR. The modified bases are converted to bulge via BER pathway involving converting modified bases to apurinic/apyrimidinic (AP) site, cleavage at AP site and break ligation. The presence of bulge induces a large change of the hybridization thermodynamics of double stranded DNA, eventually enhancing the specificity of COLD-PCR. Besides, we used the free energy of hybridization as reference to optimize the critical denaturation temperature (Tc) of COLD-PCR obtaining more specific amplification than empirical Tc. Taking advantage of the proposed bulge enhanced COLD-PCR, we are able to identify the presence of DNA lesions-containing strands at low abundance down to 0.01%. This method also exhibits high sensitivity for glycosylase with a detection limit of 10-4 U/mL (3S/N) that is superior than some recently reported methods. With the design of repair guide probe, the level of oxidative damage in genomic DNA caused by chemicals and photodynamic therapy (PDT) can be evaluated heralding more applications in clinical diagnosis and epigenetic study.

Keywords DNA Lesion; COLD-PCR; glycosylase; Base Excision Repair; Reactive Oxygen Species.

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Introduction DNA lesions that result from persistent endogenous and environmental stress are becoming increasingly attractive due to their mutagenic potential.1,2 Guanine can be easily oxidized to 8-oxoguanine (8-oxoG) under the oxidative stress, such as reactive oxygen species (ROS).3 The level of 8-oxoG emerges as a typical biomarker of human DNA damage. The presence of 8-oxoG can lead G:C to A:T mutation eventually resulting in transcriptional error.4 The aberrant accumulation of 8-oxoG is believed associated with a variety of diseases, such as cancers,5 neurological disorders,6 and heart disease.7 Uracil as another common lesion in DNA is mainly formed via deamination of cytosine.8 The resultant U:G mismatch is mutagenic because it can lead to C to T transition. The intracellular level of uracil is regulated by activation-induced cytidine deaminase (AID).9 Enzyme-catalyzed C to U of single-stranded DNA (ssDNA) has detrimental effects on immunity and cancer.10,11 In this regard, to develop methods for accurately detecting DNA lesions is crucial for the biomarker based diagnosis and the evaluation of environmental stress. Moreover, identifying these lesions at low abundance is critical in several fields of early stage diagnosis and genetic toxicity of chemicals. Much effort has been made to analyze the level of DNA lesions including HPLC,12 electrochemical sensors,13,14 single-molecule sequencing,15 nanopore approaches,16,17 and methods based on fluorescent organic probes.18 The success of these approaches allow researchers to access the level of DNA lesions in biological samples. Nevertheless, the detection of DNA lesions particularly at low abundance is still challenging because the presence of modified bases in long DNA sequence does not significantly alter the observable property of DNA. Base excision repair (BER) pathway is a highly regulated network of enzymes involved in the maintenance of gene damage and the prevention of human disease.19 The steps of BER commonly consist of (1) the excision of the modified base by DNA glycosylase to form apurinic/apyrimidinic (AP) site, (2) the cleavage of AP site by AP endonuclease. After the cleavage, the final repair can be completed through long-patch and short-patch which differ based on the involvement of flap endonuclease. Burrows and co-workers demonstrated that the gap can be directly sealed by T4 ligase in vitro to form a bulge in double stranded DNA (dsDNA).20 Sanger sequencing was employed as downstream analysis to determine the site of DNA lesion. This smart design provides the address information of DNA lesions. However, due 3



