Site-Specific One-Pot Dual Labeling of DNA by Orthogonal

Communication pubs.acs.org/bc

Site-Specific One-Pot Dual Labeling of DNA by Orthogonal Cycloaddition Chemistry Juliane Schoch,† Markus Staudt,† Ayan Samanta,† Manfred Wiessler,‡ and Andres Jas̈ chke*,† †

Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69120 Heidelberg, Germany German Cancer Research Center, Division of Medical Physics in Oncology E020, 69120 Heidelberg, Germany

‡

S Supporting Information *

ABSTRACT: Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide−alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels−