DOI: 10.1002/chem.201304292
Communication
& Aptamers
Arrest of Rolling Circle Amplification by Protein-Binding DNA Aptamers Lida Wang,[a, b] Kha Tram,[a] Monsur M. Ali,[a] Bruno J. Salena,[c] Jinghong Li,*[b] and Yingfu Li*[a]
Chem. Eur. J. 2014, 20, 2420 – 2424
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Communication Abstract: Certain DNA polymerases, such as f29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super-long single-stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand-displacement ability of these polymerases. In this work, the ability of f29DNAP to carry out RCA over circular templates containing a protein-binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein-binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.
Rolling circle amplification (RCA) is an isothermal DNA amplification process that generates extremely long DNA molecules from a single-stranded (ss) DNA circle.[1]In this unique DNA synthesis reaction, a special DNA polymerase (such as f29 DNA polymerase, f29DNAP) makes repetitive rounds of DNA copying over a circular ssDNA template using a short DNA strand as the primer and deoxyribonucleotide 5’-triphosphates (dNTPs) as the building blocks. RCA is widely exploited as a powerful signal amplification technique for bioanalysis as it converts a single primer–template recognition event into thousands of tandem DNA repeats that can be easily detected.[2] The power of this technique reflects the high processivity and the strand-displacement ability of f29DNAP: this enzyme is able to make more than 70 000 nucleotide additions before its dissociation from the DNA template,[3] and unwind doublestranded DNA on its own without requiring the assistance of any DNA helicases.[4] It has also been reported that f29DNAP is able to unwind stable secondary structures in the circular template.[3, 5] It is interesting to ask if f29DNAP can read [a] L. Wang,+ K. Tram,+ Dr. M. M. Ali, Prof. Dr. Y. Li Department of Biochemistry and Biomedical Sciences and Department of Chemistry and Chemical Biology, McMaster University 1280 Main St. W., Hamilton, ON, L8S 4K1 (Canada) E-mail: [email protected] [b] L. Wang,+ Prof. J. Li Department of Chemistry and Beijing Key Laboratory for Microanalytical Methods and Instrumentation Tsinghua University, Beijing, 100084 (P. R. China) E-mail: [email protected] [c] Prof. B. J. Salena Division of Gastroenterology, Department of Medicine McMaster University, 1280 Main St. W. Hamilton, ON, L8S 4K1 (Canada) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201304292. Chem. Eur. J. 2014, 20, 2420 – 2424
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through a ligand-bound aptamer domain that is incorporated into a circular template. In other words, can ligand–aptamer interaction arrest RCA over an aptameric circular template? Aptamers are ligand-binding single-stranded DNA or RNA sequences that can be isolated from random-sequence libraries using in vitro selection techniques.[6] It has been well documented that aptamers can be evolved to possess high affinity and specificity towards their targets[7] because aptamers and their cognate targets can form well-defined tertiary structures.[8] Thus, the formation of a tight tertiary structure might make a target-bound aptameric circle unsuitable as the template for f29DNAP (Figure 1 A). To test the above hypothesis, we designed an aptameric circular template (named ACT1) that contains a well-characterised aptamer known to bind human thrombin (Figure 1 B).[7a] In addition to the aptamer domain, ACT1 also has a primer binding site required for the RCA reaction, a TaqI restriction enzyme recognition site to be used for the analysis of the RCA product (RCAP), and a peptide nucleic acid (PNA) binding site to be used later for colorimetric reporting of the RCAP. A mutant ACT (ACT1M) was also designed in which several guanine residues in the aptamer domain critical to the target recognition were mutated into adenines. RCA reactions with ACT1 and ACT1M were carried out in the presence of [a-32P]dGTP to allow the incorporation of radioactive phosphorus into the RCAP so that it can be identified through partial digestion with TaqI, followed by denaturing polyacrylamide gel electrophoresis (dPAGE) analysis. The TaqI/dPAGE procedure was expected to produce a characteristic DNA banding pattern, which is made of monomeric, dimeric and other higher-ordered DNA repeats (Figure 1 C), as a measurement of a successful RCA reaction. First, we examined the RCA reaction with ACT1. In the absence of thrombin, ACT1 was able to function as a regular circular template for f29DNAP, based on the appearance of the expected characteristic DNA banding pattern (ACT1 Th, Figure 1 C). This indicates that the inclusion of an aptamer sequence in circular template did not disrupt the RCA reaction. In sharp contrast, the addition of thrombin (2 mm) completely arrested the RCA reaction, as the DNA banding signature was now absent (ACT1 + Th, Figure 1 C). This observation strongly suggests that thrombin engaged the aptamer domain into a rather stable complex so that f29DNAP was unable to read through. To provide further evidence to support the notion that the lack of RCA reaction was a result of specific binding between thrombin and the DNA aptamer, we carried out two control experiments. First, ACT1 was replaced by ACT1M. Since several nucleotides crucial for target recognition were mutated in ACT1M, it was expected that the inhibition on the RCA reaction by thrombin would be completely eliminated. This was confirmed by the experimental observations (ACT1M Th and + Th, Figure 1 C): the presence and absence of thrombin had no effect on the outcome of the RCA reaction. In the second experiment, bovine serum albumin (BSA) was used to substitute thrombin. Since BSA has been shown to have no affinity for the anti-thrombin aptamer, it was anticipated that the RCA reaction would proceed as usual. The observation of the char-