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to the low sensitivity of sequencing, it is difficult to identify the presence of the lesions at low abundance. Coamplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) selectively enriches ‘minority alleles’ from mixtures of wild-type and mutation-containing sequences, irrespective of the type and location of mutations.21 COLD-PCR serves as a powerful tool for asymmetrically enriching mutation-containing sequences because any mutation can cause the change of melting temperature (Tm) of dsDNA. The Tm changes induced by different types of mutations can vary, resulting in different amplification efficiency.22 Enhanced-ice-COLD-PCR23 and temperature tolerant COLD-PCR (TT-COLD-PCR)24 are new formats that enhance the robustness and efficiency of COLD-PCR. COLD-PCR does not directly detect mutations, but relies on endpoint detection methods like high resolution melting (HRM) or sequencing. These methods were exploited here for downstream analysis. Herein, we report a highly sensitive, simple and low-cost method for detecting DNA lesions by combining in vitro BER pathway and the selective enrichment of target by COLD-PCR. As previously reported,20 the modified base can be removed by BER pathway resulting in single strand break which can be further sealed by ligase, yielding a bulge at the modified base-containing strand. The bulge conformation in dsDNA is not preferable in energy particularly at high temperature. The large difference of hybridization thermodynamics between bulge-containing strand and normal strand allows efficient and selective amplification of target at relatively lower denaturation temperature, therefore reducing nonspecific signal and enhancing the specificity of COLD-PCR. Thanks to the high specific amplification of bulge enhanced COLD-PCR, high sensitivity of detecting DNA lesions is achieved with a detection limit of 0.01%. With this method, glycosylase that is responsible for maintaining the genome integrity can also be detected with high sensitivity. With the design of repair guide probe, the 8-oxoG in genomic DNA can be accessed allowing the evaluation of the level of chemicals and photodynamic therapy (PDT) mediated oxidative damage. This approach holds a great potential on studying the cellular genomic toxicity of various external stimulus. Experimental Section Materials. All of the oligonucleotides used in this work were synthesized and purified by HPLC (Sangon Co., Shanghai, China) and their sequences are listed in Table S1. Uracil-DNA Glycosylase (UDG), Human apurinic/apyrimidinic endonuclease (APE1), T4 DNA ligase, 4


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8-oxoguanine DNA glycosylase (hOGG1) lambda exonuclease, and ThermoPol reaction buffer were obtained from NEB (MA, USA). Fast Universal SYBR Green Master (ROX) was purchased from Roche (CA, USA). DNase/RNase free deionized water from Tiangen Biotech Co. (Beijing, China) was used in all experments. Bulge generation in DNA lesion-containing synthetic oligonucleotides via BER pathway. In a typical assay, to a 200 µL sealed PCR tube, 200 nM of the double stranded synthetic oligonucleotides and glycosylase (20 U/mL UDG or 2 U/mL hOGG) were mixed in 50 µL of 1×ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4 and 0.1% Triton X-100, pH 8.8) at 37 °C for 50 min. Note that for hOGG, 1×ThermoPol buffer was compensated with 50 mM NaCl and 1% BSA (w/v). Next, APE1 was added to reach a final concentration of 4 U/mL and incubated at 37 °C for another 50 min and heated to 90 °C for 10 min to inactivate glycosylase and endonuclease. 5 µL of DMSO, 1 µL of 3 mM ATP, and 800 U of T4 ligase were added and held at 16 °C for 2 h and then heated to 65 °C for 10 min. COLD-PCR for determining the presence of DNA lesions. The product of bulge generation step was diluted to 20 nM and mixed with 1×SYBR Green (Life Technologies). The fluorescence of dye was recorded ranging from 60 °C to 90 °C with an increment of 0.5 °C on a real-time PCR thermal cycler (qTOWER, Analytik Jena, Germeny). 20 nM of primers, 8 pM of the product of bulge generation step, 1×Fast Universal SYBR Green Master (ROX) were mixed thoroughly to reach a final volume of 20 µL. Prior to COLD-PCR, the normal PCR amplification was carried out on the real-time PCR thermal cycler for 10 cycles with a program of 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 30 s. For full-COLD-PCR, the procedure was as follows: 95 °C, 15 s for denaturation; 70 °C, 8 min for cross hybridization; Tc, 3 s for selective denaturation; 55 °C, 30 s for primer annealing; 72 °C, 1 min for extension. The procedure of fast-COLD-PCR was as follows: Tc, 15 s for denaturation; 55 °C, 30 s for primer annealing; 72 °C, 1 min for extension. The fluorescence was recorded at 55 °C for two types of COLD-PCR. Detection of DNA Lesions caused by ROS species. Hydroxyl radical (•OH) was generated by Fenton reaction (Fe2+ + H2O2 → Fe3+ + •OH + OH−) by mixing ferrous ions with H2O2 at a molar ratio of 1:10 at 37 °C for 30 min.25 Singlet oxygen (1O2) was generated by NaOCl/H2O2 in 5



