Bioorganic & Medicinal Chemistry Letters 23 (2013) 6851–6853
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Polycation-assisted DNA detection by reduction triggered fluorescence amplification probe Hisao Saneyoshi a,e, Naohiko Shimada b, Atsushi Maruyama b, Yoshihiro Ito a,e,⇑, Hiroshi Abe a,c,d,e,⇑ a
Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan Institute for Materials Chemistry and Engineering, Kyushu University, 744-CE11 Motooka, Nishi, Fukuoka 819-0395, Japan c Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan e Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1, Hirosawa, Wako-Shi, Saitama 351-0198, Japan b
a r t i c l e
i n f o
Article history: Received 7 June 2013 Revised 28 September 2013 Accepted 2 October 2013 Available online 11 October 2013
a b s t r a c t We have developed a fluorescence detection system for DNA, assisted by a comb-type cationic polymer (PLL-g-DX), for accelerating the reaction turnover. The combination of fluorogenic DNA probes with a comb-type cationic polymer has been demonstrated to be an effective means of signal amplification during the detection process. The method described herein represents a simple and enzyme-free detection. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: DNA Fluorescence detection Polymer Catalyst Reduction
Techniques aimed at the detection of nucleic acids have recently been the subject of considerable levels of attention because of their potential application in the early diagnosis for a variety of different diseases, including HIV, leukemia and tuberculosis.1–3 Hybridization-based analysis has been reported as an operationally simple method of detection and likened to a molecular beacon.4–9 Unfortunately, however, the amplification of the target DNA/RNA using this technique is less sensitive than other methods such as the polymerase chain reaction (PCR). Signal amplification has been used as a powerful solution to the sensitivity issues associated with the detection of nucleic acids, providing significant gains in sensitivity. The application of a chemical reaction catalyzed by the target nucleic acid is emerging as a powerful method to amplify signals.10–34 It is noteworthy that no enzymes are required for this reaction-based detection process, and this therefore represents a significant advantage from the perspective of point-of-care testing techniques in the field of biomedical research. The mechanism of the reaction-based detection process is shown in Figure 1. The first step involves the hybridization of the two reactive probes (Azm and TPP) with the target nucleic acid and their subsequent reaction to generate a fluorescence signal. The second step involves the participation of the target nucleic acid in the catalytic cycle during the detection process. During this process, strand exchange ⇑ Corresponding authors. Tel.: +81 (0)48 467 5749; fax: +81 (0)48 467 2827. E-mail address: [email protected] (H. Abe). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.005
occurs between the probes and the target strands, and this step is essential for catalytic turnover resulting signal amplification. Cationic comb-type copolymers (PLL-g-DX), which consist of a cationic poly(L-lysine) backbone and water-soluble dextran side chains, have been reported in the literature as good strand exchange promoters.35–42 Michaelis et al.43 recently applied PLLg-DX to a fluorophore transfer reaction and confirmed its overall utility based on the specific benefit of PLL-DX over spermine in terms of the catalytic turnover of an oligonucleotide-templated reaction. Furthermore, PLL-DX did not disturb the mismatch discrimination during the detection process. Herein, we report the development of a new system providing a high catalytic turnover number to amplify a fluorescence signal based on the combination of fluorogenic reactive probes with a combo-type cationic polymer, as shown in Figure 1. Our optimized system offered better results in terms of higher turnover number, shorter reaction time or lower detection limit compared with previous reports.43 We have recently reported the development and application of fluorogenic oligonucleotide probes consisting of oligonucleotides bearing fluorogenic-type azide methyl fluorescein (Azm) and triphenylphosphine (TPP) that could successful detect the target sequences without the requirement for any additional chemicals or enzymes. These so-called reduction triggered fluorescence (RETF) probes have been demonstrated to possess signal amplification ability.44–47 With the aim of making further improvements to the catalytic turnover, we recently shifted our
6852
H. Saneyoshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6851–6853
Figure 1. DNA-templated fluorogenic reaction assisted with PLL-g-DX.
