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Nucleic acid aptamers
Methods 97 (2016) 1–2
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Editorial
Nucleic acid aptamers
Nucleic acid aptamers are synthetic oligonucleotides identified from complex randomly synthesized pools containing up to 1015 different sequences [1]. The procedure first described by Tuerk and Gold [2] and named SELEX (for Systematic Evolution of Ligands by EXponantial enrichment) enables the selection of aptamers against a wide range of targets: low molecular weight molecules, peptides, nucleic acids, intact viruses and live cells (for reviews see [3–5]). The binding properties of an aptamer to its cognate molecule, characterized by a strong affinity and a high specificity, are related to its three-dimensional shape resulting from intramolecular folding of the oligonucleotide, dictated by the primary sequence. Watson Crick base pairing, G-quartet formation among other nucleotide-nucleotide interactions generate the scaffold that displays key functional groups in an optimal position for interacting with the target (hydrogen bonds, pi–pi and electrostatic interactions). The potential of aptamers for diverse applications comprising therapeutic, diagnostic and analytical purposes is nowadays widely recognized. From the early days, 25 years ago, new methodological developments have been described for the selection, the identification, the optimization and the characterization of aptamers that can be subsequently converted into biotechnological tools or integrated to various devices. In this issue of ‘‘Methods” a series of articles illustrate various aspects of the aptamer field. From the pioneering work [1,2] developments have been suggested for the selection of aptamers, making the procedure faster and cheaper (capillary electrophoresis SELEX, microfluidics functional selection, . . .). One of the recent improvement deals with the analysis of the sequence pool. Taking advantage of High Throughput Sequencing it is now possible to monitor the population at successive rounds. The power of HTS for aptamer identification has for long been limited by the lack of efficient bioinformatics tools. In this issue Thiel and Giangrande describe how the Galaxy project initially developed for genome analysis can be adapted to aptamer HTS data [6]. The use of unnatural nucleobases is also increasingly used to identify aptamers with new properties. Azéma and Chaou describe the selection of aptamers with improved hydrophobicity by incorporating 5-(octa1,7-diynyl)-20 -deoxyuridine during the selection of DNA aptamers [7]. This allowed selecting an adenine-aptamer in the presence of 25% of methanol and provides preliminary insight on the adaptability of aptamers in organic solvents. Once identified aptamers should be characterized with respect to their binding properties. Numerous methods were used: affinity chromatography, filter retention, fluorescence emission, etc. In this issue O’Sullivan and co-workers describe results obtained by http://dx.doi.org/10.1016/j.ymeth.2016.02.015 1046-2023/Ó 2016 Published by Elsevier Inc.
Surface Plasmon Resonance imaging (SPRi) for characterizing aptamers raised against beta-conglutin. SPRi is a real time label-free method allowing the simultaneous analysis of several samples [8]. However it requires the immobilisation of aptamers (in this case thiol-modified oligonucleotides). In contrast MicroScale Thermophoresis (MST) allows monitoring aptamer–target complexes in solution. This technology based on the differential movement of free and bound molecules in a temperature gradient requires a small amount of ligands but one of the component in the complex should be fluorescently labeled. Entzian and Schubert provide a protocol for the determination of ATP aptamer binding parameters by MST [9]. Membrane biomarkers are attractive targets as specific aptamers can then be used for cell-labeling, cell-sorting or for the delivery of various cargoes. The measurement of binding affinity in this case cannot be reliably achieved with purified membrane proteins as the proper functional protein structure generally requires its membrane environment. Quang et al. describe an automated procedure for the determination of the equilibrium dissociation constant and the number of binding sites on a 32P radio-labeled aptamer on a target cell [10]. RNA-based regulatory elements, named riboswitches, have been discovered in all three kingdoms of life. Riboswitches conditionally control gene expression through internal or external stimuli: binding of a small molecule to an RNA motif triggers its conformational change that translates into differential expression. Since aptamer-target complexes formation very generally involves structural re-arrangement, aptamers are functionally equivalent to riboswitches and can be engineered for artificially controlling genetic elements. However, it is still difficult to predict in silico how to transmit an aptamer conformational change