Click Reaction on Solid Phase Enables High Fidelity Synthesis of

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Click reaction on solid phase enables high fidelity synthesis of nucleobase-modified DNA Günter Mayer, Fabian Tolle, Franziska Pfeiffer, and Malte Rosenthal Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00668 • Publication Date (Web): 05 Feb 2016 Downloaded from http://pubs.acs.org on February 6, 2016

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Bioconjugate Chemistry

Figure 2. Binding analysis of different C12-I clickmer deriva-tives towards C3-GFP by flow cytometry. Shown is the mean fluorescence of Cy5-labelled DNA bound to C3-GFP-modified beads. The previously described reference C12-I-PCR (grey squares) reveals concentration dependent binding to C3-GFP, whereas the scrambled sequence C12sc-I-PCR (grey diamonds) does not. The C12-I clickmer, synthesized according to method SPS-B (black dots) exhibits binding to C3-GFP that is very similar to the PCR-synthesized reference. In contrast, the vari-ant synthesized according to method SPS-A (black triangles) shows a strongly reduced binding affinity. The data shown are the mean of two independent experiments. 73x48mm (300 x 300 DPI)

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Scheme 2. Overview on three synthesis routes for the generation of CuAAC functionalized DNA, e.g. the clickmer C12-I. 173x51mm (300 x 300 DPI)


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Click reaction on solid phase enables high fidelity synthesis of nucleobase-modified DNA Fabian Tolle, Malte Rosenthal, Franziska Pfeiffer and Günter Mayer* Life and Medical Sciences Institute, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn (Germany) KEYWORDS: aptamer, clickmer, CuAAC, solid phase synthesis ABSTRACT: The post-synthetic functionalization of nucleic acids via click chemistry (CuAAC) has seen tremendous implementation, extending the applicability of nucleobase-modified nucleic acids in fields like fluorescent labeling, nanotechnology and in vitro selection. However, the production of large quantities of high-density functionalized material via solid phase synthesis has been hampered by oxidative by-product formation associated with the alkaline work up conditions. Herein, we describe a rapid and cost-effective protocol for the high fidelity large-scale production of nucleobase-modified nucleic acids, exemplified on a recently described nucleobase-modified aptamer.

Modified nucleic acids have gained strong interest and widespread use in the past decades, as they extend the capabilities of natural nucleic acids, e.g. programming interactions by base pairing with novel unprecedented functionality and applicability.1-4 Especially the post-synthetic functionalization by CuAAC with clickable nucleotides, i.e. 5-ethynyl-2’deoxyuridine (EdU) has become very attractive for the modular, quantitative and rapid modification of DNA.5 Today, many azide-derivatized reaction partners are available, allowing the introduction of a plethora of chemical groups into DNA. This approach opens entirely novel routes for the labeling of nucleic acids6 and the generation of nucleic acid-based molecular tools such as nano-architectures7, DNA-based wires8 or, as shown recently, aptamers containing novel chemical entities9. In the recently described click-SELEX process, EdU is used for the introduction of chemical functionalizations, e.g. indoleresidues into a DNA library (IndU) (Scheme 1a,c).9 This modified DNA library is then used for the generation of nucleobase-modified DNA aptamers, so-called ‘clickmers’ with novel recognition properties. However, Seela and co-workers showed that the workup of solid phase synthesized EdUcontaining DNA under alkaline conditions results in the partial oxidation of the ethynyl-moiety, yielding a ketone containing C5-acetyl residue (KdU) (Scheme 1b,c).10 The resulting KdU residues will no longer be available for functionalization by click chemistry and, thus, lead to an inhomogeneous product. Homogenous products are possible as long as the functionalized DNA is synthesized by PCR, followed by single-strand generation, e.g. using λ exonuclease digestion (Scheme 2, PCR).11 Unfortunately, the amount of material that can be generated by PCR is limited and, due to the approx. 30 times higher price for the triphosphate compared to the respective phosphoramidite, associated with high production costs (Table 1).