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a ratio of 1:1 for 20 min. Due to their short lifetime, ROS (1O2 H2O2 HClO, and •OH) were adjusted to 100 µM and immediately incubated with DNA for 30 min. Prior to bulge generation, the lesions-containing DNA was annealed with 5’-phosphorylated repair guide probe (1000-fold excess) by 95 °C for 2 min, 70 °C for 1 min, 50 °C for 1 min, and 37 °C for 5 min. The bulge generation step was then conducted. Lambda exonuclease (final concentration, 0.1 U/µL) was added to digest the repair guide probe. Full-COLD-PCR was finally carried out to selectively enrich the bulge-containing sequence. Cell culture, genomic DNA extraction and oxidative lesion detection. HEK-293T cell line was cultivated in DMEM medium supplemented with 1% Penn/Strep and 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air. For investigating the effect of ROS generated by chemicals, the cell was cultured in H2O2-containing 1×PBS for 30 min. To study the effect of ROS generated by PDT, prior to UVA irradiation for 20 min, the cell was incubated in TiO2 (200 µg/mL) containing culture medium for 12 h. After the treatments for cell, genomic DNA was extracted by using QIAamp DNA Micro Kit (QIAGEN, Germany) for the analysis of the oxidative lesions in the interested regions. Results and Discussion The Principle of Detection of DNA Lesions by Bulge Enhanced COLD-PCR As depicted in Figure 1, the modified base is converted to bulge (single nucleotide deletion) via in vitro BER pathway. Synthetic oligonucleotides of the codon 12 of the KRAS gene containing modified base (uracil and 8-oxoG) were used as model target. The AP site is generated by glycosylases: uracil DNA glycosylase (UDG) for uracil and human 8-oxoguanine glycosylase (hOGG) for 8-oxoG, respectively. UDG as a monofunctional glycosylase does not catalyze the cleavage of phosphodiester.26 In contrast, hOGG as a bifuncational glycosylase exhibits glycosylase and AP-lyase activity leaving a 5’ phosphate and a 3’-phospho-α, β-unsaturated aldehyde.27 The glycosylase treated strand is recognized by APE1 that cleaves 5’ to the AP site to generate single strand break. For UDG treated strand, the terminus is 3’-hydroxyl and 5’-deoxyribosephosphate (5’-dRP). This 5’-dRP residue can be removed by AP lyase enzymes via β-elimination mechanism.28 In this case, T4 ligase serves as AP lyase to remove the dRP and then seal the break.29,30 Due the AP lyase activity of hOGG, the 5’-dRP is removed in 6


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the AP endonuclease step The break is therefore directly sealed by T4 ligase (left panel of Figure 1). Prior to COLD-PCR, normal PCR was conducted for 10 cycles to symmetrically amplify the two single strands resulting in two dsDNAs that differ by one nucleotide in length. Full-COLD-PCR was chosen to selectively enrich the heteroduplex (bulge-containing sequence) formed by annealing the two dsDNAs at 70 °C . In the meantime, homoduplexes are also formed. When the system is heated to the optimized critical denaturation temperature (Tc), the bulge-containing heteroduplex is de-hybridized whereas the homoduplexes remain as double-strand form. Consequently, the selective amplification of bulge-containing duplex was achieved (right panel of Figure 1). As shown in Figure 2A, the uracil containing sequence is selectively amplified within 25 cycles; however, the normal sequence is not amplified effectively because no heteroduplex is available during COLD-PCR. As expected, 8-oxoG-containing strand exhibits similar result as uracil-containing strand (Figure 2B). The high specificity of bulge enhanced COLD-PCR brings high discrimination for lesions-containing sequence and normal sequence. The selectivity of COLD-PCR dominates the performance of this approach. The choice of the type of COLD-PCR is important for the enrichment selectivity. COLD-PCR can be categorized into two types: full-COLD-PCR and fast-COLD-PCR which are referred to as “five-step PCR” and “three-step PCR”, respectively.22 In full-COLD-PCR, the procedure includes standard denaturation,

intermediate

hybridization

step

(normally

at

70

°C )

that

allows

cross-hybridization or heteroduplexes formation, critical denaturation step at Tc, primer annealing and polymerase extension. Full-COLD-PCR is suitable for all types of mutations because it relies on the lower melting temperature of heteroduplexes. In fast-COLD-PCR, the intermediate hybridization is not included, alternatively, three steps are applied including denaturation at Tc, primer annealing, and polymerase extension. The preferential amplification of fast-COLD-PCR relies on the Tm difference of single-nucleotide mismatch-containing sequence and wild type. Different types of single-nucleotide mismatch leads different changes to the hybridization thermodynamics. Some changes are not big enough to lead significant Tm reduce, restricting its application for all types of mutations. The simpler procedure of fast-COLD-PCR makes it faster than full-COLD-PCR. As shown in Figure 1, full-COLD-PCR was employed to preferentially enrich the bulge-containing sequence in our method. As described above, 7



