Báo cáo khoa học: Determination of the reopening temperature of a DNA hairpin structure in vitro pptx

Determination of the reopening temperature of a DNA hairpin structure in vitro Xuefeng Pan Institute of Microbiology, The Chinese Academy of Sciences, Beijing, China A novel method, based upon primer extension, has been developed for measuring the reopening temperature of a single type of DNA hairpin structure. Two DNA oligo- nucleotides have been utilized and designated as primers 1 and2.Primer1,withits5-and3¢-termini fully comple- mentary to the hairpin flanking sequences, was used to evaluate primer extension conditions, and primer 2, with its 3¢-end competing with the DNA hairpin stem, was used to detect the DNA hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure w as p redicted to prevent the annealing of the 3¢-end of primer 2 with the template DNA, which leads to no pri- mer extension. By incubating at different temperatures, the DNA hairpin structure can be reopened at a particular temperature where the primer extension can be carried out. This resulted in the a ppearance of double-stranded DNA that was detected on an agarose gel. This temperature is defined here as the hairpin reopening temperature. Keywords: DNA hairpin; non-B DNA secondary structure; primer extension; reopening temperature; T m . The significance of DN A folding into non-B secondary structures (e.g. pseudohairpin, hairpin, palindromic, tri- plex and G-tertraplex DNA molecules) is twofold. Firstly, DNA non-B secondary structures play important physiological roles. For example, some GC-rich DNA sequences, that fold into G-tertraplex structures, partici- pate in the regulation of gene expression [1] and the maintenance of telomere structures [2–4], and some AT- rich DNA sequences in the DNA replication origins of bacterial plasmids that can adopt non-H bounded conformations control DNA replication initiation [5]. Moreover, certain types of non-B DNA conformations may also b e n eeded for the proper organization of genetic material in chromosomes [6]. Secondly, non-B DNA secondary structures can be aberrant DNA folds, increasing the likelihood of genomic instability in DNA replication, transcription, recombination or repair [7,8]. For example, non-B DNA secondary structures formed on the lagging strand template of a DNA replication fork have been found to increase the probability of DNA replication impairment, and enhance DNA rear- rangements through recombination and repair [8]. Recently, some trinucleotide repeats in the human genome (e.g. CAG, CGG, GAA, and CGA) have been found to generate repeat expansion and contraction instabilities, responsible for the occurrence of more than 14 human genetic diseases a nd cancers [9–14]. Interest- ingly, most of these disease-causing trinucleotide repeats have been demonstrated to be capable of forming non-B DNA secondary structures, such as hairpins, pseudohair- pins, triplex and G-tertraplex DNA molecules in vitro, which have been proposed to serve as intermediates for producing expansion and contraction instabilities v ia DNA replication, recombination or repair [10–12]. Moreover, in some DNA and RNA related molecular experimental manipulations, such as DNA amplification by PCR, primer extension on a DNA or RNA template, DNA sequencing, and site-directed mutagenesis, various effects of non-B DNA secondary structure formation have also been reported [15–17]. These nucleic acid manipulations are closely related to molecular hybridization of DNA or RNA molecules, or DNA replication, reverse transcription and RNA transcription, during which DNA or RNA molecules should be unfolded [18]. Any folded DNA or RNA conformation, if remaining unden atured o r reformed through a reannealing step, may interfere with the recog- nition of the molecules, o r affect the subsequent DNA polymerization, RNA reverse transcription into DNA, or RNA transcription [18]. All the replicative DNA polymerases so far character- ized use single-stranded DNA as a template. However, some DNA polymerases, such as the DNA polymerase of bacteriophage U29 and thermostable Bacillus stearother- mophilus DNA polymerase etc., have DNA double-strand displacement activity, and enable the double-stranded DNA segment in a non-B DNA structure (e.g. a DNA hairpin) to open during DNA replication [19–21]. These DNA polymerases can either remove a DNA hairpin structure through their