- Email: [email protected]
Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: Concept and biomedical applications
Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: Concept and biomedical applications Youngeun Choi, Gavin Metcalf, Mazen Haj Sleiman, Douglas Vair-Turnbull, Sylvain Ladame ⇑ Department of Bioengineering, Imperial College London, London SW7 2AZ, UK
a r t i c l e
i n f o
Article history: Received 21 March 2014 Revised 28 May 2014 Accepted 30 May 2014 Available online xxxx Keywords: Peptide Nucleic Acids Oligonucleotide templated reaction Biomarkers Cell-free nucleic acids Cancer
a b s t r a c t Sensing technologies based on Peptide Nucleic Acids (PNAs) and oligonucleotide-templated chemistry are perfectly suited for biomedical applications (e.g., diagnosis, prognosis and stratification of diseases) and could compete well with more traditional amplification technologies using expensive dual-labelled oligonucleotide probes. PNAs can be easily synthesised and functionalised, are more stable and are more responsive to point-mutations than their DNA counterpart. For these reasons, fluorogenic PNAs represent an interesting alternative to DNA-based molecular beacons for sensing applications in a cell-free environment, where cellular uptake is not required. Ó 2014 Published by Elsevier Ltd.
1. Introduction Two decades after the pioneering work of Kool,1 Letsinger2 and Orgel,3 the concept of oligonucleotide-templated reaction (OTR)4,5 has found valuable applications in programmed synthetic chemistry, imaging and also more recently in the engineering of new technologies for the diagnosis, stratification and management of diseases, most notably Cancer. Widespread in nature, the concept of OTR uses a DNA or RNA strand as a template to catalyse an unfavourable bimolecular chemical reaction by increasing the effective molarity of two monomers otherwise present in solution at a too low concentration. Although most common approaches for sensing DNA or RNA use as probes two DNA strands functionalised at their 50 or 30 ends, recent advances have replaced chemically unstable standard deoxyribo-oligonucleotides probes with more robust synthetic analogues. Peptide Nucleic Acids6 (PNAs) in particular have received a lot of attention due to their remarkable chemical and enzymatic stability. PNAs are synthetic mimics of DNA (or RNA) in whom the negatively charged ribose-phosphate backbone has been replaced by a charge-free polyamide scaffold (Scheme 1), thus allowing them to hybridise to either DNA or RNA in a sequence specific manner and with high affinity. They can be easily synthesised and functionalised on solid support. When compared to DNA
⇑ Corresponding author. Tel.: +44 02075945308. E-mail address: [email protected] (S. Ladame).
or RNA, PNAs also show greater ability to discriminate between targets with high sequence homology.7 Very poor solubility in aqueous solutions and poor cellular uptake remain however two key obstacles to a broader use of PNAs, in particular for applications in cellulo. Water-solubility can easily be achieved by either modification of the main PNA scaffold or conjugation to cationic (e.g., arginine, lysine) or anionic (glutamic acid, aspartic acid) amino acids, thus making (otherwise charge-free) PNAs suitable for OTRs. In some cases, such modifications (e.g., guanidinium-functionalised peptide nucleic acid or GPNA8) were also shown to improve cellular uptake, thus enabling the imaging of specific cellular RNAs in live human cells (e.g., HeLa and MCF7).9 However, it is fair to say that most common applications of PNA-based OTRs are carried out in vitro. The extreme stability of PNAs and their ease of functionalisation make them perfectly suited for programmed synthetic chemistry. Recent successful applications include the RNA-catalysed transfer of a biotin tag from one PNA to another via native chemical ligation10 or the DNA-templated synthesis of bioactive peptides via the same reaction.11 In this article however, we will be focusing on the most advanced applications of PNA-based OTRs in optical sensing of nucleic acids biomarkers (e.g., Single Nucleotide Polymorphism-SNP-detection or microRNA-miR-sensing. . .) in vitro. In such cases, the specific nucleic acid sequence of interest is typically used as the template that catalyses a reaction that can be easily monitored by fluorescence spectroscopy.12 Most representative examples include (i) the use of fluorescence energy transfer (FRET probes) where the DNA (or
http://dx.doi.org/10.1016/j.bmc.2014.05.071 0968-0896/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Choi, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.071
2
Y. Choi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Scheme 1. General structures of PNA, DNA and GPNA strands.
RNA)-templated fluorescent enhancement relies on a resonance energy transfer (RET) between a fluorophore and a quencher13–24 or (ii) fluorogenic reactions where the DNA-catalysed reaction between two non-fluorescent precursors leads to the formation of a fluorescent dye. In the following section we will mainly be focusing on the latter, using examples from our groups but also from others. 2. Fluorogenic OTRs for the optical sensing of nucleic acids in vitro A general sensing strategy (used by us and others) is illustrated in Figure 1. Briefly, two water-soluble Peptide Nucleic Acids (PNAs) are functionalised at their N- or C-terminus with non-fluorescent moieties. Upon simultaneous hybridisation of the synthetic probes to a complementary nucleic acid target, both probe-heads are brought in close enough proximity to react with each other, thus generating the fluorescent product (Fig. 1).25 In the absence of DNA (or RNA) template, the fluorogenic probes are present in solution at such low concentrations that their probability to react with each other is very low. This accounts for extremely low background fluorescence and higher signal-to-noise ratios when compared to more traditional approaches using dual-labelled molecular beacons and FRET-based detection. The approach also allows for quantitative sensing of specific nucleic acid targets. The intensity of the characteristic fluorescent signal emitted can indeed be directly (and quantitatively) linked to the amount of nucleic acid of interest present in the analyte. In case of a partial or incomplete hybridisation of (at least) one of the modified oligonucleotides (e.g., as a consequence of a single nucleotide polymorphism), a significant decrease in reaction efficiency (i.e., a weaker fluorescence) is observed.26,27
3. A toolbox of fluorogenic probes heads A limited range of chemical reactions have been reported that are fluorogenic (formation of a fluorescent dye from two non-fluorescent moieties) and are compatible with the concept of OTR (i.e., can be carried out in water under near physiological conditions of pH and salt concentration).12 Among them, reactions of cyanine dye formation have been developed in our group that show great efficacy and versatility. Trimethine cyanine dyes (C3) can indeed be synthesised via a reaction of aldolisation–elimination between a 2-methylene-indoline and a Fisher’s base aldehyde (Fig. 2).25 Interestingly, this system is very versatile and the maximum fluorescence excitation (kexc) and emission (kem) wavelengths of the reporter dye can be finely tuned by modifying the structure of one or both heterocyclic precursors, as shown in Figure 2 (first two entries). In an effort to shift the fluorescence excitation and emission wavelengths toward the red/infrared part of the spectrum, we have expanded our toolbox of fluorogenic probes to pentamethine cyanine dyes (C5)28 and more recently to squaraine dyes (SQ).29 Pentamethine cyanine dyes are synthesised via the same aldolisation–elimination reaction (although using an ‘extended’ Fisher’s base aldehyde). Squaraine dyes are obtained via carbodiimide-mediated in situ activation of a non-fluorescent semi-squaraine cyanine dye. More recent work from our group has led to the development of additional probe heads covering an even broader spectrum towards the blue–green (400–500 nm) and will be reported in due course. Optical sensing/monitoring of nucleic acids using fluorogenic OTRs goes well beyond the above-mentioned examples from our group and focusing on oligonucleotide-templated cyanine dye syntheses. Many groups before and after us have reported highly successful technologies that used fluorogenic PNAs. An exhaustive list of all the fluorogenic reactions successfully applied for OT sensing of nucleic acids can be found in three independent reviews published recently by the groups of Winssinger4 and Abe12 and from our own group.5 Herein, we would just like to emphasise that the most commonly used reaction is without any doubt the Staudinger reaction using phosphines to release the optical properties of masked fluorescent (e.g., coumarin27 or rhodamin30) or luminescent (lanthanide complex31) probes. A more recent application (although using DNA probes) is based on fluorogenic tetrazine ligation where methyl-cyclopropene groups react with tetrazine to release the fluorescence of an otherwise quenched fluorescein (Fig. 3).32 4. Cell-free nucleic acid biomarkers: the future of PNA-based OTR sensing?
