Precise Site-Selective Termination of DNA Replication by Caging The

Sep 25, 2009 - We have examined the detailed termination efficiency of TNPOM as .... good termination of the KOD reaction in front of TNPPOM as in lan...
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Precise Site-Selective Termination of DNA Replication by Caging The 3-Position of Thymidine and Its Application to Polymerase Chain Reaction Akinori Kuzuya,* Fuminori Okada, and Makoto Komiyama* Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. Received June 9, 2009

A new caged thymidine, 3-N-(2-(2-nitrophenyl)propyloxymethyl)thymidine (TNPPOM) was synthesized and used as a site-selective terminator of DNA-polymerase reaction in light-assisted cohesive-ending PCR (LACE-PCR), which directly gives sticky-ended PCR products after brief UVA irradiation. Primer-extension experiments using a template involving TNPPOM have shown that this caged nucleotide efficiently and site-selectively blocks reactions of a variety of polymerases commonly used in PCR. Misincorporation of nucleobases, observed with the use of other previously reported caged thymidines, scarcely occurred. It has turned out that a slight structural difference of caging groups can significantly improve the termination yield of polymerase reactions. A LACE-PCR product coding GFP gene was prepared by using primers containing TNPPOM and was ligated with a vector fragment prepared using restriction enzymes. The resulting recombinant vector successfully transformed E. coli.

INTRODUCTION Photocaging of oligonucleotides has become an attractive topic because of its potential to control genetic events simply by irradiating light without changing either chemical nor physical conditions (1, 2). Various oligonucleotides protected with photocleavable groups (caged oligonucleotides) have been reported. Recently, we have developed a quite simple and effective new PCR system for easily preparing sticky-ended products utilizing caged nucleotides (3, 4). In current molecular biology and biotechnology, vectors are usually constructed by digesting plasmid DNA with restriction enzymes, followed by connection of this vector with predetermined gene fragment using ligase (5). In many cases, those gene fragments are prepared by PCR from various sources, and the blunt ends of the PCR products are converted to cohesive ends by the restriction enzymes for ligation with the vector. Although this technology has been mostly successful, two problems still remain unsolved for further developments of the field. First, recognition sites of commonly used restriction enzymes are mostly limited to some palindromic sequences, and it is sometimes difficult to find appropriate enzyme to digest DNA at (or near) the target manipulation site. Second, most of these enzymes recognize only 4 to 8 DNA-base sequences; thus, precise manipulation of large vectors such as adenovirus (30-38 kbp) with restriction enzymes is not very practical since their digestion occurs at too many sites in such large DNA. In lightassisted cohesive-ending (LACE) PCR (4), however, products with desired sticky ends on both ends are directly prepared after standard PCR procedures following a brief UVA irradiation, and thus no restriction enzyme treatment is necessary (Scheme 1). In order to terminate the polymerase reaction at a desired position, a caged nucleotide is incorporated into PCR primers. Within PCR cycles, elongation of the nascent strand (5′f3′ direction) is site-selectively terminated at the 3′ side of the caged nucleotide. Accordingly, the 5′ portion in the primer from the caged nucleotide remains single-stranded throughout the cycles, and predetermined sticky ends are obtained after the removal of the protecting group by brief UVA irradiation. In the previous * (+81) 3 5452 5200; FAX: (+81) 3 5452 5209; E-mail: kuzu@ mkomi.rcast.u-tokyo.ac.jp, [email protected].

Scheme 1. Outline of the Light-Assisted-Cohesive-Ending PCR (LACE-PCR) Using a Caged Nucleotide

studies, we have used a caged nucleotide 4-O-(2-(2-nitrophenyl)propyl)thymidine developed by Heckel et al. (TNPP in Figure 1) as the site-selective terminator (6). We found that TNPP is stable enough to survive repetitive thermal cycles for PCR and indeed terminates polymerase reaction site-selectively. The LACE-PCR products coding green fluorescent protein (GFP) gene was successfully prepared, directly ligated to vectors, and used to transform E. coli. Although it was successful for an initial trial, some difficulties in using TNPP as the terminator have come to light. Particularly, some of the polymerases misincorporate dG in front of TNPP almost quantitatively, probably because the hydrogen-bonding property of TNPP is similar to that of dC. Ligase is known to be a very strict enzyme; thus, such mismatches at the ends of a PCR product are unfavorable for making recombinant plasmids for standard cloning. Another difficulty of TNPP is the necessity

10.1021/bc900254e CCC: $40.75  2009 American Chemical Society Published on Web 09/25/2009

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Figure 1. Structures of the caged thymidines (TNPPOM, TNPOM, and TNPP) and oligonucleotides (10-15) used in the present work.

of using special phosphoramidite monomers in automated DNA synthesis. Chemically synthesized DNA is usually treated with conc aqueous ammonia at elevated temperatures (typically 55 °C for 8 h) to deprotect nucleobases. However, TNPP under such vigorous condition is easily transformed to dC via ammonolysis of the substituted 4-O. Thus, in combination with TNPP, it is essential to use ultramild dG and dA phosphoramidite monomers that can be deprotected under much milder conditions. This is not a very convenient situation for wider use of LACE-PCR. To solve these problems, we decided to introduce an NPP group to the 3-N position of the thymine base instead of 4-O, and have designed a new caged thymidine in this study. The 3-N-(2-(2-nitrophenyl)propyloxymethyl)thymidine (TNPPOM, Figure 1) bears a 2-(2-nitrophenyl)propyloxymethyl (NPPOM) group on the 3-N position. This group was stable enough to survive vigorous deprotection conditions in standard DNA synthesis, and thus can be used with normal phosphoramidite monomers. Primer-extension experiments using a template involving TNPPOM have shown that this caged nucleotide in fact efficiently and site-selectively blocks the reaction of a variety of polymerases commonly used in PCR. An application of LACE-PCR using TNPPOM as the site-selective terminator for constructing recombinant plasmid was also examined. While we were preparing this paper, Young et al. proposed the usage of 3-(6-nitropiperonyloxymethyl)thymidine (TNPOM) residues, which also possess a caging group on the 3-N, in light regulating mutagenesis (7). We have examined the detailed termination efficiency of TNPOM as well.

EXPERIMENTAL PROCEDURES Syntheses of Oligonucleotides Containing Caged Thymines. The phosphoramidite monomer of TNPPOM was synthesized from thymidine as shown in Scheme 2 (see Supporting Information for the details). Phosphoramidite monomers of TNPP and TNPOM were synthesized according to the literature (6, 8). The oligonucleotides containing TNPPOM (10, 12, and 15) or TNPOM (13) were prepared using standard phosphoramidite monomers on an automated DNA synthesizer. An extended 10 min coupling step was employed for TNPPOM incorporation. After deprotection with conc aqueous ammonia at 55 °C for 8 h, they were purified using denaturing PAGE (except for 10) and reverse-phase (RP) HPLC, and characterized by ESI (for 10, Hitachi M-8000) and MALDI-TOF/MS (Bruker AutoFLEX). The oligonucleotide containing TNPP (11) was prepared using a set of ultramild cyanoethyl phosphoramidites (phenoxyacetyl protected dA and 4-isopropyl-phenoxyacetyl protected dG monomers from Glen Research), and the deprotection was performed with conc aqueous ammonia at room temperature for 24 h. Their sequences are shown in Figure 1.

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Analyses of Uncaging Oligonucleotides. UVA irradiation (300 < λ < 400 nm) on a mixture containing the caged oligonucleotide substrate 10 (1 mM) was performed by using UV Spot Light Source (Hamamatsu Photonics; 200 W) through a UTVAF-50S-36U filter (Sigma Koki) at 2.5 mW/cm2. After predetermined times, 10 µL aliquots were analyzed on RPHPLC with a linear gradient of acetonitrile from 5% to 50% in 45 min in the presence of ammonium formate (50 mM). The peaks at 12.8, 17.0, and 17.3 min in Figure 2A were collected and further analyzed by ESI-MS in negative ion mode for identification. Primer Extension Reaction Using Caged Oligonucleotides as the Template. A mixture containing the caged oligonucleotide substrate 14 (2 µM), the primer oligo 11-13 (1 µM), and dNTPs (1.35 mM for each) was prepared. Unintended light exposure was carefully avoided throughout the process. After ExTaq polymerase (Takara) was added to the solution (0.1 U/µL), the following cycles were repeated 30 times on a thermal cycler: denaturation, 94 °C (15 s); annealing, 50 °C (30 s); elongation, 60 °C (70 s). The product was then analyzed on 20% denaturing PAGE. Extension with other polymerases were also examined under the following concentration without UV irradiation: [KOD -Plus- polymerase (Toyobo)] ) 0.02 U/µL, [KOD Dash polymerase (Toyobo)] ) 0.05 U/µL, and [Pfu DNA polymerase (Stratagene)] ) 0.05 U/µL. The thermal cycles for KOD -Plusand KOD Dash were the same as for ExTaq. For Pfu: denaturation, 94 °C (30 s); annealing, 55 °C (30 s); elongation, 72 °C (70 s). Imaging and quantification of gel electrophoresis were carried out on a FLA-3000G fluorescent imaging analyzer (Fujifilm, Japan). Light-Assisted Cohesive-Ending PCR (LACE-PCR). Using pQBI T7-GFP (Wako, 0.96 ng/µL) as the template, PCR was performed under the following conditions: [12 and 15] ) 300 nM, [dNTPs] ) 200 µM, and [Pfu Ultra polymerase] ) 0.05 U/µL. Thermal cycles are the same as described in the primer extension experiments. After purification with a QIAquick PCR Purification kit (Qiagen), the product in TE buffer (pH 8.5) was irradiated with UVA for 30 min at room temperature. Construction of a Recombinant Plasmid (Scheme 3). The vector for the LACE-PCR product was prepared by cutting pUC18 with EcoRI and HindIII. The product was then dephosphorylated with alkaline phosphatase. To a 1:3 mixture of this vector and LACE-PCR product, the same volume of DNA Ligation Kit (Mighty Mix) solution was added. After incubation overnight at 16 °C, JM109 (Toyobo) was transformed with the ligation product and cultured on LB-agar media containing ampicillin (50 µg/mL) and X-gal (40 µg/mL). White colonies were subjected to colony direct PCR, and a positive colony was picked up and cultured in LB media. The recombinant vector was purified with a QIAprep spin Miniprep Kit (Qiagen), and its sequence starting 150 bp away from each conjunction was determined on a 3130× Genetic Analyzer (Applied Biosystems). For both the colony direct PCR and sequencing, 5′-TCGCCATTCAGGCTGCGCAAC-3′ (243-263 bp region of pUC18 plasmid) and 5′-TGGAAAGCGGGCAGTGAGC-3′ (590-608 bp region of pUC18 plasmid) were used as the primers. In order to observe the fluorescence from expressed GFP, the purified vector was introduced into BL21-Gold (DE3) (Stratagene). The picture of the culture was taken on a Safe Imager blue-light transilluminator equipped with a Safe Imager amber filter unit (Invitrogen).

RESULTS AND DISCUSSION Synthesis of Phosphoramidite Monomer of TNPPOM and the Caged Oligonucleotides. The structures of the caged thymidines and oligonucleotides used in this study are shown

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Scheme 2. Synthetic Route of the Phosphoramidite Monomer of TNPPOM

in Figure 1. TNPP is the caged thymidine we used in our previous studies, and bears an NPP group on the 4-O position. TNPPOM is the newly designed caged thymidine that bears an NPP group on the 3-N position via a methoxy group. TNPOM is another caged thymidine that bears a caging group on the 3-N position. All of the oligonucleotides were synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry. The phosphoramidite monomer of TNPPOM was synthesized from thymidine and 2-ethylnitrobenzene as shown in Scheme 2 (see Supporting Information for the details). Phosphoramidite monomers for TNPOM and TNPP were prepared according to the literature (6, 8). For the synthesis of 11 containing TNPP, a set of ultramild cyanoethyl phosphoramidite monomers were used for dA and dG, and the deprotection was done with conc aqueous ammonia at room temperature for 24 h. Other oligonucleotides bearing TNPPOM or TNPOM were synthesized with standard monomers and protocols. The short oligonucleotide

Figure 2. Uncaging of TNPPOM. (A) RP-HPLC analyses of 10 before (upper) and after (lower) 30 min UVA irradiation. The peak indicated by the hollow square is 10, the filled circle is the desired T5, and those indicated by the hollow triangle are the byproduct bearing nitrosospecies. (B) Proposed structure of the byproduct. (C) Time-course of the uncaging.

10 was purified using reversed-phase (RP) HPLC and characterized by ESI and MALDI-TOF/MS analyses. Oligonucleotides 11-15 were purified using denaturing polyacrylamide gel electrophoresis (PAGE) and RP-HPLC, and characterized by MALDI-TOF/MS analyses (see Supporting Information). Photoremoval of NPPOM Group. Photoremoval of NPPOM group from thymine ring was examined by irradiating UVA light of 300 < λ < 400 nm on a buffered solution of 10 and analyzing it on RP-HPLC. Figure 2A shows RP-HPLC charts of the mixture before and after 30 min UVA irradiation. Before UVA irradiation, a sharp peak of the caged 10 was observed at 22 min. After UVA irradiation, on the other hand, the peak at 21 min completely disappeared, and three new peaks were observed at 12.8, 17.0, and 17.3 min. MALDI-TOF/MS analyses of the isolated peak at 12.8 min showed that this is the desired product, T5. The two peaks around 17 min, the areas of which are always equivalent to each other, were also separately isolated and subjected to ESI-MS analyses. The observed masses for them are the same in relation to each other (824.19 [M-2H]2-, 549.85 [M-3H]3-, 412.05 or 412.25 [M-4H]4-), showing that these species are the diastereomers bearing a nitrosobenzene formed via the side reaction of photoremoval pathway of NPP group (Figure 2B) (9). Figure 2C shows the time dependence of the yield of each species. Photoremoval of NPPOM (and also the side reaction) rapidly proceeds, and almost saturates after 15 min UVA irradiation. The average yield of the desired T5 was around 34% in five independent reactions. Considering the mechanism of NPP photoremoval that proceeds via an ionic nitronic acid intermediate (9), the low yield of the desired T5 may be caused by pH dependence of NPP removal. However, the yield did not improve much even when the pH was raised up to 9.0 or an Mg2+ ion was added to the solution. Higher pH is not favorable for the present system, since it elevates the risk of oxidative base damages (10), so we decided to fix the pH at 8.5 for UVA irradiation. Site-Selective Termination of Polymerase Reaction by the Three Caged Thymines in the Template under PCR Conditions. Although the yield of photoremoval is unexpectedly low for TNPPOM, its ability to terminate site-selective replication

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Scheme 3. Construction of a Recombinant Plasmid Coding GFP Gene Using LACE-PCR

turned out to be significantly superior compared to the other two caged thymidines. Site-selective termination of polymerase reaction by the three caged thymines was first examined by primer-extension experiments under PCR thermal cycle conditions in Figure 3A. By using various polymerases commonly used for PCR, FAM-labeled primer 14 (17 nt, FAM ) 6-fluoresceinamide) was extended on the modified oligonucleotide template 11-13 (24 nt), which contains one TNPP, TNPPOM, or TNPOM at the fourth position from the 5′-end. When 11 was used as the template and Ex Taq was used as the polymerase, the longest product formed after predetermined thermal cycles was 20 nt, showing that primer extension was efficiently terminated at the 3′-side of TNPP (lane 2 in Figure 3B). This result is completely consistent with our previous report (4). Similar efficient termination was observed for another template 12, in which TNPPOM is introduced in place of TNPP (lane 3). The yields of the 20-mer product for the systems employing 11 and 12 was 94% and 94%, respectively. MALDI-TOF/MS analysis of the reaction mixture also confirmed that d(GAC) was correctly added to the 3′-end of 12 (Figure 4A). When TNPOM was introduced in place of TNPPOM, on the other hand, significant amounts of further elongated products were obtained (lane 4). The yields of the 20-mer, 21-mer, and 23-mer products in lane 4 were 65%, 25%, and 10%, respectively. No undesired removal of NPPOM (or NPOM) was observed in the control HPLC analysis of the template 12 (or 13) even after the corresponding PCR thermal cycles (Supporting Figure 1 in Supporting Information). Thus, the difference between TNPPOM and TNPOM cannot be ascribed to the difference in the thermal stability of the caging group, but may be related to their structural aspects.

Similar differences in termination ability were observed for other polymerases such as KOD and Pfu. Figure 3C,D shows primer-extension experiments using Pfu polymerases. Two commercially available cocktails of this polymerase, which are optimized to the amplification of different length of the templates, were examined. Both of the cocktails showed fairly good termination for all of the three caged thymidines. Particularly, the yield of the 20-mer product for both of the NPPOM systems was around 92-94%, which is one of the best numbers we have observed so far. For NPP and NPOM systems, on the other hand, ca. 30% formation of 21-mer product was observed. Primer-extension experiments were performed with KOD polymerase and its related cocktails, as well. In our previous study, this proofreading polymerase was found to introduce dG in front of TNPP almost quantitatively as in lane 3 in Figure 3E. As a result, the major product using KOD polymerase in combination with TNPP is 21-mer (the yield is 43%), and significant amount (up to 40%) of longer products was also produced. In contrast, introduction of TNPPOM in the template resulted in fairly good termination of the KOD reaction in front of TNPPOM as in lane 4. The yield of the desired 20-mer product was 77%, and that of the longer byproduct was only 20%. Quite interestingly, the desired 20-mer product was obtained only with 16% yield by use of the template bearing TNPOM (13) even if the hydrogen bonding face of thymine is completely blocked by TNPOM. The major product was the 21mer (58% yield). MALDI-TOF/MS analysis of the reaction mixture revealed that both 20- and 21-mer products are a mixture of two or more species with different sequences around the end (Figure 4D). The mass difference between the smaller peaks corresponding to 21-mer and 20-mer is 200-230, which

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Figure 5. Analyses of the recombinant vector. (A) Agarose gel electrophoresis patterns for the LACE-PCR product and the recombinant vector. Lane 1, LACE-PCR product; lane 2, pUC18 plasmid treated with EcoRI and HindIII; lane 3, the recombinant plasmid extracted from JM109 and linearized with EcoRI; lane M, 1 kbp ladder. (B) The fluorescence of GFP expressed from the recombinant vector in BL21Gold (DE3) cells. (C) Sequencing of the cloned recombinant vector extracted from JM109 around the EcoRI conjunction and the HindIII site conjunction.

Figure 3. 20% denaturing PAGE patterns for primer extension reaction using caged oligonucleotides 11-13 as a template. (A) Structures of the oligonucleotides. (B) Lane 1, unreacted 14; lane 2, 11 + 14; lane 3, 12 + 14; lane 4, 13 + 14. (C-G) Lane 1, unreacted 14; lane 2, 11 + 14 with ExTaq (the same as (B) lane 2); lane 3, 11 + 14; lane 4, 12 + 14; lane 5, 13 + 14; M, size markers (20-, 25-, and 30-mer singlestranded DNA). The 20-mer products are indicated by the arrows.

Figure 4. MALDI-TOF/MS spectra of the reaction mixture of (A) 12 + 14 with ExTaq, (B) 12 + 14 with Pfu Turbo, (C) 12 + 14 with KOD polymerase, (D) 13 + 14 with KOD polymerase.

is significantly smaller than the minimum mass increase of native DNA elongation (addition of one dC is +289). Considering that multiple peaks were observed even for the 20-mer products, it is presumed that KOD polymerase misincorporates the wrong nucleotide also in front of the adjacent residues in the 3′ side of TNPOM (marginally for TNPPOM as well), which is upstream of elongation. It is possible that the present finding is related to the mutation mechanism induced by 3-Me-thymidine lesion in cells (11). KOD Dash, which is a cocktail of KOD polymerase and KOD exo(-) variant gave very similar PAGE patterns to KOD polymerase (Figure 3F). The yields of the 20mer product with TNPP, TNPPOM, and TNPOM were 8%, 65%, and 26%, and the yields of the 21-mer were 54%, 17%, and 52%, respectively. Undesired additional incorporation of the 21st nucleotide in front of TNPP and TNPOM was rather marginal for KOD -Plus-, another cocktail of KOD polymerase with customized buffer contents (Figure 3G). The yield of the desired 20mer product using TNPP improved to 73% (lane 3), which is almost the same as 74% yield with the use of TNPPOM (lane 4). However, the major product for the reaction using TNPOM was still the 21-mer (47%), and only 42% of the product was the desired 20-mer. Construction of a Recombinant Vector Coding GFP Gene with LACE-PCR Using Primers Containing TNPPOM. To check whether the site-selective termination practically occurs and desired sticky ends can be obtained after LACEPCR using TNPPOM as the site-selective terminator, PCR products bearing sticky ends complementary to EcoRI and HindIII termini were prepared. By using 12 and 15 as the primers, the 121-1170 bp region of pQBI T7-GFP plasmid was amplified. This portion of the plasmid involves GFP gene with T7 promoter. For this experiment, Pfu Ultra was used as the polymerase for its high fidelity and excellent termination by TNPPOM observed above. Deprotection of NPPOM groups in the PCR product was performed with UVA irradiation for 30 min on the purified product solution. Considering the yield of NPP removal (ca. 40% for each NPP), approximately 16% of the product is expected to have fully deprotected, desired sticky ends at both ends. As shown in Figure 5A, a product of desired length (1 kbp) was successfully amplified (lane 1). The insert obtained above was ligated to a vector fragment, which was prepared by digesting pUC18 with EcoRI, HindIII, and was dephosphorylated with alkaline phosphatase. Then, JM109 competent cells were transformed with the ligation

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product. After overnight incubation on a culture plate containing ampicillin and X-gal, ca. 500 white colonies were obtained (only one blue colony was observed). A total of eight colonies were subjected to direct PCR using primers complementary to pUC18 plasmid 150 bp away from each of the digestion site, and one colony gave the expected amplicon of 1.4 kbp from the designed recombinant. The size of the extracted plasmid from this colony (linearized with EcoRI, lane 3 in Figure 5A) was significantly larger than the parent pUC18 fragment and consistent with the expected 3689 bp. The other seven colonies gave no apparent amplicon. Taking into account that all of these colonies are white, the latter seven colonies are presumed to have pQBI T7GFP, which was the template for LACE-PCR. When the extracted plasmid was introduced into BL21-Gold (DE3) competent cells, in which T7 polymerase is expressed, emission of green fluorescence from the expressed GFP was clearly observed (Figure 5B), showing that no undesired and critical DNA mutation was induced in the gene in this clone by UVA irradiation. Precise construction of the recombinant vector was directly confirmed by sequencing the extracted plasmid (Figure 5C). The sequences near the EcoRI site and the HindIII site were both completely consistent with the expected ones. Successful formation of both of the sticky ends by LACE-PCR using TNPPOM as the site-selective replication terminator, and its practical use has been evidenced. Recent studies have shown that UVA radiation as well as UVB causes various oxidative damages on DNA, which result in DNA degradation or mutation (12). According to the calculation in the previous study, ca. 4% of the population can contain one damage bases such as cyclobutane pyrimidine dimer (CPD) formation, purine oxidative damage, pyrimidine oxidative damage, and abasic damage in total, and the other 96% are expected to be intact after the UVA irradiation. However, the competent cells commonly used today lack the RecA system, which is mainly responsible for repairing such damage. Thus, the cells transformed with plasmids containing such damaged bases cannot multiply themselves, and consequently, only the intact ones will be selected. We have not yet found any mutation in more than twenty kinds of recombinant plasmids we have obtained with LACE-PCR so far (data not shown). Similarly, it is strongly expected that the byproduct formed after UVA irradiation is not ligated to the vector fragment because of the strict selectivity of the ligase, and nothing to do with the recombinants.

CONCLUSIONS The newly synthesized TNPPOM residue site-selectively and efficiently blocks polymerase reaction under PCR conditions, and the selectivity was the best among the three caging group examined in this study. The caged primers involving TNPPOM can be synthesized in combination with conventional phosphoramidite monomers on an automated DNA synthesizer, and no change in deprotection conditions is necessary. Although the yield of light triggered deprotection is not very high under the conditions employed in this study, the 1-kbp LACE-PCR products using the caged primers involving TNPPOM were successfully ligated with a vector fragment prepared by restriction-enzyme treatments, and the GFP coded in the resulting recombinant plasmid was efficiently expressed in the transformed bacteria. The present findings in primer-extension reactions using three kinds of caged thymidines have shown that the blocking of hydrogen bonding between nucleobases is not the only require-

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ment to achieve efficient and selective termination of the polymerase reaction. A significant difference in termination efficiency and selectivity was observed between TNPPOM and TNPOM. A noticeable difference between these two caging groups is the position of just one methylene: in the main chain or on the benzene ring. Structural aspects of the caging group may play an important role here in a close connection with the enzymatic mechanisms of substrate recognition for each of the polymerases. Future detailed study on this topic may be relevant to understanding the mechanism of mutation in vivo as well.

ACKNOWLEDGMENT This work was partially supported by a Grant-in-Aid for Specially Promoted Scientific Research (18001001) and Grantin-Aid for Young Scientists (B) (20750126) from the Ministry of Education, Science, Sports, Culture and Technology, Japan. The support from the Global COE Program for Chemistry Innovation is also acknowledged. Supporting Information Available: Detailed synthetic procedures, MALDI-TOF/MS analyses of the caged oligonucleotides (Supporting Table 1) and RP-HPLC analyses on the thermostability of TNPPOM and TNPOM in the oligonucleotides (Supporting Figure 1). This material is available free of charge via the Internet at http://pubs.acs.org.

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