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Communication We investigated whether the ligand-promoted RCA arresting was a general property of an aptameric DNA circle; a DNA aptamer that binds human plateletderived growth factor (PDGF)[7d] was thus chosen as the second test case. The 35 nt PDGF aptamer was incorporated into an 80 nt circular template (named ACT6, Figure S1A in the Supporting Information), which was then subjected to RCA reactions in the absence and presence of PDGF. As expected, in the absence of PDGF, ACT6 can be copied by f29DNAP; however, the RCA reaction was arrested by the addition of PDGF (0.4 mm, Figure S1B). Control experiments Figure 1. A) Schematics of arresting RCA reactions by aptamer–target interactions. B) Sequences of the aptameric with a mutant circular template circular template (ACT1), ACT1 with mutations in the aptamer domain (ACT1M), and primer 1 (P1). The segments (ACT6M, Figure S1A, in which shown in bold, underlined, italic and italic-bold letters are the aptamer for thrombin (Th), the TaqI recognition seseveral guanosines were mutatquence, the primer binding site and the PNA binding site, respectively. C) Analysis of TaqI-digested RCA product ed into thymidines) and BSA by denaturing polyacrylamide gel electrophoresis (dPAGE). M: marker (linear ACT1). demonstrated that the inhibition of RCA was dependent on the specific aptamer sequence as well as the matching target for the aptamer (Figure S1B). acteristic DNA banding signature (ACT1-BSA, Figure 1 C) veriThese results strongly suggest that the observed RCA arresting fied this prediction. Taken together, our data indicate that the appears to be a general phenomenon with, at least, proteinanti-thrombin aptamer forms a highly stable complex with binding DNA aptamers. Future experiments will be directed at thrombin, which is able to block f29DNAP from copying the investigating if the same inhibition can be achieved with apcircular DNA template. tamers that bind small molecules. To rule out the possibility that the observed inhibition might Next, we examined the possibility of exploiting RCA arrestalso be dependent on the other non-aptameric sequence eleing for biosensing applications. We chose a colorimetric reportments in ACT1, we tested a new ACT, ACT2, in which 15 nucleing platform that can report the presence or absence of RCA otides (nt) before the aptameric domain and five nucleotides products through the use of a peptide nucleic acid (PNA) and after it were chosen for substitution (Figure 2 A). The same an organic dye known as DiSC2(5) (diethylthiadicarbocyaniRCA arrest was observed with ACT2 (Figure 2 C), suggesting that the inhibition was solely linked to the presence of the apne).[2g, 9] It has been shown that DiSC2(5) can bind to a PNA– tameric sequence in the ACT. DNA duplex and change its colour from blue to purple (FigWe next examined if the RCA arrest required a specific size ure 3 A). When the RCA reaction is carried out in the absence of ACT. Using ACT2, which contained 80 nt, as the reference, of the target for the aptamer, the production of the RCAP and we progressively removed 10, 20, and 30 nt, resulting in ACT3subsequent hybridisation of the PNA and binding of DiSC2(5) 5 that contained 70, 60 and 50 nt, respectively (Figure 2 A). are expected to produce a purple solution. In the presence of Similar RCA arrests were observed for each shortened ACT (Figthe target, however, RCAP will not be produced and thus, the ure 2 C), indicating the observed inhibition is not restricted to reaction mixture will be blue. As shown in Figure 3 B, the RCA a given size of ACT. reaction mixture obtained with ACT1 in the presence of thromWe also investigated whether the location of the primerbin produced a blue colour, whereas the reaction mixture in binding site can influence the outcome of RCA arresting. ACT1 the absence of thrombin showed a purple colour (Figure 3 B). was chosen as the aptameric circle for this experiment. ACT1 In addition, the control reaction mixtures (ACT1M with and was broken down into four 20 nt elements labelled as S-I to Swithout thrombin as well ACT1 with BSA) also produced the IV (Figure 2 B); matching primers, P1 to P4, were then designed expected purple colour (Figure 3 B). and used for the individual RCA reactions in the absence and Finally, we investigated the feasibility of performing quantipresence of thrombin (Figure 2 D). The RCA arrest was obtative analysis of protein targets using the above colorimetric served for each primer, indicating that the location of the detection method. For this reason, we obtained the absorption primer binding site does not interfere with the target-promotspectra of the sensing reaction mixtures, which are provided in ed inhibition of RCA. Figure 3 C. In the presence of thrombin, the sensing mixture Chem. Eur. J. 2014, 20, 2420 – 2424
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Figure 2. RCA reactions with various templates containing anti-thrombin DNA aptamer. A) Sequences of ACT2-5. The aptamer sequence is shown in bold letters. The “-” signs in the sequences of ACT3-5 indicate the nucleotides removed from ACT2. B) The sequence of ACT1 with four 20 nt sequence elements labelled as S-I to S-IV. Four primers, P1 to P4, were designed to bind each of these elements. C) PAGE analysis of TaqI-digested RCA products obtained with ACT2-5. D) PAGE analysis of TaqI-digested RCA products obtained with P1 to P4; Th: without thrombin; + Th: with thrombin.
