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One-pot Isolation of Desired Human Genome Fragment by Using Biotinylated pcPNA/S1 Nuclease Combination Arivazhagan Rajendran, Narumi Shigi, Jun Sumaoka, and Makoto Komiyama Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00202 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018
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Biochemistry
One-pot Isolation of Desired Human Genome Fragment by Using Biotinylated pcPNA/S1 Nuclease Combination Arivazhagan Rajendran,*,†,‡ Narumi Shigi,†,§ Jun Sumaoka,†,¶ and Makoto Komiyama*,†,§ †
Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8577, Japan
Supporting Information Placeholder ABSTRACT: Scission of human genome at predetermined sites and isolation of a particular fragment are of great interest for the analysis of lesion/modification sites, proteomics, and for gene therapy. However, methods for human genome scission and specific fragment isolation are limited. Here, we report a novel onepot method for the site-specific scission of DNA by using the combination of biotinylated pcPNA/S1 nuclease, and isolation of a desired fragment by streptavidin coated magnetic beads. The proof-of-concept was initially demonstrated for the clipping of plasmid DNA and isolation of required fragment. Our method was then successfully applied for the isolation of a fragment from cellderived human genome.
Human genome is highly susceptible to various modifications, lesions and damages.1 These alterations significantly affect their functions and also how cells read these genes. DNA methylation is one such example that occurs through epigenetic modification. Other lesions that commonly occur in the human genome are oxidation (8-oxoguanine2 and thymine glycol),3 abasic site,4,5 replication errors,6 interstrand crosslinking,7,8 light-induced pyrimidine dimers,9 and single- and double-strand breaks.10 The molecular level mechanisms of these lesions and modifications are not well known. Further, the identification of such lesion sites in human genome and their consequences are important to understand the diseases caused by them and also for their treatments. In addition to the DNA modifications, analysis of proteins bound to a particular DNA region is of great interest in proteomics. To analyze these lesions and bound proteins, human genome should be fragmented at desired sites and the particular region of interest should be isolated. Till date, there are several methods reported for the isolation of the desired human genome region.11-22 For example, the isolation of specific genes from high molecular weight chromatin was performed by nucleoprotein hybridization method in which an enzymatic digest of the genome was treated with an exonuclease, and the single-strand (ss) portion formed at the end of the target fragment was conjugated with complementary DNA probe.12 Alternatively, a long CAG triplet repeats containing fragment was isolated by one strand of peptide nucleic acid (PNA).16 Triplex affinity capture method was used to obtain the fragments involving homopyrimidine/homopurine sequences.13,14 Some of the recent methods are the proteomics of isolated chromatin segments (PICh)20 that use locked nucleic acids (LNAs), insertional chromatin immunoprecipitation (iChIP)19 and engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).21,22 However, these meth-
ods are either specific for a particular sequence or require tedious and complicated experimental procedures. Further, there is no singly accepted technology of choice to efficiently isolate genomic fragments without critical limitations in their sequences. Regarding the DNA scission, several protein-based DNA cutters such as the hybrid restriction enzymes generated by the fusion of the cleavage domain of Fok I with zinc finger proteins (ZFN)23 and transcription activator-like effectors (TALEN),24,25 homing endonucleases,26 RNA-guided clustered regularly interspaced short palindromic repeat-associated enzyme 9 (CRISPR-Cas9) system,27,28 and the DNA-guided artificial restriction enzyme based on the Pyrococcus furiosus Argonaute (PfAgo)29 were reported. We have been working on the site-specific double-strand break by artificial restriction DNA cutter (ARCUT)30 in which a pair of pseudo-complementary PNAs (pcPNAs)31 and ssDNAcleaving Ce(IV)/EDTA complex were combined. In the present study, we replaced the metal complex by ssDNA-cleaving enzyme S1 nuclease for the DNA scission, and biotin was conjugated at the terminus of one of the pcPNAs, so that streptavidin (STV)coated magnetic beads can be directly used for the isolation of the desired DNA fragment. The sequences of the pcPNAs and their binding regions in DNA are given in Figure 1a. These pcPNAs are composed of the poly(N-(aminoethyl)glycine) backbone