attention towards polycation assisted DNA detection. The combination of fluorogenic probes with a polymer allowed us to gain the level of sensitivity required for detection by increasing the number of catalytic cycles. To apply the polymer to the DNA-templated fluorogenic reaction using our system, we initially tested the influence of cationic polymer on the bimolecular reaction in the absence of the target DNA strand. In general, the cationic polymer accommodates negatively charged strands on nucleic acid through a series of strongly interactions. It is noteworthy that 20 lM of the cationic polymer induced a bimolecular reaction (unspecific reaction). Our preliminary experiments revealed that 5 lM of polymer had no discernible impact on the reaction system and provided results similar to those of the control experiment involving no polymer at all as shown in Figure 3. Next, to determine the appropriate polymer concentration for DNA detection, fluorogenic sensing against 100 pM of the target DNA was performed. Fluorogenic sensing of a model DNA sequence (50 -TAAGCAGAGTTCAAAAGCCCTTCAGCG-30 ) was performed on the bcr/abl target gene originating from the human bcr/abl junction site, which is related to chronic myelogenous leukemia
Figure 3. Negative effect of polymer concentration against unspecific reaction. Conditions: Azm probe (100 nM), TPP probe (200 nM), PLL-g-DX (0–20 lM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 40 °C, 1 h. ⁄Yield was calculated from the fluorescence intensity of the Azm probe reduced by dithiothreitol (DTT).
A
B
Figure 4. Effect of temperature on the DNA detection. Conditions: (A) (no polymer); Azm probe (50 nM), TPP probe (50 nM), target DNA (10 pM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 25 °C, 40 °C, 55 °C 1 h. (B) (with polymer); Az probe (50 nM), TPP probe (50 nM), Target DNA (10 pM), PLL-g-DX (1 lM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 25 °C, 40 °C, 55 °C 1 h. ⁄Yield was calculated from the fluorescence intensity of the Azm probe reduced by dithiothreitol (DTT).
Figure 2. Chemical structure of reactive moiety in fluorogenic DNA probes and cationic-comb type polymer (PLL-g-DX, 30K90D poly-lysine 30 K dextran 90 wt %, amino group 0.78 mmol/mg).
(K562).48,49 The lengths of the Azm and the TPP probes were 8mer and 10-mer, respectively, as shown in Figure 2. The melting temperature of the probe was 46.7 °C, according to our previous study.45 The effective polymer concentration was found to be 1 lM when the two probes in the RETF system were both used at a concentration of 50 nM. The N/P ratio was estimated to be approximately 1 (data not shown). The effect of the temperature on the detection process was determined to be an important factor for the hybridization and the polymer assisted strand exchange reaction. Comparable experiments were performed to evaluate changes in the temperature and polymer effect, as shown in Figure 4. As a result, the polymer was found to work well at 40 °C with a target concentration of 10 pM. At a temperature of 55 °C, the results were similar to those obtained at 25 °C and showed the lowest background signal. Unfortunately, however, no effective signal was observed at
H. Saneyoshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6851–6853 Table 1 Enhanced turnover number in the presence of PLL-g-DX
No polymer With polymer
6853
References and notes
25 °C
40 °C
55 °C
— —
— 127.8
— 46.8
Conditions: left; Azm probe (50 nM), TPP probe (50 nM), Target DNA (10 pM), PLLg-DX (1 lM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 40 °C, 1 h.
25 °C. In contrast, in the absence of the polymer under all of the conditions, no effective signal was observed, as shown in Figure 4. According to a report by Seitz and colleagues,43 the addition of PLL-g-DX to the reaction mixture was particularly effective for stabilizing the duplex compared with the system containing no PLL-gDX. It was therefore assumed that the duplex was too stable for the catalytic cycle at 25 °C. In contrast, temperatures of 40 and 55 °C were found to be satisfactory for the catalytic cycle to give multiple chemical reactions. We then proceeded to estimate the turnover number from the reaction yield. The maximum turnover number was estimated to be 127.8 at 40 °C, with the next highest turnover estimated to be 46.8 at 55 °C, as shown in Table 1. These data represented a remarkable improvement and a higher class of catalytic activity compared with the no polymer conditions and data that we had reported 53.6 against 50 pM target after 4 h previously.45 In addition, a reaction time of only 1 h was required to detect the target DNA at a concentration of 10 pM. These results indicated that the addition of polymer effectively enhanced the sensitivity of the promoter in the detection cycle. To obtain higher levels of sensitivity and sequence selectivity, the effects of several other factors on the system need to be considered, including the development of an RETF probe incorporating a modified nucleoside such as 2-thiothymidine, as well as the optimization of the pH and salt concentration. Work towards further refining the detection process is currently underway in our laboratory. In summary, we have developed a polycation assisted DNA detection system that effectively enhanced the reaction turnover to amplify the fluorescence signal from the target nucleic acid. It is envisaged that polycation-assisted detection will become an essential tool for nucleic acid detection. Acknowledgments H.A. and Y.I. were financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the New Energy, Industrial Technology Development Organization (NEDO) and Precursory Research for Embryonic Science and Technology (PREST). We are grateful for the support received from the Brain Science Institute (BSI) Research Resource Center for performing the mass spectral analyses. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.10. 005.