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bulge-containing sequence is amplified effectively due to the heteroduplex formation at intermediate hybridization step. In contrast, formation of heteroduplex is not allowed if fast-COLD-PCR applied in this system (Figure S1A). In fast-COLD-PCR, the discrimination of bulge-containing sequence and normal sequence relies on the differential Tm of the two perfectly matched dsDNAs differing by 1 bp in length. Unfortunately, there is no significant difference in Tm for the two dsDNAs. As expected, despite the denaturation temperatures used, fast-COLD-PCR does not provide efficient discrimination of bulge-containing sequence and normal sequence (Figure S1B). Free Energy Guided Denaturation Temperature Determination Tc is another critical factor for the success of COLD-PCR. The empirical Tc is usually 0.5-1.5

°C lower than the Tm of the preferentially amplified target. Tm is always measured by fluorescent dyes. The melting curves of bulge-containing sequence and normal sequence are presented in Figure 3A. The difference in Tm is ~2 °C which is slightly lower than the predicted result (~3 °C ) by NUPACK (Figure 3B). The hybridization yield of DNA is concentration dependent so that Tm can vary at different concentrations. The ratio of de-hybridization increases as the decrease of strand concentration. The initial concentration of targets in COLD-PCR is much lower than that used for Tm measurement. The empirical Tc is therefore not accurate to reflect the hybridization status. As predicted by NUPACK, the Tm of the targets at the concentration for amplification are both 7.5 °C lower than that at the concentration for Tm measurement. Unfortunately, the fluorescent dye is not sufficiently sensitive to indicate the melting of dsDNA at that low concentration. To determine the Tc accurately, we established the curve of de-hybridization ratio versus concentration adjusted-free energy change by NUPACK (Figure 3C, for free energy calculation, see supporting note1). As reported previously, the highest discrimination factor for single nucleotide mismatched strand (including deletion and insertion) and perfectly matched strand can be achieved when the free energy of one of them approaches zero.31 As shown in Figure 3C, the free energy of bulge-containing sequence and normal strand is close to zero at 74 and 77 °C . Full-COLD-PCRs with the Tc marked in Figure 3C were performed. All of them exhibit differential threshold cycles (Ct) for bulge-containing strand and normal strand in amplification curves (Figure S2). By the comparison of the ∆Ct (∆Ct=Ct, Bulge-Ct, Normal) in Figure 3D, COLD-PCR at 77 °C provides the 8


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most effective discrimination and the fastest enrichment for bulge-containing sequence than others that is consistent with the prediction. As aforementioned results, the selectivity of COLD-PCR is determined by the hybridization thermodynamics of the targets at Tc. Different types of single nucleotide changes possess varying free energy changes. By using the thermodynamic parameters from NUPACK, we calculated the free energies of the amplicons containing G:C, G:A, G:T, G:G, and G:□ (bulge), respectively. As shown in Figure 3E, the bulge exhibits larger change in free energy than others probably because the conformational change caused by bulge significantly weakens the stability of dsDNA. The bulge-containing sequence is enriched more efficiently than other types of single nucleotide changes in terms of its larger free energy change (Figure 3F and Figure S3). Accordingly, COLD-PCR is advantageous for enriching bulge-containing sequence. Detection of DNA Lesions at Low Abundance and Quantification of DNA Glycosylase The level of DNA lesions serve as a biomarker for related diseases and the risk of ROS exposure. To detect DNA lesions at low abundance is therefore advantageous for diagnosing diseases at early stage and evaluating the potential risk of ROS. Due to the high selectivity of bulge enhanced COLD-PCR, we speculated that this approach can be employed for the detection of lesions at low abundance. As shown in Figure 4A and C, the COLD-PCR can clearly distinguish the single uracil/8-oxoG containing sequences at different fractions from the normal sequence. For both types of the lesions, the Ct value shows good linear relation with the logarithm of the fraction of lesion-containing DNA in a broad range of 0.01% to 100% (Figure 4B and 4D). To the best of our knowledge, this is the lowest abundance of single nucleotide change that can be reached by COLD-PCR without any auxiliary probe and downstream analysis.32,33 Glycosylase plays a crucial role in BER pathway.34 The expression level of glycosylase is associated with various diseases including inflammation,35 Alzheimer's disease,36 and cancers.37 Conventional glycosylase assays include radioactive methods, HPLC, and mass spectrometry. Recent advances of DNA nanosensors allow the detection of glycosylase (e.g. UDG and hOGG) with high sensitivity.38-40 The success of detecting low abundance lesions suggests that this method may be used for quantifying glycosylases with high sensitivity. The conversion rate of 9



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modified base to bulge is determined by the amount of glycosylase when the amounts of other enzymes are fixed. We amplified the synthetic oligonucleotides repaired by varying amounts of hOGG via full-COLD-PCR at the optimal Tc. As shown in Figure 4E, the sequences are selectively enriched in the presence of hOGG (>10-5 U/mL) within 25 cycles. Similarly, the Ct shows good linear relation with the logarithm of the hOGG concentration ranging from 10-4 to 10-1 U/mL. The detection limit of 10-4 U/mL (Figure 4F) was determined with a signal/noise ratio of 3 which is superior than some recent reported methods.41,42 Detection of 8-oxoG caused by ROS species Continuous exposure to ROS can cause oxidative damage of DNA. The level of 8-oxoG in genomic DNA reflects the stress of ROS. The challenge for detecting 8-oxoG caused by ROS in genomic DNA via bulge enhanced COLD-PCR is that the repair of the primer-binding region may weaken the binding of primer at annealing temperature, resulting in poor amplification efficiency. To address this challenge, we firstly introduced repair guide probe which hybridizes with the interested region excluding primer-binding region. As illustrated in Figure 5A, the repair guide probe which is 5’-phosphorylated and in large excess (1000 fold) was annealing with the ROS treated dsDNA resulting in strand displacement. The 8-oxoGs in the probe-binding region are then repaired by BER pathway because APE1 is a dsDNA specific endonuclease and does not cleave the AP site at single strand DNA. Subsequently, the two long strands re-hybridize upon the repair guide probe is digested by lambda exonuclease. Consequently, the bulge can be generated in the interested region except for the primer binding region. We used the synthetic oligonucleotide of the codon 12 of the KRAS gene as a proof of concept to verify the feasibility of the repair guide probe. The synthetic oligonucleotide was incubated with 100 µM H2O2. 8-oxoG can be generated and distributes in the entire strand. As shown in the Figure 5B, the amplification efficiency is low without the probe-guiding repair. In contrast, 8-oxoG-containing sequence is amplified via COLD-PCR with the probe-guiding repair (Figure 5C). Therefore, the usefulness and efficiency of repair guide probe were proven. As expected, the Ct value decreases with increasing the amount of H2O2, suggesting that our method is able to reflect the 8-oxoG level in the H2O2 treated synthetic oligonucleotides (Figure 6A and Figure S4A). The results of different ROS including singlet oxygen (1O2), hypochlorite (HClO), hydroxyl free radical (•OH) of 100 µM are presented in Figure 6B (for amplification curves, see 10