to an expression platform. Schneider and Suess developed an in vivo fluorescence screen in yeast for the identification of novel riboswitches from pre-selected RNA-aptamer libraries [11]. This allowed the identification of a neomycin-sensitive riboswitch that can increase up to 8-fold the production of Green Fluorescent Protein upon ligand addition. Identification of aptamers for the detection of pathogens or markers of biomedical interest met a tremendous interest over the last few years. Hamula et al. selected aptamers against live M-type 11 of Streptococcus pyogenes [12]. Serotyping these bacteria is currently laborious. Using counter-selection steps and screening against other bacteria, several aptamers were identified that bind preferentially to the M-type 11 of S. pyogenes with an equilibrium dissociation constant Kd in the nanomolar range i.e. with affinity similar to those of monoclonal antibodies. Lamberti et al. report the selection of RNA aptamers directed against the cancer Antigen
2
Editorial / Methods 97 (2016) 1–2
125 biomarker that is the most widely used marker for ovarian cancer [13]. One of the isolated sequences that exhibits a Kd value in the nanomolar range, might potentially lead to the development of a diagnostic tool. The binding properties of aptamers as well as the versatility of their synthesis and ease for modification make them exquisite candidates for the development of biosensors. Goux et al. describe a fluorescence anisotropy assay related to the structure-switching concept [14]. Fluorescently labelled peptide nucleic acid (PNA) is used as an alternative probe to conventional dye-tagged DNA to form a hybrid duplex with the ATP aptamer. The target-induced release of the PNA probe leads to a signal decrease that provides this ATP sensor with a detection limit of 3 lM. Aptamer–polyelectrolyte materials can be engineered for biosensing and delivery. Mastronardi et al. reviewed the key steps for preparing aptamerembedded polyelectrolyte multilayer films and microcapsules as well as the characterization methods of target binding functionality and responsiveness of these smart material systems [15]. Zhou et al. reported the design of a transmembrane protein CD63 aptamer-based electrochemical biosensor for the detection of exosomes that may constitute tumor biomarkers [16]. The structure-switching aptasensor can detect 1 106 particles/mL of exosomes, which represents 100-fold decrease in the limit of detection compared to commercial immunoassays. Several aptamers were recently selected against cell surface biomarkers that are overexpressed at the surface of cancer cells. These aptamers can be used for developing new tools for cancer diagnostics. Dickey and Giangrande discussed recent progresses about the isolation and detection of circulating tumor cells (CTCs) by aptamers [17]. The detection of CTCs remains a critical challenge and the review presents different methods for improving the detection sensitivity and to deal with the heterogeneity of tumor cells. With the same goal i.e. increased detection sensitivity of cancer cells Civit et al. developed caged aptamers against
lymphoma cancer cells, using an apta-PCR assay [18]. Caged aptamers bear photo-labile groups that enable to control their recognition properties using light. The reported method allows achieving the detection of as few as 77 cells. The articles assembled in this issue provide fascinating examples of the rapidly expanding field of nucleic acid aptamers that nowadays rival antibodies for many applications. References [1] A.D. Ellington, J.W. Szostak, Nature 346 (1990) 818–822. [2] C. Tuerk, L. Gold, Science 249 (1990) 505–510. [3] M. Mascini, I. Palchetti, S. Tombelli, Angew. Chem. Int. Ed. Engl. 51 (2012) 1316–1332. [4] A. Cibiel, C. Pestourie, F. Ducongé, Biochimie 94 (2012) 1595–1606. [5] E. Dausse, S. Da Rocha Gomes, J.J. Toulmé, Curr. Opin. Pharmacol. 9 (2009) 602–607. [6] W. Thiel, P. Giangrande, Methods 97 (2016) 3–10. [7] T. Chaou, B. Vialet, L. Azéma, Methods 97 (2016) 11–19. [8] M. Jauset Rubio, M. Svoboda, T. Mairal, C. O’Sullivan, Methods 97 (2016) 20–26. [9] C. Entzian, T. Schubert, Methods 97 (2016) 27–34. [10] N. Nguyen Quang, C. Pestourie, A. Cibiel, F. Ducongé, Methods 97 (2016) 35–43. [11] C. Schneider, B. Suess, Methods 97 (2016) 44–50. [12] C.L.A. Hamula, H. Peng, Z. Wang, G.T. Tyrell, X.-F. Li, X.C. Le, Methods 97 (2016) 51–57. [13] I. Lamberti, S. Scaran, C.L. Esposito, A. Antoccia, G. Antonini, C. Tanzarella, V. De Francicis, M. Minunni, Methods 97 (2016) 58–68. [14] E. Goux, Q. Lespinasse, V. Guieu, S. Perrier, C. Ravelet, E. Fiore, E. Peyrin, Methods 97 (2016) 69–74. [15] E. Mastronardi, P.K. Tsae, X. Zhang, A. Pach, Y. Sultan, M. DeRosa, Methods 97 (2016) 75–87. [16] Q. Zhou, A. Rahumain, K. Son, D.S. Shin, T. Patel, A. Revzin, Methods 97 (2016) 88–93. [17] D.D. Dickey, P. Giangrande, Methods 97 (2016) 94–103. [18] L. Civit, A. Pinto, A. Rodrigues-Correia, A. Heckel, C. O’Sullivan, G. Mayer, Methods 97 (2016) 104–109.