1. Tang, W.; Chow, W. H. A.; Ying, L.; Kong, H.; Tang, Y.-W.; Lemieux, B. J. Infect. Dis. 2010, 201, S46. 2. Nashed, A. L.; Rao, K. W.; Gulley, M. L. J. Mol. Diagn. 2003, 5, 63. 3. Dheda, K.; Ruhwald, M.; Theron, G.; Peter, J.; Yam, W. C. Respirology 2013, 18, 217. 4. Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303. 5. Piatek, A. S.; Tyagi, S.; Pol, A. C.; Telenti, A.; Miller, L. P.; Kramer, F. R.; Alland, D. Nat. Biotechnol. 1998, 16, 359. 6. Fang, X.; Li, J. J.; Tan, W. Anal. Chem. 2000, 72, 3280. 7. Tyagi, S.; Marras, S. A. E.; Kramer, F. R. Nat. Biotechnol. 2000, 18, 1191. 8. Bratu, D. P.; Cha, B.-J.; Mhlanga, M. M.; Kramer, F. R.; Tyagi, S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13308. 9. Vargas, D. Y.; Raj, A.; Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17008. 10. Li, X.; Liu, D. R. Angew. Chem., Int. Ed. 2004, 43, 4848. 11. Silverman, A. P.; Kool, E. T. Chem. Rev. 2006, 106, 3775. 12. Grossmann, T. N.; Strohbach, A.; Seitz, O. ChemBioChem 2008, 9, 2185. 13. Pianowski, Z. L.; Winssinger, N. Chem. Soc. Rev. 2008, 37, 1330. 14. Kolpashchikov, D. M. Chem. Rev. 2010, 110, 4709. 15. Shibata, A.; Abe, H.; Ito, Y. Molecules 2012, 17, 2446. 16. Ma, Z.; Taylor, J.-S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11159. 17. Ma, Z.; Taylor, J.-S. Bioorg. Med. Chem. 2001, 9, 2501. 18. Ma, Z.; Taylor, J.-S. Bioconjugate Chem. 2003, 14, 679. 19. Sando, S.; Sasaki, T.; Kanatani, K.; Aoyama, Y. J. Am. Chem. Soc. 2003, 125, 15720. 20. Cai, J.; Li, X.; Yue, X.; Taylor, J. S. J. Am. Chem. Soc. 2004, 126, 16324. 21. Cai, J.; Li, X.; Taylor, J. S. Org. Lett. 2005, 7, 751. 22. Dose, C.; Ficht, S.; Seitz, O. Angew. Chem., Int. Ed. 2006, 45, 5369. 23. Grossmann, T. N.; Seitz, O. J. Am. Chem. Soc. 2006, 128, 15596. 24. Pianowski, Z. L.; Winssinger, N. Chem. Commun. 2007, 3820. 25. Dose, C.; Seitz, O. Bioorg. Med. Chem. 2008, 16, 65. 26. Franzini, R. M.; Kool, E. T. ChemBioChem 2008, 9, 2981. 27. Franzini, R. M.; Kool, E. T. Org. Lett. 2008, 10, 2935. 28. Pianowski, Z.; Gorska, K.; Oswald, L.; Merten, C. A.; Winssinger, N. J. Am. Chem. Soc. 2009, 131, 6492. 29. Li, H.; Franzini, R. M.; Bruner, C.; Kool, E. T. ChemBioChem 2010, 11, 2132. 30. Prusty, D. K.; Herrmann, A. J. Am. Chem. Soc. 2010, 132, 12197. 31. Arian, D.; Kovbasyuk, L.; Mokhir, A. Inorg. Chem. 2011, 50, 12010. 32. Röthlingshöfer, M.; Gorska, K.; Winssinger, N. J. Am. Chem. Soc. 2011, 133, 18110. 33. Röthlingshöfer, M.; Gorska, K.; Winssinger, N. Org. Lett. 2011, 14, 482. 34. Prusty, D. K.; Kwak, M.; Wildeman, J.; Herrmann, A. Angew. Chem., Int. Ed. 2012, 51, 11894. 35. Maruyama, A.; Katoh, M.; Ishihara, T.; Akaike, T. Bioconjugate Chem. 1997, 8, 3. 36. Ferdous, A.; Watanabe, H.; Akaike, T.; Maruyama, A. Nucleic Acids Res. 1998, 26, 3949. 37. Maruyama, A.; Watanabe, H.; Ferdous, A.; Katoh, M.; Ishihara, T.; Akaike, T. Bioconjugate Chem. 1998, 9, 292. 38. Torigoe, H.; Ferdous, A.; Watanabe, H.; Akaike, T.; Maruyama, A. J. Biol. Chem. 1999, 274, 6161. 39. Ferdous, A.; Akaike, T.; Maruyama, A. Bioconjugate Chem. 2000, 11, 520. 40. Ferdous, A.; Akaike, T.; Maruyama, A. Biomacromolecules 2000, 1, 186. 41. Kim, W. J.; Akaike, T.; Maruyama, A. J. Am. Chem. Soc. 2002, 124, 12676. 42. Kim, W. J.; Sato, Y.; Akaike, T.; Maruyama, A. Nat. Mater. 2003, 2, 815. 43. Michaelis, J.; Maruyama, A.; Seitz, O. Chem. Commun. 2013, 49, 618. 44. Abe, H.; Wang, J.; Furukawa, K.; Oki, K.; Uda, M.; Tsuneda, S.; Ito, Y. Bioconjugate Chem. 2008, 19, 1219. 45. Furukawa, K.; Abe, H.; Hibino, K.; Sako, Y.; Tsuneda, S.; Ito, Y. Bioconjugate Chem. 2009, 20, 1026. 46. Furukawa, K.; Abe, H.; Tamura, Y.; Yoshimoto, R.; Yoshida, M.; Tsuneda, S.; Ito, Y. Angew. Chem., Int. Ed. 2011, 50, 12020. 47. Tamura, Y.; Furukawa, K.; Yoshimoto, R.; Kawai, Y.; Yoshida, M.; Tsuneda, S.; Ito, Y.; Abe, H. Bioorg. Med. Chem. Lett. 2012, 22, 7248. 48. Melo, J.; Gordon, D.; Cross, N.; Goldman, J. Blood 1993, 81, 158. 49. Dobson, M. G.; Galvin, P.; Barton, D. E. Expert Rev. Mol. Diagn. 2007, 7, 359.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Polycation-assisted DNA detection by reduction triggered fluorescence amplification probe Hisao Saneyoshi a,e, Naohiko Shimada b, Atsushi Maruyama b, Yoshihiro Ito a,e,⇑, Hiroshi Abe a,c,d,e,⇑ a
Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan Institute for Materials Chemistry and Engineering, Kyushu University, 744-CE11 Motooka, Nishi, Fukuoka 819-0395, Japan c Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan e Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1, Hirosawa, Wako-Shi, Saitama 351-0198, Japan b
a r t i c l e
i n f o
Article history: Received 7 June 2013 Revised 28 September 2013 Accepted 2 October 2013 Available online 11 October 2013
a b s t r a c t We have developed a fluorescence detection system for DNA, assisted by a comb-type cationic polymer (PLL-g-DX), for accelerating the reaction turnover. The combination of fluorogenic DNA probes with a comb-type cationic polymer has been demonstrated to be an effective means of signal amplification during the detection process. The method described herein represents a simple and enzyme-free detection. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: DNA Fluorescence detection Polymer Catalyst Reduction
Techniques aimed at the detection of nucleic acids have recently been the subject of considerable levels of attention because of their potential application in the early diagnosis for a variety of different diseases, including HIV, leukemia and tuberculosis.1–3 Hybridization-based analysis has been reported as an operationally simple method of detection and likened to a molecular beacon.4–9 Unfortunately, however, the amplification of the target DNA/RNA using this technique is less sensitive than other methods such as the polymerase chain reaction (PCR). Signal amplification has been used as a powerful solution to the sensitivity issues associated with the detection of nucleic acids, providing significant gains in sensitivity. The application of a chemical reaction catalyzed by the target nucleic acid is emerging as a powerful method to amplify signals.10–34 It is noteworthy that no enzymes are required for this reaction-based detection process, and this therefore represents a significant advantage from the perspective of point-of-care testing techniques in the field of biomedical research. The mechanism of the reaction-based detection process is shown in Figure 1. The first step involves the hybridization of the two reactive probes (Azm and TPP) with the target nucleic acid and their subsequent reaction to generate a fluorescence signal. The second step involves the participation of the target nucleic acid in the catalytic cycle during the detection process. During this process, strand exchange ⇑ Corresponding authors. Tel.: +81 (0)48 467 5749; fax: +81 (0)48 467 2827. E-mail address: [email protected] (H. Abe). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.005