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Figure S4B). As can be seen in Figure 6B, 1O2 as molecular oxygen in the lowest excited state is more reactive than other ROS. HEK-293T cell was cultured in H2O2 containing medium for 30 min potentially yielding intercellular oxidative damage of DNA. The genomic DNA was extracted and repaired by aforementioned probe-guided BER pathway. COLD-PCR was carried out to selectively enrich the bulge-containing species. As shown in Figure 6C and S4C, Ct value decreases with the concentration of H2O2 indicating that the level of oxidative guanine lesion is proportional with the ROS amount. Various nanoparticles have been used for enhancing PDT due their advantages of rapid ROS generation. Titanium oxide nanoparticle (TiO2) which is a widely used photosensitizer for PDT was investigated for oxidative guanine lesion during PDT.43 As presented in Figure 6D and S4D, TiO2 enhanced PDT results in a higher intercellular level of 8-oxoG than nanoparticle-free PDT. This result suggests that the use of nanoparticles in PDT potentially increase the risk of mutagenic lesions. Conclusion In summary, a simple method for detecting DNA lesions with high sensitivity is successfully developed by employing the high amplification specificity of bulge enhanced COLD-PCR. Uracil and 8-oxoG containing sequences can be detected at abundance down to 0.01% with high confidence. The large change in hybridization thermodynamics induced by bulge conformation makes a highly specific COLD-PCR that allows direct detection of modified bases at low abundance down to 0.01% without any downstream analysis. The remarkable sensitivity for repaired products also enables us to quantify the glycosylase with high sensitivity down to 10-4 U/mL. With the design of repair guide probe, the level of DNA oxidative lesions at unknown site can be evaluated. On this basis, DNA oxidative lesions by ROS which are caused by chemicals and PDT are measured. As the merits discussed above, we anticipate that this method would find broad applications in expanding the use of COLD-PCR for detecting various types of epigenetic modifications and in coupling with various downstream analysis to provide deep insight of DNA lesions based diagnosis.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (31600687 and 21606013) and Fundamental Research Funds for the Central Universities (12060070031, 12060090029, 12060072011, 12060026025, and 12130001003). Supporting Information The Supporting Information is available free of charge on the website. The sequences of oligonucleotides used in this work are listed in table S1. The detail of concentration-adjusted free energy calculation are in supporting note 1. The supporting figures are included.

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FIGURES AND CAPTIONS

Figure 1. Schematic illustration of the detection of DNA Lesions by bulge enhanced COLD-PCR. The uracil and 8-oxoG lesions are converted to bulge via different in vitro BER pathways followed by the amplification of normal PCR and COLD-PCR enhanced selective amplification of bulge-containing sequence.

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Figure 2. Amplification curves by COLD-PCR in the presence/absence of single lesion site, (A) Uracil modification, (B) 8-oxoG modification. The sequences are listed in supporting information. 200 nM oligonucleotides was used for repair, the concentration of target for COLD-PCR is 8 pM.

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Figure 3. (A) Melting curves of bulge-containing sequence and normal sequence. (B) Comparison of the Tm of bulge-containing sequence and normal sequence (red: measured by SYBR green, dark and yellow: predicted by NUPACK at 20 nM and 8 pM). (C) Relation of the ratio of de-hybridization and free energy. The bulge-containing sequence and normal sequence at different temperatures are labeled in the curve. (D) ∆Ct of bulge-containing sequence and normal sequence in COLD-PCR with different Tc. (E) Comparison of de-hybridization ratio and free energy for bulge (G:□), single nucleotide mutation, and normal sequence at 77 °C . (F) Comparison of the ∆Ct to normal sequence of single nucleotide mutation and bulge-containing sequence at 77 °C .

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Figure 4. COLD-PCR curves of uracil (A) and 8-oxoG (C) containing sequences at different ratios. Ct of COLD-PCR versus the logarithm of the fraction of lesion-containing sequence, uracil (B) and 8-oxoG (D). (E) COLD-PCR curves of 8-oxoG-containing sequence repaired by different amounts of hOGG. (F) Ct of COLD-PCR versus the logarithm of the concentration of hOGG. All experiments were repeated three times.

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Figure 5. (A) Scheme of the probe-guided repair. The repaired bases are those on the red strand in the repaired region. The bulge is eventually present in the initial dsDNA upon the repair guide probe digested by lambda exonuclease. COLD-PCR curves of single 8-oxoG-containing sequence and normal sequence in the absence/presence (B/C) of repair guide probe.

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Figure 6. (A) Normal synthetic oligonucleotides treated with different concentrations of H2O2. Ct of COLD-PCR versus the concentrations of H2O2. (B) Normal synthetic oligonucleotides treated with different types of ROS of 100 µM. Ct of COLD-PCR versus different ROS. (C) HEK-293T cell was treated with different concentrations of H2O2. Ct of COLD-PCR versus the concentrations of H2O2 in culture medium. (D) Oxidative lesions in the genomic DNA of HEK-293T cell caused by the ROS from PDT. The combination of TiO2 nanoparticle and UVA irradiation causes more oxidative lesion. Note that for cell assay, the codon 12 of KRAS gene was tested. All experiments were repeated for three times.

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For TOC only

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Sensitive Detection of DNA Lesions by Bulge Enhanced Highly

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