Scheme 1. Product and by-product formation of EdUfunctionalized DNA and nucleosides.

Table 1. Comparison of per batch DNA production scales and the associated expenses for consumables for clickmer production via PCR and solid phase synthesis (SPS). Production method Production scale Approx. cost for 1 nmol


PCR < 100 pmol > 3500 €

SPS > 5 nmol ca. 100 €

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Scheme 2. Overview on three synthesis routes for the generation of CuAAC functionalized DNA, e.g. the clickmer C12-I.

For many interesting applications of EdU containing ‘clickable’ DNA, larger amounts (> 1 nmol) are required, which are simply not practicably accessible by PCR. In this case, solid phase synthesis is the only method of choice, but will, following the traditional procedures, lead to inhomogeneous products bearing the undesired KdU by-product (Scheme 2, SPS-A). As a solution to this problem, we propose the solid phase synthesis of EdU containing DNA, followed by a click-reaction with the DNA still attached to the solid phase and subsequent deprotection and purification according to the standard procedures (Scheme 2, SPS-B). To test this approach, we synthesized the clickmer C12, which is known to interact with C3-GFP.9 This DNA contains six EdU residues, three of which have been shown to be essential for maintaining C3-GFP binding. To this end, we developed a novel protocol for the large-scale functionalization of nucleic acids on solid support, enabling us to synthesize this molecule according to the three procedures detailed in Scheme 2; enzymatically (PCR), or by solid-phase synthesis, introducing the indole-residue either after (Scheme 2, SPS-A), or prior to (Scheme 2, SPS-B) the alkaline workup step. We analyzed the resulting clickmer variants on an agarose gel (Fig. 1a) and by HPLC/MS (Fig. 1b). Both chemically synthesized clickmers revealed a clear and distinct band on the agarose gel, demonstrating the production of pure full length DNA. However, the HPLC retention profile of the variant modified with indole-azide after DNA workup (C12-I-SPS-A) was broader (Fig. 1b, top) compared to the one that has been modified on the solid phase prior to the workup (C12-I-SPS-B, Fig. 1b, bottom). Also, the identified mass of the variant syn-

thesized according to strategy SPS-A differed significantly from the calculated mass (Fig. 1b, top), indicating an incomplete functionalization of the DNA under these conditions. In turn, C12-I synthesized according to method SPS-B (click reaction on the solid phase) showed a sharp retention profile and matched the calculated mass precisely, indicating full conversion to the desired product (Fig. 1b, bottom). To verify the full functionalization of the DNA synthesized according to the above detailed procedures, we further analyzed the clickmer variants on the nucleoside level. Therefore, we enzymatically digested the respective DNA molecules to individual nucleosides and analyzed the resultant mixture by HPLC/MS. As a control, C12 DNA, synthesized on solid phase and worked up under alkaline conditions, but not modified with the indole-azid (C12-SPS-A) was analyzed, revealing the two characteristic peaks for EdU and the expected KdU by-product (Fig. 1c, top). Next, the C12 DNA generated by the same strategy, but further reacted with the indole-azide via CuAAC in solution (C12-I-SPS-A), was analyzed. As expected, the peak corresponding to EdU vanished, whereas the peak reflecting the indole-modified product (IdU) emerged (Fig. 1c, middle). Notably, the KdU related peak was still present, underlining the heterogeneity of the resultant DNA molecules. In turn, the modification of the solid-phase synthesized C12 by indole-azides on the solid matrix prior to the alkaline workup procedure (C12-I-SPS-B) resulted in a homogeneous DNA population without any KdU contamination, showing a 77% increased yield of the desired IndU residues. (Fig. 1c, bottom).


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Figure 1. Comparison of the two alternative solid phase based DNA production strategies (SPS-A and SPS-B). a) Ethidium bromide stained 4% agarose gel showing the production of pure full length DNA for both synthesis conditions. b) HPLC-MS based analysis of the functionalized DNA. C12-I-SPS-A (top) shows a broad retention profile and a deviation from the calculated mass, indicating an incomplete formation of the indole functionalized product. C12-I-SPS-B (bottom) shows a sharp retention profile with the expected mass of the fully functionalized product. c) HPLC (left) and ESI-MS (right) based analysis of the nucleoside mixture resulting from the enzymatic digestion of the DNA. For both click reaction strategies full conversion of EdU to IndU is observed. However, the SPS-A produced DNA is contaminated with KdU (middle), whereas the SPS-B produced DNA is free of the undesired KdU by-product. Thus, SPS-B produced DNA contains a 77% higher yield of the desired IndU product.


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Having demonstrated the full functionalization of the solid phase synthesized clickmer, we next went a step further and analyzed the impact of the synthesis strategies detailed in Scheme 2 on the binding behavior of C12-I. Therefore, we performed a flow cytometry analysis utilizing Cy5labelled C12-I variants and the target protein C3-GFP immobilized on magnetic beads.9 As a reference, we included PCR-synthesized C12-I, and a scrambled variant thereof (C12sc-I), which has previously been shown to not interact with C3-GFP.9 As depicted in Fig. 2, the PCR synthesized C12-I (C12-I-PCR) behaved as predicted and revealed a concentration dependent binding to C3-GFP modified beads (KD-value ~216 ± 5 nM, grey squares). In contrast, the PCRsynthesized scrambled variant (C12sc-I-PCR, Fig. 2, grey diamonds) did not show interaction with C3-GFP. The C12I clickmer, synthesized according to method SPS-B (C12-ISPS-B, Fig. 2, black dots) exhibited a concentration dependent binding to C3-GFP, similar to the PCR-synthesized reference (KD-value ~494 ± 44 nM). In contrast, the variant synthesized according to method SPS-A (C12-I-SPS-A Fig. 2, black triangles) showed a strongly reduced binding affinity towards C3-GFP functionalized magnetic beads. These findings underline the quality of the fully functionalized clickmers produced by the novel synthesis route.

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ized nucleic acids in numerous fields such as nanotechnology, catalysis, and (bio)medicine.

ASSOCIATED CONTENT Supporting Information A detailed description of all experimental procedures is given in the Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Prof. Dr. G. Mayer, Life and Medical Sciences Institute, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn (Germany) E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources This work was made possible by funds from the German Research Council (Deutsche Forschungsgemeinschaft) to G.M. (MA 3442/4-2 and MA 3442/4-1).

ACKNOWLEDGMENT The authors want to thank Dr. Tim Gehrke and Ella Biotech GmbH for the fruitful collaboration and the excellent support regarding all aspects of solid phase synthesis.

REFERENCES Figure 2. Binding analysis of different C12-I clickmer derivatives towards C3-GFP by flow cytometry. Shown is the mean fluorescence of Cy5-labelled DNA bound to C3-GFP-modified beads. The previously described reference C12-I-PCR (grey squares) reveals concentration dependent binding to C3-GFP, whereas the scrambled sequence C12sc-I-PCR (grey diamonds) does not. The C12-I clickmer, synthesized according to method SPS-B (black dots) exhibits binding to C3-GFP that is very similar to the PCR-synthesized reference. In contrast, the variant synthesized according to method SPS-A (black triangles) shows a strongly reduced binding affinity. The data shown are the mean of two independent experiments.

To demonstrate the generality of our method we synthesized oligonucleotides with the widespread biotin functionalization. Similar to the indole functionalization (Fig. S1a), product yields only up to 76% could be observed for the SPS-A synthesis route, whereas full conversion was observed for the SPS-B route (Fig. S1b). In summary, we describe a general strategy for the high fidelity large-scale production of nucleobase-modified nucleic acids. This method is not only indispensable for the production of clickmers, but can also be applied for numerous other applications involving alkyne modified nucleic acids suffering from oxidative by-product formation.10 The strategy can be used for the production of oligonucleotides with most of the frequently used functionalizations such as fluorophores and biotin, however may not be suitable for highly base-labile functionalizations. The increase in quality and dramatic reduction of production cost will facilitate the widespread application of clickmers and other functional-