double-strand displacement activ- ities or be stalled by the DNA hairpin structure [21,22]. In the latter case, the stalled DNA polymerase leaves a 3¢-end on the growing strand, which m ay subsequently search out a short region of homology along the nearby downstream template and allow the DNA polymerase to re-assemble at the 3¢-end and continue the DNA replication (strand-slippage) [21–24]. Correspondence to X. Pan, Institute of Microbiology, The Chinese Academy of Sciences, Beijing 100080, China. E-mail: xpan@staffmail.ed.ac.uk or xuefengpancam@yahoo.com.cn Abbreviations: RF, replicative form; X-gal, 5-bromo-4-chloroindol- 3-yl b- D -galactoside. (Received 5 March 2004, revised 5 July 2004, accepted 23 July 2004) Eur. J. Biochem. 271, 3665–3670 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04301.x The stability of a non-B DNA conformation (e.g. DNA hairpin structure) can be characterized by its melting temperature ( T m ). Determination o f T m is essential f or many nucleic acid-related experimental manipulations, such as primer extensions with DNA or RNA, DNA or RNA hybridization and oligonucleotide DNA primer design [16,18,25,26]. So far, experimental methods, such as UV, circular dichroism and NMR, and theoretical calculations, such as nearest-neighbour analysis, have been widely used to obtain thermodynamic parameters, including the T m ,andto detect nucleic acid structural transitions [27–35]. Amongst these, UV spectroscopy is the most common experimental method. H owever, under certain circumstances where the critical transitions between DNA folding and unfolding are invisible to UV [30], alternative m ethods, such as circular dichroism, are required. On the other hand, the thermody- namic parameters obtained by experimental methods reflect only overall information on a DNA or RNA population, not a local one (e.g. a partial sequence of a DNA or RNA molecule [18,26]). Although theoretical calculations, such as nearest-neighbour analysis, can be applied for a local sequence analysis (e.g. of a single hairpin structure) it will become inaccurate for certain DNA hairpin loops. For example, a CG closing base pair enhances stability over o ther closing base pairs and cannot be explained by the current nearest-neighbour model [27–29,33,35]. A n experimental method for m easuring a single type of DNA hairpin’s reopening temperature embedded in a DNA molecule has been developed in this work. The method utilizes the knowledge of DNA hairpin structure formation in vitro and the effects of DNA hairpin structure on a s ite-specific oligonucleotide DNA-mediated primer extension reaction. Materials and methods Bacterial strains, media, DNA and biochemicals E. coli strains TG1 [SupE hsdD5 thi D(lac-proAB)F¢]and JM101 [F¢ traD36 lacIq D(lacZ)M15 proAB]wereusedin this work [18,26]. Luria–Bertani broth was used for the bacterial cultivation and M9 minimal medium was used to maintain F¢ factors in E. coli strains [18]. All manipulations were following standard methods [18], unless indicated. Plasmid pTac5 [37] and M13mp19 [18] were stocks of this laboratory; the M13 single-stranded DNA construct was the M13-E-E derivative with a 102 bp DNA deletion in an inverted repeat region [38]. Oligonucleotide primer 1: 5¢-CCACCCTCG*T*C*GGCCAC-3¢ (where asterisks refer to mismatched bases and G*T*C* is the marker); primer 2: 5¢-ATGGCCTGAG*AGCCACCC-3¢ (G* as the marker); and primer 3: 5¢-TCAG*AGGCCACAAACCA CAC-3¢ (G* as the marke r) w ere s ynthesized using an Applied Biosystems DNA synthesiser. The markers indica- ted in each oligonucleotide primer were applied when DNA sequencing to confirm the primer extension products. Restriction enzymes, Eco RI, PstI, BamHI, T4 DNA ligase, T4 DNA polymerase, and isopropyl thio- b- D -galactoside, 5- bromo-4-chloroindol-3-yl b- D -galactoside ( X-gal) stock solutions were purchased from Promega ( Beijing, China). dNTPs, ATP and dithiothreitol were from Biochemicals (San Diego, CA, USA). Subcloning the template DNA and prepare ssDNA Cloning of an 800 bp sequence from a prochymosin expression plasmid p Tac5 onto M13mp19 was carried out as the follows: pTac5 was digested by EcoRI restriction enzyme, and the 800 bp EcoRI fragment was recovered by using the low-melting agarose gel method [18]. This EcoRI fragment was then subcloned into the EcoRI site in M13mp19. The ligated DNA was used to transform TG1 competent cells as prepared by a CaCl 2 method [18]. Replicative form (RF) DNA from the white plaques as selected on Luria–Bertani agarose plates containing isopro- pyl thio-b- D -galactoside and X-gal was isolated, and the orientation of the insert in M13mp19 was determined by PstI digestion. Single-stranded M13 DNA was prepared as described by Sambrook et al. [18]. Annealing of the primer–DNA template and primer extension analysis Formation of the primer–single-strand DNA template and the subsequent primer extension w ere performed based on the method established by Kunkel [18,24], modified as follows: primer was mixed with the dUTP-containing M13- E-E ssDNA (extracted from RZ1032) at a ratio of 3 : 1 in annealing buffer (10·) containing 200 m M Tris/Cl (pH 7.5), 20 m M MgCl 2 ,500m M NaCl, respectively. These mixtures were then kept at 70 °C for 5 mins and cooled to 12 °C, 22 °Cand30°C, respectively, in two different ways. One way was to allow the annealing reaction to proceed at room temperature, allowing slow annealing to 12 °C, 22 °Cand 30 °C, respectively. The other was to transfer the annealing reaction into a waterbath held at 12 °C, 22 °Cor30°Cafter the denaturing reaction at 70 °C (fast annealing), and then store on ice. Primer extensions (second strand synthesis) were carried out in a 20 lL reaction by adding buffer (10·) containing 5 m M of each dNTP, 10 m M ATP, 100 m M Tris/ Cl (pH 8.0), 50 m M MgCl 2 ,20m M dithiothreitol, one unit of T4 DNA polymerase and three units of T4 DNA ligase, and incubating at different temperatures for 90 mins. The primer extension products (synthesized double-stranded RF DNA) were analysed by running agarose gels. Plating the M13 bacteriophage and DNA sequencing To further confirm that the RF DNA was produced through the DNA oligonucleotide primed primer extension reaction, TG1 c ompetent cells prepared with the CaCl 2 method [18] were transformed with RF DNA from each primer extension reaction. M13 bacteriophage carrying primer 2: 5¢-ATGGCCTGAG*AGCCACCC-3¢ were pla- ted on E. coli TG1 and the mutants were screened by DNA sequencing of the DNA i n the plaques by t he method described in [18]. The oligonu cleotide 5¢-GGTTGTC GGCGTCGATAATCAAACT-3¢ was used as the sequen- cing primer. Determination of the hairpin reopening temperature Oligonucleotide primer 2 was mixed with single-stranded template DNA at a ratio of 3 : 1 in the annealing buffer. This mixture was denatured at 70 °C for 5 mins, and then 3666 X. Pan (Eur. J. Biochem. 271) Ó FEBS 2004 slowly cooled to 12 °C (slow annealing). After these treatments, one unit of T4 DNA polymerase was added and the mixture was equally divided into a liquots that were incubated at different temperatures for primer extension. Following 90 mins of primer extension each aliquot was analysed by agarose gel electrophoresis. Results and Discussion Experimental rationale In order t o determine the r eopening temperature of an individual secondary structure in a single DNA molecule (Fig. 1 A,B), knowledge of DNA replication, DNA folding and oligonucleotide-mediated primer extension has been a pplied to establish a method. The molec ular mechanism underlying the method is explained in Fig. 1B,C. As can be seen in Fig. 1B,C, a DNA hairpin structure can be adopted by a small region in the template DNA sequence. Such a hairpin structure was designed to abolish primer extension by interfering with the annealing of the 3¢ bases of the DNA oligonucleotide primer with the template DNA (Fig. 1B). However, primer extension is possible as long as the hairpin structure is melted and the DNA primer can anneal with its complementary region in the DNA template. As can be seen in Fig. 1A, two primers were d esigned as follows: primer 1, with its 5 ¢-and3¢-ends fully complementary to the hairpin flanking sequences, was used to monitor the primer extension reaction conditions; and primer 2, with its 3¢-terminus located in the hairpin stem, was used to detect the DNA hairpin reo pening temperature (Fig. 1C). It was expected that the primer–template annealing and subsequent extension reactions from the primer 1–DNA template hybrids could be used a s positive controls to establish the conditions of the DNA polymerase-cata- lysed primer extension reactions. Under the same condi- tions, if the primer 1–DNA template hybrid appeared to be extended by DNA polymerase while the primer 2–DNA template hybrid appeared not to be, i t could