Figure 1. General sensing strategy based on the concept of oligonucleotidetemplated reaction and using two non-fluorescent PNAs to detect the presence of a nucleic acid target.
Considering the poor cellular uptake of PNAs (unless modified), the most promising biomedical applications for PNA probes may well be the detection of nucleic acid biomarkers in a cell free
Please cite this article in press as: Choi, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.071
Y. Choi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Figure 2. Selected examples of fluorogenic reactions of cyanine dye formation compatible with the principle of OTR.
Figure 3. Selected examples of fluorogenic reactions suitable for OT sensing.
environment. The search for non-invasive tools for the diagnosis, stratification and management of cancer has long been a goal of cancer research that has led to great interest in the field of circulating nucleic acids (cfNAs) in biological fluids such as plasma and serum.33 The detection of cfNAs in clinical samples, including blood, saliva, and urine, could serve as a potential ‘liquid biopsy’ achieved by minimally or non-invasive techniques. Such technique would represent a valuable alternative to highly invasive tumour tissue biopsies. Additionally, levels of cfNAs can reflect tumour development, with increasing concentrations being observed in patients upon cancer growth and maturation.34 The precise
physiological events that result in heightened cfNAs during the development and progression of cancer are still not fully understood. However, collective research in the field suggests that circulating DNA analyses allow the detection of tumour-related genetic and epigenetic alterations, including methylation and mutations.35 Developing profiling methods that are efficient and quantitative has proven to be challenging for most circulating nucleic acids because of their small size (e.g., miRNA36), small amounts and the sequence similarity among family members. Such profiling therefore requires reliable, highly sensitive and quantitative procedures that can be easily multiplexed and require minimal quantity
Please cite this article in press as: Choi, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.071
4
Y. Choi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
of patient sample. New sensing technologies based on PNAs and OTR could help address this urgent need and provide an alternative to the current high-throughput profiling techniques that generally require a relatively large volume of serum and are therefore commonly used in initial screening processes only. 5. Conclusion To summarise, high reaction efficiency leading to the formation of a reaction product with high fluorescence quantum yield accounts for extremely sensitive sensors. The extremely responsive nature of PNAs to point mutations also guarantees greater sensing specificity than traditional DNA-based probes and make them perfectly suited for detection of nucleic acids in vitro and in a cell-free environment. Finally, when compared to molecular beacons/Taqman probes, the nature of the sensing process (formation of a fluorescent product from non-fluorescent precursors) offers the advantage of a greater signal-to-noise ratio and a significantly lower cost. However, as recently highlighted in an excellent review by Seitz and co-workers37 OTRs are traditionally characterised by limited (up to 1000) catalytic turnovers because of template/product inhibition. New strategies to overcome these intrinsic limitations are necessary to further establish OTR-based sensing as a viable, more reliable and more affordable alternative to commonly used FRET-based molecular beacons. Acknowledgements This research was supported by the Leverhulme Trust (Grant RPG-2012-603), a studentship from Cancer Research UK and by the EU FP7 (Career Integration Grant CIG 293981). References and notes 1. Wang, S. H.; Kool, E. T. Nucleic Acids Res. 1994, 22, 2326.