(1) Perrin, D. M., Garestier, T., and Hélène, C. (2001) Bridging the gap between proteins and nucleic acids: A metal-independent RNAseA mimic with two protein-like functionalities. J. Am. Chem. Soc. 123, 1556–1563. (2) Vaught, J. D., Bock, C., Carter, J., Fitzwater, T., Otis, M., Schneider, D., Rolando, J., Waugh, S., Wilcox, S. K., and Eaton, B. E. (2010) Expanding the chemistry of DNA for in vitro selection. J. Am. Chem. Soc. 132, 4141–4151. (3) Tolle, F., and Mayer, G. (2013) Dressed for success– applying chemistry to modulate aptamer functionality. Chem. Sci. 4, 60–67. (4) Diafa, S., and Hollenstein, M. (2015) Generation of Aptamers with an Expanded Chemical Repertoire. Molecules 20, 16643–16671. (5) El-Sagheer, A. H., and Brown, T. (2010) Click chemistry with DNA. Chem. Soc. Rev. 39, 1388. (6) Salic, A., and Mitchison, T. J. (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U. S. A. 105, 2415–2420. (7) Cassinelli, V., Oberleitner, B., Sobotta, J., Nickels, P., Grossi, G., Kempter, S., Frischmuth, T., Liedl, T., and Manetto, A. (2015) One-Step Formation of “Chain-Armor-”Stabilized DNA Nanostruc-tures. Angew. Chem., Int. Ed. Engl. 54, 7795–7798. (8) Burley, G. A., Gierlich, J., Mofid, M. R., Nir, H., Tal, S., Eichen, Y., and Carell, T. (2006) Directed DNA metallization. J. Am. Chem. Soc. 128, 1398–1399. (9) Tolle, F., Brändle, G. M., Matzner, D., and Mayer, G. (2015) A Versatile Approach Towards Nucle-obase-Modified Aptamers. Angew. Chem., Int. Ed. Engl. 54, 10971–10974. (10) Ingale, S. A., Mei, H., Leonard, P., and Seela, F. (2013) Ethynyl side chain hydration during synthesis and workup of “clickable” oligonucleotides: bypassing acetyl group formation by triisopropylsilyl protection. J. Org. Chem. 78, 11271–11282. (11) Gierlich, J., Gutsmiedl, K., Gramlich, P. M. E., Schmidt, A., Burley, G. A., and Carell, T. (2007) Synthesis of Highly Modi-


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Bioconjugate Chemistry fied DNA by a Combination of PCR with Alkyne Bearing Triphosphates and Click Chemistry. Chemistry 13, 9486–9494.


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Figure 1. Comparison of the two alternative solid phase based DNA production strategies (SPS-A and SPSB). a) Ethidium bromide stained 4% agarose gel showing the production of pure full length DNA for both synthesis conditions. b) HPLC-MS based analysis of the functionalized DNA. C12-I-SPS-A (top) shows a broad retention profile and a deviation from the calculated mass, indicating an incom-plete formation of the indole functionalized product. C12-I-SPS-B (bottom) shows a sharp retention profile with the expected mass of the fully functionalized product. c) HPLC (left) and ESI-MS (right) based analysis of the nucleoside mixture resulting from the enzymatic digestion of the DNA. For both click reaction strategies full conversion of EdU to IndU is observed. However, the SPS-A produced DNA is contaminated with KdU (middle), whereas the SPS-B produced DNA is free of the undesired KdU by-product. Thus, SPS-B produced DNA contains a 77% higher yield of the desired IndU product. 177x202mm (300 x 300 DPI)



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Scheme 1. Product and by-product formation of EdU-functionalized DNA and nucleosides. 84x82mm (300 x 300 DPI)


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Click Reaction on Solid Phase Enables High Fidelity Synthesis of

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