and contains 2,6diaminopurine (D) and 2-thiouracil (Us) instead of adenine and thymine respectively, thus termed pseudo-complementary. The presence of these unnatural nucleobases are necessary to avoid the PNA-PNA duplex. In addition, lysine residues (K) and phosphoserine (Sp) were introduced to improve the solubility and increased binding affinity to the negatively charged DNA backbone. The detailed structure and synthesis of pcPNAs were described in our previous report.31 At first, our one-pot isolation method was tested for plasmid DNA. The detailed protocol of the pcPNAs assisted formation of single-stranded region, double-strand break by S1 nuclease, and isolation of the desired fragment by DNA-pcPNA2Bio/STV-coated magnetic beads is given in supporting information. Note that a biotin is attached only to pcPNA2, which is complementary with the long protruding portion formed in the target fragment upon the present site-selective scission. In brief, the blue fluorescent protein gene integrated plasmid (pBFP-N1)32 was linearized by StuI and used for our study. The linearized plasmid was incubated with a pair of pcPNAs (one pcPNA contained biotin in the C-terminal lysine connected through a PEG2 linker). Since the pcPNA/DNA complex is more stable than the native DNA/DNA base pairing, the pcPNAs invade the DNA and form the invasion complex
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Figure 1. One-pot isolation of a 3 kbp fragment from a linearized plasmid. a) The sequences of the pcPNAs and their binding sites in DNA are given. The underlined and boxed regions indicate the pcPNA-DNA base-pairing and single-stranded (ss) regions, respectively. The pseudo-complementary bases 2,6-diaminopurine, 2-thiouracil were denoted as D and U (U instead of Us for simplicity), respectively. Lysine, phosphoserine, and polyethylene glycol linker are denoted as K, Sp, and PEG2, respectively. b) Formation of the invasion complex and ss regions, S1 scission, and isolation of the target fragment by STV-coated magnetic beads are illustrated. c) The characterization of the S1 scission and target isolation at various pH. d) Control experiment in the absence of biotin in both the pcPNAs. Experimental conditions: Invasion: 0.5 mM HEPES, pH 7.0, 50ºC, 1 h. S1 scission: 300 mM NaCl, 10 mM MES, pH 5.5-7.0, at 25ºC for 3 h. Recovery: 25 µL of 30 mM biotin at 95ºC for 10 min. Final concentrations: [Linear pBFP-N1] = 2.62 nM; [pcPNAs] = 100 nM; [Dynabeads] = 1150 µg; [S1] = 125 U. [Agarose] = 1%, [TBE] = 1X, 100 V, Staining = Gelstar. (Figure 1b). These pcPNAs were designed in such a way that they are 5 bases laterally shifted from each other when they invade the DNA (boxed sequences in Figure 1 a). This in turn leads to the formation of ss region in both the strands of DNA (Figures 1a and 1b). Then, NaCl was added to the reaction mixture to stabilize and protect the duplex region from S1 scission. The ss regions were then cleaved by S1 nuclease, leading to double-strand break for the formation of two DNA fragments (3 kbp target and 1.7 kbp). Each of them is binding to the corresponding pcPNA at their termini (Figure 1b). To stop the S1 scission, EDTA was added. The reaction solution was then treated with STV-coated magnetic beads, and removed the unbound fragments. The beads were washed, and the target fragment was eluted by boiling the magnetic beads in the presence of free biotin solution. The selective isolation of the target fragment was characterized by the agarose gel electrophoresis. At first the S1 scission and the target fragment isolation were carried out at pH 5.5 where S1 scission is most efficient. As shown in Figure 1c, S1 scission successfully yielded two new bands at around 3 and 1.7 kbp in addition to the uncleaved substrate at around 5 kbp (see lane marked S1 cut). Other minor bands were the fragments by non-specific scission, as S1 nuclease somewhat cleaves even the duplex DNA at lower pH. At this condition, the amount of S1 cleavage was estimated to be 73%. Next, the affinity pulldown of the target fragment by magnetic beads was carried out. The solution containing the unbound fragment (lane marked unbound) and the recovered target fragment (lane marked recovered) were loaded into the same gel. As can be seen from the gel, the band intensity of the 3 kbp target and the invasion complex were decreased in the unbound solution when compared to the S1 cut alone, while the intensity of the 1.7 kbp was unaffected. Moreover, in the recovered solution the target fragment was successfully found. When compared to the initial