2. Gryaznov, S. M.; Schultz, R.; Chaturvedi, S. K.; Letsinger, R. L. Nucleic Acids Res. 1994, 22, 2366. 3. Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 7963. 4. Gorska, K.; Winssinger, N. Angew. Chem., Int. Ed. 2013, 52, 6820. 5. Percivalle, C.; Bartolo, J. F.; Ladame, S. Org. Biomol. Chem. 2013, 11, 16. 6. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365, 566. 7. Igloi, G. L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8562. 8. Dragulescu-Andrasi, A.; Rapireddy, S.; He, G.; Bhattacharya, B.; Hyldig-Nielsen, J. J.; Zon, G.; Ly, D. H. J. Am. Chem. Soc. 2006, 128, 16104. 9. Pianowski, Z.; Gorska, K.; Oswald, L.; Merten, C. A.; Winssinger, N. J. Am. Chem. Soc. 2009, 131, 6492. 10. Grossmann, T. N.; Roglin, L.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 7119. 11. Erben, A.; Grossmann, T. N.; Seitz, O. Angew. Chem., Int. Ed. 2011, 50, 2828. 12. For a recent review see Shibata, A.; Abe, H.; Ito, Y. Molecules 2012, 17, 2446. 13. Kleinbaum, D. J.; Kool, E. T. Chem. Commun. 2010, 8154. 14. Roloff, A.; Seitz, O. Bioorg. Med. Chem. 2013, 21, 3458. 15. Roloff, A.; Seitz, O. Chem. Sci. 2013, 4, 432. 16. Chen, X. H.; Roloff, A.; Seitz, O. Angew. Chem., Int. Ed. 2012, 51, 4479. 17. Bethge, L.; Jarikote, D. V.; Seitz, O. Bioorg. Med. Chem. 2008, 16, 114. 18. Cai, J.; Li, X.; Taylor, J. S. Org. Lett. 2005, 7, 751. 19. Cai, J.; Li, X.; Yue, X.; Taylor, J. S. J. Am. Chem. Soc. 2004, 126, 16324. 20. Ma, Z.; Taylor, J. S. Bioconjugate Chem. 2003, 14, 679. 21. Sadhu, K. K.; Winssinger, N. Chem. Eur. J. 2013, 19, 8182. 22. Sadhu, K. K.; Eierhoff, T.; Römer, W.; Winssinger, N. J. Am. Chem. Soc. 2012, 134, 20013. 23. Gorska, K.; Manicardi, A.; Barluenga, S.; Winssinger, N. Chem. Commun. 2011, 4364. 24. Gorska, K.; Keklikoglou, I.; Tschulena, U.; Winssinger, N. Chem. Sci. 2011, 2, 1969. 25. Meguellati, K.; Koripelly, G.; Ladame, S. Angew. Chem., Int. Ed. 2010, 49, 2738. 26. Meguellati, K.; Koripelly, G.; Ladame, S. J. Anal. Mol. Tech. 2013, 1, 5. 27. Pianowski, Z. L.; Winssinger, N. Chem. Commun. 2007, 3820. 28. Koripelly, G.; Meguellati, K.; Ladame, S. Bioconjugate Chem. 2010, 21, 2103. 29. Sleiman, M. H.; Ladame, S. Chem. Commun. 2014, 5288. 30. Abe, H.; Wang, J.; Furukawa, K.; Oki, K.; Uda, M.; Tsuneda, S.; Ito, Y. Bioconjugate Chem. 2008, 19, 1219. 31. Saneyoshi, H.; Ito, Y.; Abe, H. J. Am. Chem. Soc. 2013, 135, 13632. 32. Seckute, J.; Yang, J.; Davaraj, N. K. Nucleic Acids Res. 2013, 41, e148. 33. Gormally, E.; Caboux, E.; Vineis, P.; Hainaut, P. Mutat. Res. 2007, 635, 105. 34. Chan, K. C.; Lo, Y. M. Br. J. Cancer 2007, 96, 681. 35. Schwarzenbach, H.; Hoon, D. S.; Pantel, K. Nat. Rev. 2011, 11, 237. 36. Jeffrey, S. S. Nat. Biotechnol. 2008, 26, 400. 37. Michaelis, J.; Roloff, A.; Seitz, O. Org. Biomol. Chem. 2014, 12, 2821.