Jean-Jacques Toulmé Eric Peyrin Frédéric Ducongé
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Editorial
Nucleic acid aptamers
Nucleic acid aptamers are synthetic oligonucleotides identified from complex randomly synthesized pools containing up to 1015 different sequences [1]. The procedure first described by Tuerk and Gold [2] and named SELEX (for Systematic Evolution of Ligands by EXponantial enrichment) enables the selection of aptamers against a wide range of targets: low molecular weight molecules, peptides, nucleic acids, intact viruses and live cells (for reviews see [3–5]). The binding properties of an aptamer to its cognate molecule, characterized by a strong affinity and a high specificity, are related to its three-dimensional shape resulting from intramolecular folding of the oligonucleotide, dictated by the primary sequence. Watson Crick base pairing, G-quartet formation among other nucleotide-nucleotide interactions generate the scaffold that displays key functional groups in an optimal position for interacting with the target (hydrogen bonds, pi–pi and electrostatic interactions). The potential of aptamers for diverse applications comprising therapeutic, diagnostic and analytical purposes is nowadays widely recognized. From the early days, 25 years ago, new methodological developments have been described for the selection, the identification, the optimization and the characterization of aptamers that can be subsequently converted into biotechnological tools or integrated to various devices. In this issue of ‘‘Methods” a series of articles illustrate various aspects of the aptamer field. From the pioneering work [1,2] developments have been suggested for the selection of aptamers, making the procedure faster and cheaper (capillary electrophoresis SELEX, microfluidics functional selection, . . .). One of the recent improvement deals with the analysis of the sequence pool. Taking advantage of High Throughput Sequencing it is now possible to monitor the population at successive rounds. The power of HTS for aptamer identification has for long been limited by the lack of efficient bioinformatics tools. In this issue Thiel and Giangrande describe how the Galaxy project initially developed for genome analysis can be adapted to aptamer HTS data [6]. The use of unnatural nucleobases is also increasingly used to identify aptamers with new properties. Azéma and Chaou describe the selection of aptamers with improved hydrophobicity by incorporating 5-(octa1,7-diynyl)-20 -deoxyuridine during the selection of DNA aptamers [7]. This allowed selecting an adenine-aptamer in the presence of 25% of methanol and provides preliminary insight on the adaptability of aptamers in organic solvents. Once identified aptamers should be characterized with respect to their binding properties. Numerous methods were used: affinity chromatography, filter retention, fluorescence emission, etc. In this issue O’Sullivan and co-workers describe results obtained by http://dx.doi.org/10.1016/j.ymeth.2016.02.015 1046-2023/Ó 2016 Published by Elsevier Inc.
Surface Plasmon Resonance imaging (SPRi) for characterizing aptamers raised against beta-conglutin. SPRi is a real time label-free method allowing the simultaneous analysis of several samples [8]. However it requires the immobilisation of aptamers (in this case thiol-modified oligonucleotides). In contrast MicroScale Thermophoresis (MST) allows monitoring aptamer–target complexes in solution. This technology based on the differential movement of free and bound molecules in a temperature gradient requires a small amount of ligands but one of the component in the complex should be fluorescently labeled. Entzian and Schubert provide a protocol for the determination of ATP aptamer binding parameters by MST [9]. Membrane biomarkers are attractive targets as specific aptamers can then be used for cell-labeling, cell-sorting or for the delivery of various cargoes. The measurement of binding affinity in this case cannot be reliably achieved with purified membrane proteins as the proper functional protein structure generally requires its membrane environment. Quang et al. describe an automated procedure for the determination of the equilibrium dissociation constant and the number of binding sites on a 32P radio-labeled aptamer on a target cell [10]. RNA-based regulatory elements, named riboswitches, have been discovered in all three kingdoms of life. Riboswitches conditionally control gene expression through internal or external stimuli: binding of a small molecule to an RNA motif triggers its conformational change that translates into differential expression. Since aptamer-target complexes formation very generally involves structural re-arrangement, aptamers are functionally equivalent to riboswitches and can be engineered for artificially controlling genetic elements. However, it is still difficult to predict in silico how to transmit an aptamer conformational change to an expression platform. Schneider and Suess