substrate concentration, the amounts of invasion complex and target recoveries were found to be 21 and 55%, respectively (Table S1). Note that, in addition to the target fragment, uncleaved substrate was also recovered due to the presence of biotinylated pcPNA in the invasion complex. However, this is not a critical problem in the isolation of target fragment from human genome. As shown below, when the reaction mixture was sufficiently fragmented with a restriction enzyme, only the target fragment was selectively obtained. Next, the same experiment was carried out at pH 6.0, 6.5, and 7.0, and in all the pH values, the double-strand break was successful. However, the cleavage yield decreased from 73 to 27% when pH was increased from 5.5 to 7.0, as S1 nuclease is not so active at higher pH. Interestingly, the amount of non-specific fragments were decreased when pH was increased and disappeared at neutral pH. In all pH values, the target fragment (between 20 to 55%) and invasion complex (between 21 to 54%) were successfully recovered with the total yield of above 70% (for detailed yield analysis, see Table S1). The overall observations indicated the interesting feature of our method that S1 scission and fragment isolation can be performed even at neutral pH. Importantly, no non-specific fragment was recovered by the pulldown assay, with the exception at pH 5.5 where tiny amount of non-specific fragment below 1.7 kbp appeared. The control experiment in the absence of biotin in both the pcPNAs indicated that S1 scission was successful while no fragment can be isolated (Figure 1d). Notably, the sitespecific scission takes place pH independently even in the presence of only pcPNA1. However, the specific scission is absent with pcPNA2Bio alone even at pH 5.5. This observation could possibly due to the difference in binding affinity and single-strand invasion of these pcPNAs (Figure S1). On the other hand, another fragment of the site-selective scission (the one in the right bottom of Figure 1b) could also be
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Biochemistry a)
qPCR primers: Target 0.86 kbp
b)
1.5 kbp
NsiI
S1
4732
859-877
BFP gene
NsiI
pBFP-N1
Human genome
2329
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Target 1
successfully isolated as shown in Figure 2, simply by using different set of pcPNAs (pcPNA3/pcPNA4Bio). Note that pcPNA4Bio is complementary with the protruding part in this 1.7 kbp fragment. In Figure 1 where pcPNA1/pcPNA2Bio was used, however, this fragment was washed away from the beads and never isolated. The recovery yield of the 1.7 kbp fragment was 69% (see Table S2). Unreacted substrate was hardly recovered in this case. Systematic experiments performed at various pH and enzyme concentrations indicated that our method is highly tunable for pH vs concentration of S1 nuclease (Figure S2 and Table S3). The optimization of other conditions indicated that elution temperatures can be varied from 60 to 95°C (Figure S3 and Table S4), the S1 scission can be performed from 3 h to overnight, biotin in the pcPNA at the terminus is better than in the middle, and the presence of phosphoserine in the biotin containing pcPNA has almost no effect (Figure S4-S5, and Table S5-S6). The isolation of the specific fragment from single copy of BFP gene integrated human genome in K562 cell line33,34 (K562-BFP, Figure 3a) was then performed. The genome was extracted from the cells using DNeasy Blood & Tissue Kit and used for our experiments. As in the case of plasmid DNA, the procedures for the S1 scission and fragment isolation were nearly the same with only minor changes (see supporting information). The pcPNAs invasion to the whole genome and S1 scission were performed. Then, the sample was purified by phenol/chloroform/isoamyl alcohol and ethanol precipitation. To make the DNA fragmentation, the purified sample was digested by NsiI. This enzyme recognizes 6 base-pairs in the genome and its scission will produce about 0.76 million restriction fragments (3.1×109/46 ≈ 0.76×106) and we aim to specifically isolate a single fragment out of it. The restriction fragments were then concentrated using Amicon ultra centrifugal filters (30 K). The affinity pulldown assay was carried out and the recovered sample was finally quantified by real-time PCR. The
Nonspecific control
8
0
Figure 2. One-pot isolation of a 1.7 kbp fragment from a linearized plasmid shown in Figure 1b. a) The sequences of the pcPNA3/pcPNA4Bio, and their binding sites in DNA are given. The sequences of pcPNA1/pcPNA2Bio are shown in Figure 1a. b) Gel images of the comparison of the selective isolation of 3 and 1.7 kbp. Experimental conditions: Invasion: 10 mM HEPES, pH 7.0, 50ºC, 1 h. S1 scission: 300 mM NaCl, 10 mM HEPES, pH 7.0, at 25ºC for 5 h. Recovery: 25 µL of 30 mM biotin at 95ºC for 10 min. Final concentration: [Linear pBFP-N1] = 2.62 nM; [pcPNAs] = 200 nM; [Dynabeads] = 1150 µg; [S1] = 450 U. [Agarose] = 1%, [TBE] = 1X, 100 V, Staining = Gelstar.
Control
10 Recovery efficiency (%)
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Control 2
Nonspecific control 3
Figure 3. One-pot isolation of target fragment (0.86 kbp) from human genome. a) Schematic illustration of the BFP gene integrated human genome, the positions of S1 nuclease and NsiI restriction sites, and primer binding sites for qPCR experiments. The pcPNA1/pcPNA2Bio were used in this case. b) The plot of recovery yields when compared to the initial substrate concentration. Colour bars indicate the triplicate experiments. Experimental conditions: Invasion: 5 mM HEPES, pH 7.0, 50ºC, 4 h. S1 scission: 300 mM NaCl, 10 mM MES, pH 5.5, at 25ºC for 3 h. NsiI cut: 37°C, overnight. Recovery: 25 µL of 10 mM biotin at 95ºC for 10 min. Final concentration: [K562-BFP] = 15 µg; [pcPNAs] = 500 nM; [Dynabeads] = 250 µg; [S1] = 2500 U, [NsiI] = 100 U. amplification of 0.86 kbp region from the isolated target was performed. As shown in Figure 3b, the triplicate experiments indicated that the target fragment can be recovered over 2 to 9% from the initial human genome (see Figure S6 for qPCR calibration curve, and Table S7 for average threshold cycle values for the samples). Nonspecific fragments were almost absent with an exception in one case, possibly due to the nonspecific binding to the magnetic beads. In a recent study by using recombinant CRISPR ribonucleoproteins, the recovery yields of 40 to 60% of target fragments from human genome with the associated contamination of nonspecific fragments of about 4 to 5% were reported.35 Due to the incompletion of the site-specific scission by pcPNAs/S1 nuclease and purification steps, the recovery yield in the present method becomes less than 10%. In order to isolate simple restriction fragments, our previous method using biotinylated pcPNA yielded above 40% of the target fragments.34 In that, the human genome was first treated with a restriction enzyme, and desired restriction fragment was picked up from the mixture. The present method entirely differs from the previous one in that the genome was first cut at a predetermined site by artificial DNA cutter which shows far higher site-specificity than naturally occurring restriction enzymes (the present system recognizes 20 bp in the genome). Then, the desired fragment was isolated in one-pot fashion. Thus, genomic fragments having desired sequence and length can be straightforwardly obtained. In concluding remarks, we have developed a novel biochemical one-pot method by the combined use of biotinylated pcPNA and S1 nuclease for the double-strand scission and isolation of a predetermined DNA sequences in kilo base length. It was successfully applied for both a plasmid DNA and cell derived human genome. This method is highly tunable for a variety of experimental conditions such as pH and concentration of S1 nuclease, and also compatible for the biological environments. In principle, this
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method requires no tedious experimental procedure, and can be applied for any sequence in human genome by designing the pcPNA pairs for each sequence. The efficiency of recovery is high enough for the downstream application of the isolated fragment, for instance, next-generation DNA sequencing. This method is superior to the PCR amplification of the whole human genome for retaining the information of the epigenetic modifications and lesion sites. The downstream application of the isolated fragment is expected to provide the molecular level information of lesions and modifications. Such kind of downstream applications of the isolated fragment, and isolation of the human DNA fragment with bound proteins are under investigation.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: . Experimental procedures, Figures S1-S6, Table S1-S7, additional discussions, and sequence of the linear pBFP-N1 (PDF)
AUTHOR INFORMATION Corresponding Author *
[email protected] (A.R.) *
[email protected] (M.K.)
Present Addresses ‡ Institute of Advanced Energy, Kyoto University, Gaokasho, Uji, Kyoto 611-0011, Japan § International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan ¶ Department of Applied Chemistry, School of Engineering, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji, Tokyo 192-0982, Japan
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was partially supported by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (No. 22000007). A.R. sincerely thanks MEXT for the Grant-in-Aid for Young Scientists (16K17934).
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Table of Contents artwork
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