Please cite this article in press as: Choi, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.071
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: Concept and biomedical applications Youngeun Choi, Gavin Metcalf, Mazen Haj Sleiman, Douglas Vair-Turnbull, Sylvain Ladame ⇑ Department of Bioengineering, Imperial College London, London SW7 2AZ, UK
a r t i c l e
i n f o
Article history: Received 21 March 2014 Revised 28 May 2014 Accepted 30 May 2014 Available online xxxx Keywords: Peptide Nucleic Acids Oligonucleotide templated reaction Biomarkers Cell-free nucleic acids Cancer
a b s t r a c t Sensing technologies based on Peptide Nucleic Acids (PNAs) and oligonucleotide-templated chemistry are perfectly suited for biomedical applications (e.g., diagnosis, prognosis and stratification of diseases) and could compete well with more traditional amplification technologies using expensive dual-labelled oligonucleotide probes. PNAs can be easily synthesised and functionalised, are more stable and are more responsive to point-mutations than their DNA counterpart. For these reasons, fluorogenic PNAs represent an interesting alternative to DNA-based molecular beacons for sensing applications in a cell-free environment, where cellular uptake is not required. Ó 2014 Published by Elsevier Ltd.
1. Introduction Two decades after the pioneering work of Kool,1 Letsinger2 and Orgel,3 the concept of oligonucleotide-templated reaction (OTR)4,5 has found valuable applications in programmed synthetic chemistry, imaging and also more recently in the engineering of new technologies for the diagnosis, stratification and management of diseases, most notably Cancer. Widespread in nature, the concept of OTR uses a DNA or RNA strand as a template to catalyse an unfavourable bimolecular chemical reaction by increasing the effective molarity of two monomers otherwise present in solution at a too low concentration. Although most common approaches for sensing DNA or RNA use as probes two DNA strands functionalised at their 50 or 30 ends, recent advances have replaced chemically unstable standard deoxyribo-oligonucleotides probes with more robust synthetic analogues. Peptide Nucleic Acids6 (PNAs) in particular have received a lot of attention due to their remarkable chemical and enzymatic stability. PNAs are synthetic mimics of DNA (or RNA) in whom the negatively charged ribose-phosphate backbone has been replaced by a charge-free polyamide scaffold (Scheme 1), thus allowing them to hybridise to either DNA or RNA in a sequence specific manner and with high affinity. They can be easily synthesised and functionalised on solid support. When compared to DNA
⇑ Corresponding author. Tel.: +44 02075945308. E-mail address: [email protected] (S. Ladame).
or RNA, PNAs also show greater ability to discriminate between targets with high sequence homology.7 Very poor solubility in aqueous solutions and poor cellular uptake remain however two key obstacles to a broader use of PNAs, in particular for applications in cellulo. Water-solubility can easily be achieved by either modification of the main PNA scaffold or conjugation to cationic (e.g., arginine, lysine) or anionic (glutamic acid, aspartic acid) amino acids, thus making (otherwise charge-free) PNAs suitable for OTRs. In some cases, such modifications (e.g., guanidinium-functionalised peptide nucleic acid or GPNA8) were also shown to improve cellular uptake, thus enabling the imaging of specific cellular RNAs in live human cells (e.g., HeLa and MCF7).9 However, it is fair to say that most common applications of PNA-based OTRs are carried out in vitro. The extreme stability of PNAs and their ease of functionalisation make them perfectly suited for programmed synthetic chemistry. Recent successful applications include the RNA-catalysed transfer of a biotin tag from one PNA to another via native chemical ligation10 or the DNA-templated synthesis of bioactive peptides via the same reaction.11 In this article however, we will be focusing on the most advanced applications of PNA-based OTRs in optical sensing of nucleic acids biomarkers (e.g., Single Nucleotide Polymorphism-SNP-detection or microRNA-miR-sensing. . .) in vitro. In such cases, the specific nucleic acid sequence of interest is typically used as the template that catalyses a reaction that can be easily monitored by fluorescence spectroscopy.12 Most representative examples include (i) the use of fluorescence energy transfer (FRET probes) where the DNA (or
http://dx.doi.org/10.1016/j.bmc.2014.05.071 0968-0896/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Choi, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.071