developed an in vivo fluorescence screen in yeast for the identification of novel riboswitches from pre-selected RNA-aptamer libraries [11]. This allowed the identification of a neomycin-sensitive riboswitch that can increase up to 8-fold the production of Green Fluorescent Protein upon ligand addition. Identification of aptamers for the detection of pathogens or markers of biomedical interest met a tremendous interest over the last few years. Hamula et al. selected aptamers against live M-type 11 of Streptococcus pyogenes [12]. Serotyping these bacteria is currently laborious. Using counter-selection steps and screening against other bacteria, several aptamers were identified that bind preferentially to the M-type 11 of S. pyogenes with an equilibrium dissociation constant Kd in the nanomolar range i.e. with affinity similar to those of monoclonal antibodies. Lamberti et al. report the selection of RNA aptamers directed against the cancer Antigen
2
Editorial / Methods 97 (2016) 1–2
125 biomarker that is the most widely used marker for ovarian cancer [13]. One of the isolated sequences that exhibits a Kd value in the nanomolar range, might potentially lead to the development of a diagnostic tool. The binding properties of aptamers as well as the versatility of their synthesis and ease for modification make them exquisite candidates for the development of biosensors. Goux et al. describe a fluorescence anisotropy assay related to the structure-switching concept [14]. Fluorescently labelled peptide nucleic acid (PNA) is used as an alternative probe to conventional dye-tagged DNA to form a hybrid duplex with the ATP aptamer. The target-induced release of the PNA probe leads to a signal decrease that provides this ATP sensor with a detection limit of 3 lM. Aptamer–polyelectrolyte materials can be engineered for biosensing and delivery. Mastronardi et al. reviewed the key steps for preparing aptamerembedded polyelectrolyte multilayer films and microcapsules as well as the characterization methods of target binding functionality and responsiveness of these smart material systems [15]. Zhou et al. reported the design of a transmembrane protein CD63 aptamer-based electrochemical biosensor for the detection of exosomes that may constitute tumor biomarkers [16]. The structure-switching aptasensor can detect 1 106 particles/mL of exosomes, which represents 100-fold decrease in the limit of detection compared to commercial immunoassays. Several aptamers were recently selected against cell surface biomarkers that are overexpressed at the surface of cancer cells. These aptamers can be used for developing new tools for cancer diagnostics. Dickey and Giangrande discussed recent progresses about the isolation and detection of circulating tumor cells (CTCs) by aptamers [17]. The detection of CTCs remains a critical challenge and the review presents different methods for improving the detection sensitivity and to deal with the heterogeneity of tumor cells. With the same goal i.e. increased detection sensitivity of cancer cells Civit et al. developed caged aptamers against
lymphoma cancer cells, using an apta-PCR assay [18]. Caged aptamers bear photo-labile groups that enable to control their recognition properties using light. The reported method allows achieving the detection of as few as 77 cells. The articles assembled in this issue provide fascinating examples of the rapidly expanding field of nucleic acid aptamers that nowadays rival antibodies for many applications. References [1] A.D. Ellington, J.W. Szostak, Nature 346 (1990) 818–822. [2] C. Tuerk, L. Gold, Science 249 (1990) 505–510. [3] M. Mascini, I. Palchetti, S. Tombelli, Angew. Chem. Int. Ed. Engl. 51 (2012) 1316–1332. [4] A. Cibiel, C. Pestourie, F. Ducongé, Biochimie 94 (2012) 1595–1606. [5] E. Dausse, S. Da Rocha Gomes, J.J. Toulmé, Curr. Opin. Pharmacol. 9 (2009) 602–607. [6] W. Thiel, P. Giangrande, Methods 97 (2016) 3–10. [7] T. Chaou, B. Vialet, L. Azéma, Methods 97 (2016) 11–19. [8] M. Jauset Rubio, M. Svoboda, T. Mairal, C. O’Sullivan, Methods 97 (2016) 20–26. [9] C. Entzian, T. Schubert, Methods 97 (2016) 27–34. [10] N. Nguyen Quang, C. Pestourie, A. Cibiel, F. Ducongé, Methods 97 (2016) 35–43. [11] C. Schneider, B. Suess, Methods 97 (2016) 44–50. [12] C.L.A. Hamula, H. Peng, Z. Wang, G.T. Tyrell, X.-F. Li, X.C. Le, Methods 97 (2016) 51–57. [13] I. Lamberti, S. Scaran, C.L. Esposito, A. Antoccia, G. Antonini, C. Tanzarella, V. De Francicis, M. Minunni, Methods 97 (2016) 58–68. [14] E. Goux, Q. Lespinasse, V. Guieu, S. Perrier, C. Ravelet, E. Fiore, E. Peyrin, Methods 97 (2016) 69–74. [15] E. Mastronardi, P.K. Tsae, X. Zhang, A. Pach, Y. Sultan, M. DeRosa, Methods 97 (2016) 75–87. [16] Q. Zhou, A. Rahumain, K. Son, D.S. Shin, T. Patel, A. Revzin, Methods 97 (2016) 88–93. [17] D.D. Dickey, P. Giangrande, Methods 97 (2016) 94–103. [18] L. Civit, A. Pinto, A. Rodrigues-Correia, A. Heckel, C. O’Sullivan, G. Mayer, Methods 97 (2016) 104–109.
Jean-Jacques Toulmé Eric Peyrin Frédéric Ducongé