occurs between the probes and the target strands, and this step is essential for catalytic turnover resulting signal amplification. Cationic comb-type copolymers (PLL-g-DX), which consist of a cationic poly(L-lysine) backbone and water-soluble dextran side chains, have been reported in the literature as good strand exchange promoters.35–42 Michaelis et al.43 recently applied PLLg-DX to a fluorophore transfer reaction and confirmed its overall utility based on the specific benefit of PLL-DX over spermine in terms of the catalytic turnover of an oligonucleotide-templated reaction. Furthermore, PLL-DX did not disturb the mismatch discrimination during the detection process. Herein, we report the development of a new system providing a high catalytic turnover number to amplify a fluorescence signal based on the combination of fluorogenic reactive probes with a combo-type cationic polymer, as shown in Figure 1. Our optimized system offered better results in terms of higher turnover number, shorter reaction time or lower detection limit compared with previous reports.43 We have recently reported the development and application of fluorogenic oligonucleotide probes consisting of oligonucleotides bearing fluorogenic-type azide methyl fluorescein (Azm) and triphenylphosphine (TPP) that could successful detect the target sequences without the requirement for any additional chemicals or enzymes. These so-called reduction triggered fluorescence (RETF) probes have been demonstrated to possess signal amplification ability.44–47 With the aim of making further improvements to the catalytic turnover, we recently shifted our
6852
H. Saneyoshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6851–6853
Figure 1. DNA-templated fluorogenic reaction assisted with PLL-g-DX.
attention towards polycation assisted DNA detection. The combination of fluorogenic probes with a polymer allowed us to gain the level of sensitivity required for detection by increasing the number of catalytic cycles. To apply the polymer to the DNA-templated fluorogenic reaction using our system, we initially tested the influence of cationic polymer on the bimolecular reaction in the absence of the target DNA strand. In general, the cationic polymer accommodates negatively charged strands on nucleic acid through a series of strongly interactions. It is noteworthy that 20 lM of the cationic polymer induced a bimolecular reaction (unspecific reaction). Our preliminary experiments revealed that 5 lM of polymer had no discernible impact on the reaction system and provided results similar to those of the control experiment involving no polymer at all as shown in Figure 3. Next, to determine the appropriate polymer concentration for DNA detection, fluorogenic sensing against 100 pM of the target DNA was performed. Fluorogenic sensing of a model DNA sequence (50 -TAAGCAGAGTTCAAAAGCCCTTCAGCG-30 ) was performed on the bcr/abl target gene originating from the human bcr/abl junction site, which is related to chronic myelogenous leukemia
Figure 3. Negative effect of polymer concentration against unspecific reaction. Conditions: Azm probe (100 nM), TPP probe (200 nM), PLL-g-DX (0–20 lM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 40 °C, 1 h. ⁄Yield was calculated from the fluorescence intensity of the Azm probe reduced by dithiothreitol (DTT).