2
Y. Choi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Scheme 1. General structures of PNA, DNA and GPNA strands.
RNA)-templated fluorescent enhancement relies on a resonance energy transfer (RET) between a fluorophore and a quencher13–24 or (ii) fluorogenic reactions where the DNA-catalysed reaction between two non-fluorescent precursors leads to the formation of a fluorescent dye. In the following section we will mainly be focusing on the latter, using examples from our groups but also from others. 2. Fluorogenic OTRs for the optical sensing of nucleic acids in vitro A general sensing strategy (used by us and others) is illustrated in Figure 1. Briefly, two water-soluble Peptide Nucleic Acids (PNAs) are functionalised at their N- or C-terminus with non-fluorescent moieties. Upon simultaneous hybridisation of the synthetic probes to a complementary nucleic acid target, both probe-heads are brought in close enough proximity to react with each other, thus generating the fluorescent product (Fig. 1).25 In the absence of DNA (or RNA) template, the fluorogenic probes are present in solution at such low concentrations that their probability to react with each other is very low. This accounts for extremely low background fluorescence and higher signal-to-noise ratios when compared to more traditional approaches using dual-labelled molecular beacons and FRET-based detection. The approach also allows for quantitative sensing of specific nucleic acid targets. The intensity of the characteristic fluorescent signal emitted can indeed be directly (and quantitatively) linked to the amount of nucleic acid of interest present in the analyte. In case of a partial or incomplete hybridisation of (at least) one of the modified oligonucleotides (e.g., as a consequence of a single nucleotide polymorphism), a significant decrease in reaction efficiency (i.e., a weaker fluorescence) is observed.26,27
3. A toolbox of fluorogenic probes heads A limited range of chemical reactions have been reported that are fluorogenic (formation of a fluorescent dye from two non-fluorescent moieties) and are compatible with the concept of OTR (i.e., can be carried out in water under near physiological conditions of pH and salt concentration).12 Among them, reactions of cyanine dye formation have been developed in our group that show great efficacy and versatility. Trimethine cyanine dyes (C3) can indeed be synthesised via a reaction of aldolisation–elimination between a 2-methylene-indoline and a Fisher’s base aldehyde (Fig. 2).25 Interestingly, this system is very versatile and the maximum fluorescence excitation (kexc) and emission (kem) wavelengths of the reporter dye can be finely tuned by modifying the structure of one or both heterocyclic precursors, as shown in Figure 2 (first two entries). In an effort to shift the fluorescence excitation and emission wavelengths toward the red/infrared part of the spectrum, we have expanded our toolbox of fluorogenic probes to pentamethine cyanine dyes (C5)28 and more recently to squaraine dyes (SQ).29 Pentamethine cyanine dyes are synthesised via the same aldolisation–elimination reaction (although using an ‘extended’ Fisher’s base aldehyde). Squaraine dyes are obtained via carbodiimide-mediated in situ activation of a non-fluorescent semi-squaraine cyanine dye. More recent work from our group has led to the development of additional probe heads covering an even broader spectrum towards the blue–green (400–500 nm) and will be reported in due course. Optical sensing/monitoring of nucleic acids using fluorogenic OTRs goes well beyond the above-mentioned examples from our group and focusing on oligonucleotide-templated cyanine dye syntheses. Many groups before and after us have reported highly successful technologies that used fluorogenic PNAs. An exhaustive list of all the fluorogenic reactions successfully applied for OT sensing of nucleic acids can be found in three independent reviews published recently by the groups of Winssinger4 and Abe12 and from our own group.5 Herein, we would just like to emphasise that the most commonly used reaction is without any doubt the Staudinger reaction using phosphines to release the optical properties of masked fluorescent (e.g., coumarin27 or rhodamin30) or luminescent (lanthanide complex31) probes. A more recent application (although using DNA probes) is based on fluorogenic tetrazine ligation where methyl-cyclopropene groups react with tetrazine to release the fluorescence of an otherwise quenched fluorescein (Fig. 3).32 4. Cell-free nucleic acid biomarkers: the future of PNA-based OTR sensing?
Figure 1. General sensing strategy based on the concept of oligonucleotidetemplated reaction and using two non-fluorescent PNAs to detect the presence of a nucleic acid target.
Considering the poor cellular uptake of PNAs (unless modified), the most promising biomedical applications for PNA probes may well be the detection of nucleic acid biomarkers in a cell free
Please cite this article in press as: Choi, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.071
Y. Choi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Figure 2. Selected examples of fluorogenic reactions of cyanine dye formation compatible with the principle of OTR.
Figure 3. Selected examples of fluorogenic reactions suitable for OT sensing.
environment. The search for non-invasive tools for the diagnosis, stratification and management of cancer has long been a goal of cancer research that has led to great interest in the field of circulating nucleic acids (cfNAs) in biological fluids such as plasma and serum.33 The detection of cfNAs in clinical samples, including blood, saliva, and urine, could serve as a potential ‘liquid biopsy’ achieved by minimally or non-invasive techniques. Such technique would represent a valuable alternative to highly invasive tumour tissue biopsies. Additionally, levels of cfNAs can reflect tumour development, with increasing concentrations being observed in patients upon cancer growth and maturation.34 The precise
physiological events that result in heightened cfNAs during the development and progression of cancer are still not fully understood. However, collective research in the field suggests that circulating DNA analyses allow the detection of tumour-related genetic and epigenetic alterations, including methylation and mutations.35 Developing profiling methods that are efficient and quantitative has proven to be challenging for most circulating nucleic acids because of their small size (e.g., miRNA36), small amounts and the sequence similarity among family members. Such profiling therefore requires reliable, highly sensitive and quantitative procedures that can be easily multiplexed and require minimal quantity
Please cite this article in press as: Choi, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.071
4
Y. Choi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
of patient sample. New sensing technologies based on PNAs and OTR could help address this urgent need and provide an alternative to the current high-throughput profiling techniques that generally require a relatively large volume of serum and are therefore commonly used in initial screening processes only. 5. Conclusion To summarise, high reaction efficiency leading to the formation of a reaction product with high fluorescence quantum yield accounts for extremely sensitive sensors. The extremely responsive nature of PNAs to point mutations also guarantees greater sensing specificity than traditional DNA-based probes and make them perfectly suited for detection of nucleic acids in vitro and in a cell-free environment. Finally, when compared to molecular beacons/Taqman probes, the nature of the sensing process (formation of a fluorescent product from non-fluorescent precursors) offers the advantage of a greater signal-to-noise ratio and a significantly lower cost. However, as recently highlighted in an excellent review by Seitz and co-workers37 OTRs are traditionally characterised by limited (up to 1000) catalytic turnovers because of template/product inhibition. New strategies to overcome these intrinsic limitations are necessary to further establish OTR-based sensing as a viable, more reliable and more affordable alternative to commonly used FRET-based molecular beacons. Acknowledgements This research was supported by the Leverhulme Trust (Grant RPG-2012-603), a studentship from Cancer Research UK and by the EU FP7 (Career Integration Grant CIG 293981). References and notes 1. Wang, S. H.; Kool, E. T. Nucleic Acids Res. 1994, 22, 2326.
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