be inferred that the failure of the primer 2–DNA template hybrid primer extension reaction was due to the effects of the hairpin structure on the process of primer 2 annealing to the DNA template. Analysis of secondary structure formation by thermal denaturation, renaturation and primer extension To determine whether the expected secondary structure can o r cannot f orm through denaturation and renatur- ation manipulations, the fast annealing (fast renaturation) and slow annealing (slow renaturation) procedures (Materials and methods) were performed after the primer 2 and th e single-stranded DNA template mixture were Fig. 1. Organization and the working mech- anism. (A) DNA template and potential hair- pin structu res (free energy va lues labelled were computed by a program [25]), and the loca- tions of the primer 1, 2 and 3 pairing. (B) Paring of primer 2 with the template when a hairpin structure has been formed through denaturation and renaturation (free energy value labelled was computed by a program [25]). (C) Illustration of the working mechan- ism for measuring the reopening temperature of a small DNA hairpin structure. Ó FEBS 2004 Reopening temperature of single DNA hairpin (Eur. J. Biochem. 271) 3667 denatured at 70 °C for 5 mins. Primer exte nsion r eactions were then started by adding one unit of T4 DNA polymerase to these renaturation mixtures. The primer extension products were compared by agarose gel electrophoresis. As can be seen in Fig. 2A, the slow renaturation reaction produced smeared DNA products (Fig. 2 A, lane 1), while the f ast annealing reaction showed two dominant bands (Fig. 2A, lane 3), indicating that the p rimer extensions with primer 2 produce different products as a function of the different annealing manipulations. This suggested that the conformations of the template DNA formed after t he two different annealing reactions were different. Heteroduplex DNA formed by p rimer 2 and the template DNA generated through fast annealing to 22 °C seemed to allow the T4 DNA polymerase to synthesize double-stranded DNA strands more completely, while the heteroduplex DNA generated through slow annealing to 22 °Cpreventedthe T4 DNA polymerase from carrying out primer extension at 22 °C. Fast annealing may decrease the likelihood of the self-folding of the template DNA, while it may increase the probability of primer 2 binding to the template DNA. By contrast, slow annealing with primer 2 may form incompletely paired heteroduplex DNA as shown in F ig. 1B. Under this condition, the two ÔGGÕ bases i n t he template may b e unavailable due to formation o f a hairpin structure, w hich leaves the two ÔCCÕ bases at the 3¢-end of primer 2 unpaired. To further confirm that the problem encountered for primer 2 mediated primer extension was due to the effects of template DNA folding (forming a hairpin structure), annealing reactions between primer 1 and single-stranded DNA were also carried out using the slow annealing manipulation. As expected, heteroduplex molecules formed by primer 1 and the template DNA allowed T4 DNA polymerase to produce double-stranded DNA, irrespective of the a nnealing temperatures ( data not shown). More impressively, this DNA re plication was detected when the reacting temperature was as low a s 12 °C (Fig. 3, lane 3), while in the same situation, the primer 2–single-strand DNA template cannot be used by T4 DNA polymerase to synthesise any double-stranded DNA products (Fig. 3, lane 5). In addition to primer 1 extension, primer 3 [designed to prevent formation of the h airpin structure (Fig. 1A)] can also enable primer extension to proceed under either slow (Fig. 2 B, lane 3) or fast (Fig. 2B, lanes 2 and 4) annealing. These data taken together indicate that the hairpin-forming region (as indicated in Fig. 1A) can indeed affect primer 2 DNA primer extension, most likely due to hairpin structure formation (Fig. 1B). Measuring the reopening temperature for a single type of DNA hairpin As can be seen in Fig. 3, primer extension with primer 1 can produce e xtended double-stranded DNA products (Fig. 3, lane 3), but primer 2 cannot (Fig. 3 , lane 5). When considering that these two reactions used the same conditions and only differed in the primer DNA, it is reasonable to believe that the f ailure of primer 2-mediated primer extension was simply due to hairpin structure formation on the template DNA, which left the ÔCCÕ bases