A
B
Figure 4. Effect of temperature on the DNA detection. Conditions: (A) (no polymer); Azm probe (50 nM), TPP probe (50 nM), target DNA (10 pM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 25 °C, 40 °C, 55 °C 1 h. (B) (with polymer); Az probe (50 nM), TPP probe (50 nM), Target DNA (10 pM), PLL-g-DX (1 lM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 25 °C, 40 °C, 55 °C 1 h. ⁄Yield was calculated from the fluorescence intensity of the Azm probe reduced by dithiothreitol (DTT).
Figure 2. Chemical structure of reactive moiety in fluorogenic DNA probes and cationic-comb type polymer (PLL-g-DX, 30K90D poly-lysine 30 K dextran 90 wt %, amino group 0.78 mmol/mg).
(K562).48,49 The lengths of the Azm and the TPP probes were 8mer and 10-mer, respectively, as shown in Figure 2. The melting temperature of the probe was 46.7 °C, according to our previous study.45 The effective polymer concentration was found to be 1 lM when the two probes in the RETF system were both used at a concentration of 50 nM. The N/P ratio was estimated to be approximately 1 (data not shown). The effect of the temperature on the detection process was determined to be an important factor for the hybridization and the polymer assisted strand exchange reaction. Comparable experiments were performed to evaluate changes in the temperature and polymer effect, as shown in Figure 4. As a result, the polymer was found to work well at 40 °C with a target concentration of 10 pM. At a temperature of 55 °C, the results were similar to those obtained at 25 °C and showed the lowest background signal. Unfortunately, however, no effective signal was observed at
H. Saneyoshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6851–6853 Table 1 Enhanced turnover number in the presence of PLL-g-DX
No polymer With polymer
6853
References and notes
25 °C
40 °C
55 °C
— —
— 127.8
— 46.8
Conditions: left; Azm probe (50 nM), TPP probe (50 nM), Target DNA (10 pM), PLLg-DX (1 lM), 20 mM Tris–HCl (pH 7.2), 100 mM MgCl2, 40 °C, 1 h.
25 °C. In contrast, in the absence of the polymer under all of the conditions, no effective signal was observed, as shown in Figure 4. According to a report by Seitz and colleagues,43 the addition of PLL-g-DX to the reaction mixture was particularly effective for stabilizing the duplex compared with the system containing no PLL-gDX. It was therefore assumed that the duplex was too stable for the catalytic cycle at 25 °C. In contrast, temperatures of 40 and 55 °C were found to be satisfactory for the catalytic cycle to give multiple chemical reactions. We then proceeded to estimate the turnover number from the reaction yield. The maximum turnover number was estimated to be 127.8 at 40 °C, with the next highest turnover estimated to be 46.8 at 55 °C, as shown in Table 1. These data represented a remarkable improvement and a higher class of catalytic activity compared with the no polymer conditions and data that we had reported 53.6 against 50 pM target after 4 h previously.45 In addition, a reaction time of only 1 h was required to detect the target DNA at a concentration of 10 pM. These results indicated that the addition of polymer effectively enhanced the sensitivity of the promoter in the detection cycle. To obtain higher levels of sensitivity and sequence selectivity, the effects of several other factors on the system need to be considered, including the development of an RETF probe incorporating a modified nucleoside such as 2-thiothymidine, as well as the optimization of the pH and salt concentration. Work towards further refining the detection process is currently underway in our laboratory. In summary, we have developed a polycation assisted DNA detection system that effectively enhanced the reaction turnover to amplify the fluorescence signal from the target nucleic acid. It is envisaged that polycation-assisted detection will become an essential tool for nucleic acid detection. Acknowledgments H.A. and Y.I. were financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the New Energy, Industrial Technology Development Organization (NEDO) and Precursory Research for Embryonic Science and Technology (PREST). We are grateful for the support received from the Brain Science Institute (BSI) Research Resource Center for performing the mass spectral analyses. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.10. 005.
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