had the highest absorption peak at 650 nm and the second highest peak at 550 nm. In the absence of thrombin, the magnitude of both peaks registered significant changes: A550 became more prominent than A650. Based on this observation, we examined the A650/A550 ratio when the concentration of thrombin was varied between 0–2000 nm (Figure S2A in the Supporting Information). A linear empirical correlation is observed when A650/A550 is plotted as a function of the thrombin concentration on a logarithmic scale (Figure S2B). The data analysis establishes a detection limit of 15 nm for thrombin (3s of blank). These results suggest that the colorimetric detection method can be used as a quantitative method to measure the concentration of the protein target. In summary, we have made a novel observation that the binding of a protein target to an aptamer in a circular DNA template can arrest RCA reactions by f29DNAP. This observation was demonstrated twice with different protein–aptamer pairs. Our findings indicate that protein-binding aptamers can form highly stable complexes with their targets in solution, consistent with results from many structural analyses showing that aptamers form very defined tertiary structures with their targets.[8] Both the thrombin and PDGF aptamers exhibit a Kd of approximately 25 and 0.1 nm, respectively. Therefore, the observation of the complete arrest of RCA at saturated target Chem. Eur. J. 2014, 20, 2420 – 2424
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Figure 3. Biosensing based on RCA arrest. A) Schematic for colorimetric detection of RCA product using peptide nucleic acid (PNA) and DiSC2(5). B) Photographs of various RCA reaction mixtures after the addition of PNA and DiSC2(5); Th: thrombin. C) Absorption spectra of the RCA reactions of ACT1 in the absence (purple) and presence (blue) of thrombin.
concentration illustrates that both of the tested aptamers appear to fold uniformly into functional (ligand-binding) structures (i.e., these aptamers do not appear to produce misfolded or alternative structures) under the tested conditions. Thus, the RCA arrest can be used as a strategy to examine the folding ability of such aptamers or to search for improved aptamers with uniform folding characteristics. We have also shown that the arrest of RCA by aptamer– target interactions can be formulated into a colorimetric assay for the detection of the target of the aptamer. The translation of RCA arrest into a simple and convenient assay continues to showcase the versatility of using aptamers as molecular recognition elements for biosensing applications. Control of the accessibility to DNA by polymerases is a common mechanism used by both eukaryotic and prokaryotic cells as a way to regulate gene expression. For example, in eukaryotic cells, histones bind and organise DNA into compact structures making DNA inaccessible to RNA polymerases;[10] post-translational modifications of histones alter their interaction with DNA, freeing it for RNA transcription.[11] In prokaryotic cells, the transcription of many metabolic enzymes is controlled by repressor proteins that bind DNA tightly and prevent its access by RNA polymerases until the metabolic enzymes are in demand.[12] In this study, we have shown that the accessibility of f29DNAP to a circular template can be controlled simply by the incorporation of a protein-binding aptameric domain into the circular template. We hope this finding can lead to the development of more advanced artificial systems that can
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Communication accurately mimic the more intricate biological mechanisms that regulate the accessibility of DNA. Finally, we are tempted to speculate that the inability of f29DNAP to read through aptamer–target complexes in a circular aptameric template might simply reflect the fact that DNA polymerases have never had an opportunity to evolve a coping mechanism to access the DNA sequence in such settings, given that DNA aptamers are man-made structures and are not known to exist in nature.
[3]
[4]
Acknowledgements This research was supported by research grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), SENTINEL Bioactive Paper Network, National Basic Research Program of China (No. 2011CB935704), the National Natural Science Foundation of China (No. 21235004, No. 21327806), the European Union Seventh Framework Programme (No. 260600). L.W. was funded by a scholarship from China Scholarship Council. K.T. is a recipient of an NSERC Postgraduate Scholarship.
[5] [6] [7]
Keywords: aptamers · biosensors · molecular competition · protein detection · rolling circle amplification [1] a) A. Fire, S. Q. Xu, Proc. Natl. Acad. Sci. USA 1995, 92, 4641 – 4645; b) D. Liu, S. L. Daubendiek, M. A. Zillman, K. Ryan, E. T. Kool, J. Am. Chem. Soc. 1996, 118, 1587 – 1594. [2] a) C. Larsson, J. Koch, A. Nygren, G. Janssen, A. K. Raap, U. Landegren, M. Nilsson, Nat. Methods 2004, 1, 227 – 232; b) P. M. Lizardi, X. Huang, Z. Zhu, P. Bray-Ward, D. C. Thomas, D. C. Ward, Nat. Genet. 1998, 19, 225 – 232; c) Y. Weizmann, M. K. Beissenhirtz, Z. Cheglakov, R. Nowarski, M. Kotler, I. Willner, Angew. Chem. 2006, 118, 7544 – 7548; Angew. Chem. Int. Ed. 2006, 45, 7384 – 7388; d) D. A. Di Giusto, W. A. Wlassoff, J. J. Gooding, B. A. Messerle, G. C. King, Nucleic Acids Res. 2005, 33, e64; e) L. Yang, C. W. Fung, E. J. Cho, A. D. Ellington, Anal. Chem. 2007, 79, 3320 – 3329; f) L. Zhou, L. J. Ou, X. Chu, G. L. Shen, R. Q. Yu, Anal. Chem. 2007, 79, 7492 – 7500; g) M. M. Ali, Y. Li, Angew. Chem. 2009, 121, 3564 – 3567; Angew. Chem. Int. Ed. 2009, 48, 3512 – 3515; h) Z. Cheglakov, Y. Weizmann, B. Basnar, I. Willner, Org. Biomol. Chem. 2007, 5, 223 – 225; i) E. J. Cho, L. Yang, M. Levy, A. D. Ellington, J. Am. Chem. Soc. 2005, 127, 2022 – 2023; j) L. Tang, Y. Liu, M. M. Ali, D. K. Kang, W. Zhao, J. Li, Anal. Chem. 2012, 84, 4711 – 4717; k) Y. Tian, Y. He, C. Mao, ChemBioChem 2006, 7, 1862 – 1864; l) W. Zhao, M. M. Ali, M. A. Brook, Y. Li, Angew.
Chem. Eur. J. 2014, 20, 2420 – 2424
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[8]
[9]
[10] [11] [12]
Chem. 2008, 120, 6428 – 6436; Angew. Chem. Int. Ed. 2008, 47, 6330 – 6337. a) L. Blanco, A. Bernad, J. M. Lazaro, G. Martin, C. Garmendia, M. Salas, J. Biol. Chem. 1989, 264, 8935 – 8940; b) D. Canceill, E. Viguera, S. D. Ehrlich, J. Biol. Chem. 1999, 274, 27481 – 27490. a) A. J. Berman, S. Kamtekar, J. L. Goodman, J. M. Lazaro, M. de Vega, L. Blanco, M. Salas, T. A. Steitz, EMBO J. 2007, 26, 3494 – 3505; b) S. Kamtekar, A. J. Berman, J. Wang, J. M. Lazaro, M. de Vega, L. Blanco, M. Salas, T. A. Steitz, Mol. Cell. 2004, 16, 609 – 618; c) S. Kamtekar, A. J. Berman, J. Wang, J. M. Lazaro, M. de Vega, L. Blanco, M. Salas, T. A. Steitz, EMBO J. 2006, 25, 1335 – 1343; d) J. A. Morin, F. J. Cao, J. M. Lazaro, J. R. AriasGonzalez, J. M. Valpuesta, J. L. Carrascosa, M. Salas, B. Ibarra, Proc. Natl. Acad. Sci. USA 2012, 109, 8115 – 8120; e) I. Rodriguez, J. M. Lazaro, L. Blanco, S. Kamtekar, A. J. Berman, J. Wang, T. A. Steitz, M. Salas, M. de Vega, Proc. Natl. Acad. Sci. USA 2005, 102, 6407 – 6412; f) M. S. Soengas, J. A. Esteban, J. M. Lazaro, A. Bernad, M. A. Blasco, M. Salas, L. Blanco, EMBO J. 1992, 11, 4227 – 4237. C. Lin, X. Wang, Y. Liu, N. C. Seeman, H. Yan, J. Am. Chem. Soc. 2007, 129, 14475 – 14481. a) A. D. Ellington, J. W. Szostak, Nature 1990, 346, 818 – 822; b) C. Tuerk, L. Gold, Science 1990, 249, 505 – 510. a) L. C. Bock, L. C. Griffin, J. A. Latham, E. H. Vermaas, J. J. Toole, Nature 1992, 355, 564 – 566; b) M. Famulok, G. Mayer, Acc. Chem. Res. 2011, 44, 1349 – 1358; c) L. S. Green, D. Jellinek, R. Jenison, A. Ostman, C. H. Heldin, N. Janjic, Biochemistry 1996, 35, 14413 – 14424; d) R. D. Jenison, S. C. Gill, A. Pardi, B. Polisky, Science 1994, 263, 1425 – 1429; e) S. M. Nimjee, C. P. Rusconi, B. A. Sullenger, Annu. Rev. Med. 2005, 56, 555 – 583; f) S. E. Osborne, I. Matsumura, A. D. Ellington, Curr. Opin. Chem. Biol. 1997, 1, 5 – 9; g) D. S. Wilson, J. W. Szostak, Annu. Rev. Biochem. 1999, 68, 611 – 647. a) J. Feigon, T. Dieckmann, F. W. Smith, Chem. Biol. 1996, 3, 611 – 617; b) T. Hermann, D. J. Patel, Structure 2000, 8, R47 – 54; c) R. K. Montange, R. T. Batey, Annu. Rev. Biophys. 2008, 37, 117 – 133; d) D. J. Patel, Curr. Opin Chem. Biol. 1997, 1, 32 – 46. a) I. Dilek, M. Madrid, R. Singh, C. P. Urrea, B. A. Armitage, J. Am. Chem. Soc. 2005, 127, 3339 – 3345; b) M. Komiyama, S. Ye, X. Liang, Y. Yamamoto, T. Tomita, J. M. Zhou, H. Aburatani, J. Am. Chem. Soc. 2003, 125, 3758 – 3762; c) L. M. Wilhelmsson, B. Norden, K. Mukherjee, M. T. Dulay, R. N. Zare, Nucleic Acids Res. 2002, 30, E3. a) K. Luger, A. W. Mader, R. K. Richmond, D. F. Sargent, T. J. Richmond, Nature 1997, 389, 251 – 260; b) D. Rhodes, Nature 1997, 389, 233. a) T. Jenuwein, C. D. Allis, Science 2001, 293, 1074 – 1080; b) B. D. Strahl, C. D. Allis, Nature 2000, 403, 41 – 45. a) W. Gilbert, B. Muller-Hill, Proc. Natl. Acad. Sci. USA 1966, 56, 1891 – 1898; b) F. Jacob, J. Monod, J. Mol. Biol. 1961, 3, 318 – 356; c) R. Schleif, Annu. Rev. Biochem. 1992, 61, 199 – 223.
Received: November 2, 2013 Revised: December 23, 2013 Published online on February 7, 2014
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& Aptamers
Arrest of Rolling Circle Amplification by Protein-Binding DNA Aptamers Lida Wang,[a, b] Kha Tram,[a] Monsur M. Ali,[a] Bruno J. Salena,[c] Jinghong Li,*[b] and Yingfu Li*[a]
Chem. Eur. J. 2014, 20, 2420 – 2424
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Communication Abstract: Certain DNA polymerases, such as f29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super-long single-stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand-displacement ability of these polymerases. In this work, the ability of f29DNAP to carry out RCA over circular templates containing a protein-binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein-binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.
Rolling circle amplification (RCA) is an isothermal DNA amplification process that generates extremely long DNA molecules from a single-stranded (ss) DNA circle.[1]In this unique DNA synthesis reaction, a special DNA polymerase (such as f29 DNA polymerase, f29DNAP) makes repetitive rounds of DNA copying over a circular ssDNA template using a short DNA strand as the primer and deoxyribonucleotide 5’-triphosphates (dNTPs) as the building blocks. RCA is widely exploited as a powerful signal amplification technique for bioanalysis as it converts a single primer–template recognition event into thousands of tandem DNA repeats that can be easily detected.[2] The power of this technique reflects the high processivity and the strand-displacement ability of f29DNAP: this enzyme is able to make more than 70 000 nucleotide additions before its dissociation from the DNA template,[3] and unwind doublestranded DNA on its own without requiring the assistance of any DNA helicases.[4] It has also been reported that f29DNAP is able to unwind stable secondary structures in the circular template.[3, 5] It is interesting to ask if f29DNAP can read [a] L. Wang,+ K. Tram,+ Dr. M. M. Ali, Prof. Dr. Y. Li Department of Biochemistry and Biomedical Sciences and Department of Chemistry and Chemical Biology, McMaster University 1280 Main St. W., Hamilton, ON, L8S 4K1 (Canada) E-mail: [email protected] [b] L. Wang,+ Prof. J. Li Department of Chemistry and Beijing Key Laboratory for Microanalytical Methods and Instrumentation Tsinghua University, Beijing, 100084 (P. R. China) E-mail: [email protected] [c] Prof. B. J. Salena Division of Gastroenterology, Department of Medicine McMaster University, 1280 Main St. W. Hamilton, ON, L8S 4K1 (Canada) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201304292. Chem. Eur. J. 2014, 20, 2420 – 2424
www.chemeurj.org
through a ligand-bound aptamer domain that is incorporated into a circular template. In other words, can ligand–aptamer interaction arrest RCA over an aptameric circular template? Aptamers are ligand-binding single-stranded DNA or RNA sequences that can be isolated from random-sequence libraries using in vitro selection techniques.[6] It has been well documented that aptamers can be evolved to possess high affinity and specificity towards their targets[7] because aptamers and their cognate targets can form well-defined tertiary structures.[8] Thus, the formation of a tight tertiary structure might make a target-bound aptameric circle unsuitable as the template for f29DNAP (Figure 1 A). To test the above hypothesis, we designed an aptameric circular template (named ACT1) that contains a well-characterised aptamer known to bind human thrombin (Figure 1 B).[7a] In addition to the aptamer domain, ACT1 also has a primer binding site required for the RCA reaction, a TaqI restriction enzyme recognition site to be used for the analysis of the RCA product (RCAP), and a peptide nucleic acid (PNA) binding site to be used later for colorimetric reporting of the RCAP. A mutant ACT (ACT1M) was also designed in which several guanine residues in the aptamer domain critical to the target recognition were mutated into adenines. RCA reactions with ACT1 and ACT1M were carried out in the presence of [a-32P]dGTP to allow the incorporation of radioactive phosphorus into the RCAP so that it can be identified through partial digestion with TaqI, followed by denaturing polyacrylamide gel electrophoresis (dPAGE) analysis. The TaqI/dPAGE procedure was expected to produce a characteristic DNA banding pattern, which is made of monomeric, dimeric and other higher-ordered DNA repeats (Figure 1 C), as a measurement of a successful RCA reaction. First, we examined the RCA reaction with ACT1. In the absence of thrombin, ACT1 was able to function as a regular circular template for f29DNAP, based on the appearance of the expected characteristic DNA banding pattern (ACT1 Th, Figure 1 C). This indicates that the inclusion of an aptamer sequence in circular template did not disrupt the RCA reaction. In sharp contrast, the addition of thrombin (2 mm) completely arrested the RCA reaction, as the DNA banding signature was now absent (ACT1 + Th, Figure 1 C). This observation strongly suggests that thrombin engaged the aptamer domain into a rather stable complex so that f29DNAP was unable to read through. To provide further evidence to support the notion that the lack of RCA reaction was a result of specific binding between thrombin and the DNA aptamer, we carried out two control experiments. First, ACT1 was replaced by ACT1M. Since several nucleotides crucial for target recognition were mutated in ACT1M, it was expected that the inhibition on the RCA reaction by thrombin would be completely eliminated. This was confirmed by the experimental observations (ACT1M Th and + Th, Figure 1 C): the presence and absence of thrombin had no effect on the outcome of the RCA reaction. In the second experiment, bovine serum albumin (BSA) was used to substitute thrombin. Since BSA has been shown to have no affinity for the anti-thrombin aptamer, it was anticipated that the RCA reaction would proceed as usual. The observation of the char-
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Communication We investigated whether the ligand-promoted RCA arresting was a general property of an aptameric DNA circle; a DNA aptamer that binds human plateletderived growth factor (PDGF)[7d] was thus chosen as the second test case. The 35 nt PDGF aptamer was incorporated into an 80 nt circular template (named ACT6, Figure S1A in the Supporting Information), which was then subjected to RCA reactions in the absence and presence of PDGF. As expected, in the absence of PDGF, ACT6 can be copied by f29DNAP; however, the RCA reaction was arrested by the addition of PDGF (0.4 mm, Figure S1B). Control experiments Figure 1. A) Schematics of arresting RCA reactions by aptamer–target interactions. B) Sequences of the aptameric with a mutant circular template circular template (ACT1), ACT1 with mutations in the aptamer domain (ACT1M), and primer 1 (P1). The segments (ACT6M, Figure S1A, in which shown in bold, underlined, italic and italic-bold letters are the aptamer for thrombin (Th), the TaqI recognition seseveral guanosines were mutatquence, the primer binding site and the PNA binding site, respectively. C) Analysis of TaqI-digested RCA product ed into thymidines) and BSA by denaturing polyacrylamide gel electrophoresis (dPAGE). M: marker (linear ACT1). demonstrated that the inhibition of RCA was dependent on the specific aptamer sequence as well as the matching target for the aptamer (Figure S1B). acteristic DNA banding signature (ACT1-BSA, Figure 1 C) veriThese results strongly suggest that the observed RCA arresting fied this prediction. Taken together, our data indicate that the appears to be a general phenomenon with, at least, proteinanti-thrombin aptamer forms a highly stable complex with binding DNA aptamers. Future experiments will be directed at thrombin, which is able to block f29DNAP from copying the investigating if the same inhibition can be achieved with apcircular DNA template. tamers that bind small molecules. To rule out the possibility that the observed inhibition might Next, we examined the possibility of exploiting RCA arrestalso be dependent on the other non-aptameric sequence eleing for biosensing applications. We chose a colorimetric reportments in ACT1, we tested a new ACT, ACT2, in which 15 nucleing platform that can report the presence or absence of RCA otides (nt) before the aptameric domain and five nucleotides products through the use of a peptide nucleic acid (PNA) and after it were chosen for substitution (Figure 2 A). The same an organic dye known as DiSC2(5) (diethylthiadicarbocyaniRCA arrest was observed with ACT2 (Figure 2 C), suggesting that the inhibition was solely linked to the presence of the apne).[2g, 9] It has been shown that DiSC2(5) can bind to a PNA– tameric sequence in the ACT. DNA duplex and change its colour from blue to purple (FigWe next examined if the RCA arrest required a specific size ure 3 A). When the RCA reaction is carried out in the absence of ACT. Using ACT2, which contained 80 nt, as the reference, of the target for the aptamer, the production of the RCAP and we progressively removed 10, 20, and 30 nt, resulting in ACT3subsequent hybridisation of the PNA and binding of DiSC2(5) 5 that contained 70, 60 and 50 nt, respectively (Figure 2 A). are expected to produce a purple solution. In the presence of Similar RCA arrests were observed for each shortened ACT (Figthe target, however, RCAP will not be produced and thus, the ure 2 C), indicating the observed inhibition is not restricted to reaction mixture will be blue. As shown in Figure 3 B, the RCA a given size of ACT. reaction mixture obtained with ACT1 in the presence of thromWe also investigated whether the location of the primerbin produced a blue colour, whereas the reaction mixture in binding site can influence the outcome of RCA arresting. ACT1 the absence of thrombin showed a purple colour (Figure 3 B). was chosen as the aptameric circle for this experiment. ACT1 In addition, the control reaction mixtures (ACT1M with and was broken down into four 20 nt elements labelled as S-I to Swithout thrombin as well ACT1 with BSA) also produced the IV (Figure 2 B); matching primers, P1 to P4, were then designed expected purple colour (Figure 3 B). and used for the individual RCA reactions in the absence and Finally, we investigated the feasibility of performing quantipresence of thrombin (Figure 2 D). The RCA arrest was obtative analysis of protein targets using the above colorimetric served for each primer, indicating that the location of the detection method. For this reason, we obtained the absorption primer binding site does not interfere with the target-promotspectra of the sensing reaction mixtures, which are provided in ed inhibition of RCA. Figure 3 C. In the presence of thrombin, the sensing mixture Chem. Eur. J. 2014, 20, 2420 – 2424
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Figure 2. RCA reactions with various templates containing anti-thrombin DNA aptamer. A) Sequences of ACT2-5. The aptamer sequence is shown in bold letters. The “-” signs in the sequences of ACT3-5 indicate the nucleotides removed from ACT2. B) The sequence of ACT1 with four 20 nt sequence elements labelled as S-I to S-IV. Four primers, P1 to P4, were designed to bind each of these elements. C) PAGE analysis of TaqI-digested RCA products obtained with ACT2-5. D) PAGE analysis of TaqI-digested RCA products obtained with P1 to P4; Th: without thrombin; + Th: with thrombin.
had the highest absorption peak at 650 nm and the second highest peak at 550 nm. In the absence of thrombin, the magnitude of both peaks registered significant changes: A550 became more prominent than A650. Based on this observation, we examined the A650/A550 ratio when the concentration of thrombin was varied between 0–2000 nm (Figure S2A in the Supporting Information). A linear empirical correlation is observed when A650/A550 is plotted as a function of the thrombin concentration on a logarithmic scale (Figure S2B). The data analysis establishes a detection limit of 15 nm for thrombin (3s of blank). These results suggest that the colorimetric detection method can be used as a quantitative method to measure the concentration of the protein target. In summary, we have made a novel observation that the binding of a protein target to an aptamer in a circular DNA template can arrest RCA reactions by f29DNAP. This observation was demonstrated twice with different protein–aptamer pairs. Our findings indicate that protein-binding aptamers can form highly stable complexes with their targets in solution, consistent with results from many structural analyses showing that aptamers form very defined tertiary structures with their targets.[8] Both the thrombin and PDGF aptamers exhibit a Kd of approximately 25 and 0.1 nm, respectively. Therefore, the observation of the complete arrest of RCA at saturated target Chem. Eur. J. 2014, 20, 2420 – 2424
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Figure 3. Biosensing based on RCA arrest. A) Schematic for colorimetric detection of RCA product using peptide nucleic acid (PNA) and DiSC2(5). B) Photographs of various RCA reaction mixtures after the addition of PNA and DiSC2(5); Th: thrombin. C) Absorption spectra of the RCA reactions of ACT1 in the absence (purple) and presence (blue) of thrombin.
concentration illustrates that both of the tested aptamers appear to fold uniformly into functional (ligand-binding) structures (i.e., these aptamers do not appear to produce misfolded or alternative structures) under the tested conditions. Thus, the RCA arrest can be used as a strategy to examine the folding ability of such aptamers or to search for improved aptamers with uniform folding characteristics. We have also shown that the arrest of RCA by aptamer– target interactions can be formulated into a colorimetric assay for the detection of the target of the aptamer. The translation of RCA arrest into a simple and convenient assay continues to showcase the versatility of using aptamers as molecular recognition elements for biosensing applications. Control of the accessibility to DNA by polymerases is a common mechanism used by both eukaryotic and prokaryotic cells as a way to regulate gene expression. For example, in eukaryotic cells, histones bind and organise DNA into compact structures making DNA inaccessible to RNA polymerases;[10] post-translational modifications of histones alter their interaction with DNA, freeing it for RNA transcription.[11] In prokaryotic cells, the transcription of many metabolic enzymes is controlled by repressor proteins that bind DNA tightly and prevent its access by RNA polymerases until the metabolic enzymes are in demand.[12] In this study, we have shown that the accessibility of f29DNAP to a circular template can be controlled simply by the incorporation of a protein-binding aptameric domain into the circular template. We hope this finding can lead to the development of more advanced artificial systems that can
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Communication accurately mimic the more intricate biological mechanisms that regulate the accessibility of DNA. Finally, we are tempted to speculate that the inability of f29DNAP to read through aptamer–target complexes in a circular aptameric template might simply reflect the fact that DNA polymerases have never had an opportunity to evolve a coping mechanism to access the DNA sequence in such settings, given that DNA aptamers are man-made structures and are not known to exist in nature.
[3]
[4]
Acknowledgements This research was supported by research grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), SENTINEL Bioactive Paper Network, National Basic Research Program of China (No. 2011CB935704), the National Natural Science Foundation of China (No. 21235004, No. 21327806), the European Union Seventh Framework Programme (No. 260600). L.W. was funded by a scholarship from China Scholarship Council. K.T. is a recipient of an NSERC Postgraduate Scholarship.
[5] [6] [7]
Keywords: aptamers · biosensors · molecular competition · protein detection · rolling circle amplification [1] a) A. Fire, S. Q. Xu, Proc. Natl. Acad. Sci. USA 1995, 92, 4641 – 4645; b) D. Liu, S. L. Daubendiek, M. A. Zillman, K. Ryan, E. T. Kool, J. Am. Chem. Soc. 1996, 118, 1587 – 1594. [2] a) C. Larsson, J. Koch, A. Nygren, G. Janssen, A. K. Raap, U. Landegren, M. Nilsson, Nat. Methods 2004, 1, 227 – 232; b) P. M. Lizardi, X. Huang, Z. Zhu, P. Bray-Ward, D. C. Thomas, D. C. Ward, Nat. Genet. 1998, 19, 225 – 232; c) Y. Weizmann, M. K. Beissenhirtz, Z. Cheglakov, R. Nowarski, M. Kotler, I. Willner, Angew. Chem. 2006, 118, 7544 – 7548; Angew. Chem. Int. Ed. 2006, 45, 7384 – 7388; d) D. A. Di Giusto, W. A. Wlassoff, J. J. Gooding, B. A. Messerle, G. C. King, Nucleic Acids Res. 2005, 33, e64; e) L. Yang, C. W. Fung, E. J. Cho, A. D. Ellington, Anal. Chem. 2007, 79, 3320 – 3329; f) L. Zhou, L. J. Ou, X. Chu, G. L. Shen, R. Q. Yu, Anal. Chem. 2007, 79, 7492 – 7500; g) M. M. Ali, Y. Li, Angew. Chem. 2009, 121, 3564 – 3567; Angew. Chem. Int. Ed. 2009, 48, 3512 – 3515; h) Z. Cheglakov, Y. Weizmann, B. Basnar, I. Willner, Org. Biomol. Chem. 2007, 5, 223 – 225; i) E. J. Cho, L. Yang, M. Levy, A. D. Ellington, J. Am. Chem. Soc. 2005, 127, 2022 – 2023; j) L. Tang, Y. Liu, M. M. Ali, D. K. Kang, W. Zhao, J. Li, Anal. Chem. 2012, 84, 4711 – 4717; k) Y. Tian, Y. He, C. Mao, ChemBioChem 2006, 7, 1862 – 1864; l) W. Zhao, M. M. Ali, M. A. Brook, Y. Li, Angew.
Chem. Eur. J. 2014, 20, 2420 – 2424
www.chemeurj.org
[8]
[9]
[10] [11] [12]
Chem. 2008, 120, 6428 – 6436; Angew. Chem. Int. Ed. 2008, 47, 6330 – 6337. a) L. Blanco, A. Bernad, J. M. Lazaro, G. Martin, C. Garmendia, M. Salas, J. Biol. Chem. 1989, 264, 8935 – 8940; b) D. Canceill, E. Viguera, S. D. Ehrlich, J. Biol. Chem. 1999, 274, 27481 – 27490. a) A. J. Berman, S. Kamtekar, J. L. Goodman, J. M. Lazaro, M. de Vega, L. Blanco, M. Salas, T. A. Steitz, EMBO J. 2007, 26, 3494 – 3505; b) S. Kamtekar, A. J. Berman, J. Wang, J. M. Lazaro, M. de Vega, L. Blanco, M. Salas, T. A. Steitz, Mol. Cell. 2004, 16, 609 – 618; c) S. Kamtekar, A. J. Berman, J. Wang, J. M. Lazaro, M. de Vega, L. Blanco, M. Salas, T. A. Steitz, EMBO J. 2006, 25, 1335 – 1343; d) J. A. Morin, F. J. Cao, J. M. Lazaro, J. R. AriasGonzalez, J. M. Valpuesta, J. L. Carrascosa, M. Salas, B. Ibarra, Proc. Natl. Acad. Sci. USA 2012, 109, 8115 – 8120; e) I. Rodriguez, J. M. Lazaro, L. Blanco, S. Kamtekar, A. J. Berman, J. Wang, T. A. Steitz, M. Salas, M. de Vega, Proc. Natl. Acad. Sci. USA 2005, 102, 6407 – 6412; f) M. S. Soengas, J. A. Esteban, J. M. Lazaro, A. Bernad, M. A. Blasco, M. Salas, L. Blanco, EMBO J. 1992, 11, 4227 – 4237. C. Lin, X. Wang, Y. Liu, N. C. Seeman, H. Yan, J. Am. Chem. Soc. 2007, 129, 14475 – 14481. a) A. D. Ellington, J. W. Szostak, Nature 1990, 346, 818 – 822; b) C. Tuerk, L. Gold, Science 1990, 249, 505 – 510. a) L. C. Bock, L. C. Griffin, J. A. Latham, E. H. Vermaas, J. J. Toole, Nature 1992, 355, 564 – 566; b) M. Famulok, G. Mayer, Acc. Chem. Res. 2011, 44, 1349 – 1358; c) L. S. Green, D. Jellinek, R. Jenison, A. Ostman, C. H. Heldin, N. Janjic, Biochemistry 1996, 35, 14413 – 14424; d) R. D. Jenison, S. C. Gill, A. Pardi, B. Polisky, Science 1994, 263, 1425 – 1429; e) S. M. Nimjee, C. P. Rusconi, B. A. Sullenger, Annu. Rev. Med. 2005, 56, 555 – 583; f) S. E. Osborne, I. Matsumura, A. D. Ellington, Curr. Opin. Chem. Biol. 1997, 1, 5 – 9; g) D. S. Wilson, J. W. Szostak, Annu. Rev. Biochem. 1999, 68, 611 – 647. a) J. Feigon, T. Dieckmann, F. W. Smith, Chem. Biol. 1996, 3, 611 – 617; b) T. Hermann, D. J. Patel, Structure 2000, 8, R47 – 54; c) R. K. Montange, R. T. Batey, Annu. Rev. Biophys. 2008, 37, 117 – 133; d) D. J. Patel, Curr. Opin Chem. Biol. 1997, 1, 32 – 46. a) I. Dilek, M. Madrid, R. Singh, C. P. Urrea, B. A. Armitage, J. Am. Chem. Soc. 2005, 127, 3339 – 3345; b) M. Komiyama, S. Ye, X. Liang, Y. Yamamoto, T. Tomita, J. M. Zhou, H. Aburatani, J. Am. Chem. Soc. 2003, 125, 3758 – 3762; c) L. M. Wilhelmsson, B. Norden, K. Mukherjee, M. T. Dulay, R. N. Zare, Nucleic Acids Res. 2002, 30, E3. a) K. Luger, A. W. Mader, R. K. Richmond, D. F. Sargent, T. J. Richmond, Nature 1997, 389, 251 – 260; b) D. Rhodes, Nature 1997, 389, 233. a) T. Jenuwein, C. D. Allis, Science 2001, 293, 1074 – 1080; b) B. D. Strahl, C. D. Allis, Nature 2000, 403, 41 – 45. a) W. Gilbert, B. Muller-Hill, Proc. Natl. Acad. Sci. USA 1966, 56, 1891 – 1898; b) F. Jacob, J. Monod, J. Mol. Biol. 1961, 3, 318 – 356; c) R. Schleif, Annu. Rev. Biochem. 1992, 61, 199 – 223.
Received: November 2, 2013 Revised: December 23, 2013 Published online on February 7, 2014
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