of primer 2 Ôflapping Õ (Fig. 1B). The DNA conformation depicted in Fig. 1B cannot be used as template by the DNA polymerase to synthesize the second strand DNA due to the unpaired 3¢-end of the primer, until the ÔGGÕ bases in the hairpin stem are freed by elevating the reaction temperature and paired with the ÔCCÕ bases at the 3¢-end of primer 2. In this work, the reopening of the hairpin structure and the subsequent primer extensions were attempted by dividing the reaction into aliquots, and incubating at different temperatures. The results of these manipulations are presented in Fig. 4. As can be seen in Fig. 4, primer extension products were detected when the reaction temperature (reopening and extension temperature) was above 19 °C. The appearance of the 12345 1234 5 6 A B Fig. 2. Primer extension reactions with primer 2 and primer 3 under fast and slow annealing conditions. (A) Primer extension with primer 2 under slow and fast annealing conditions: lanes 1 and 2, primer extension product and primer-free control under slow anne aling; lanes 3 and 4, primer exte nsion product and primer-free control under fast annealing; lane 5, ssDNA control. (B) Primer extension product with primer 3 unde r slow and fast annealing: lane 1, RF DNA con trol; lanes 2 and 4, primer extension products with p rimer 3 under fast annealing; lane 3, the primer extension product under slow annealing with ATP; lane 5, primer-free control and lane 6, the ssDNA template control. 1234567 RFDNA Products RNA Fig. 3. Primer extension reactions with primer 1 and primer 2. Primer extension products using primer 1 and primer 2 have been indicated. Lanes1and2,RFDNAcontrols;lane3,primerextensionwithprimer 1at12 °C u nder slow annealing; lane 4, the primer 1-free cont rol; lan e 5, primer extension with primer 2 at 12 °C under slow annealing; lane 6, primer 2-free cont rol; lane 7, single-stranded DNA template control. 3668 X. Pan (Eur. J. Biochem. 271) Ó FEBS 2004 double-stranded DNA products on the agarose gel was taken to indicate the temperature at which the hairpin structure had been reopened and where the 3 ¢ ÔCCÕ of p rimer 2 had formed a fully hydrogen bonded heteroduplex with the single- stranded DNA template. Further confirmation th at the double-stranded DNA molecules seen in Figs 2B, 3 and 4 weremadethroughprimer1,2and3-mediatedprimer extensions was obtained by transformation. E. coli TG1 competent cells were transformed with these double-stran- ded DNA products and single-stranded DNA molecules isolated from the M13 plaques were sequenced (data not shown) to check the DNA markers carried by primers 1, 2 and 3. Comparison between the experimentally obtained reopening temperature and the T m values with the nearest-neighbour thermodynamic calculation As indicated in Fig. 4, the reopening temperature of the hairpin (Fig. 1B) was  19 °C. This temperature has been compared with the T m values calculated b y using a nearest- neighbour thermodynamics based software [25,39]. The theoretical T m , when calculated based on the folding temperatures of 12 °C, 19 °C, 22 °Cand37°C, and a DNA folding condition of 1.0 M Na + ,are27.5°C, 27.9 °C, 28.7 °C and 28.1 °C, respectively. H owever, as the actual experimental DNA folding concentration of Na + is 50 m M , the correspo nding T m calculated at those folding temper- atures are 20.4 °C, 21 °C, 19.9 °Cand20.2°C, respectively, which are fairly close to the experimentally obtained reopening temperatures. Conclusion Based on the effects of a hairpin structure formed in template DNA on DNA primer annealing and DNA polymerase-catalysed primer extension, a novel method for measuring the reopening temperature for a single type of hairpin structure in DNA has been established. The reopening temperature obtained e xperimentally was fairly close to the T m values obtained by a nearest-neighbour thermodynamics calculation, suggesting that it can be useful for evaluating the reopening temperature for a DNA hairpin in a local region of DNA, and for comparing the reopening temperatures for a group of hairpin structures when the reaction system is defined. 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Biochem. 271) Ó FEBS 2004 . reopening temperature for a DNA hairpin in a local region of DNA, and for comparing the reopening temperatures for a group of hairpin structures when the reaction. hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure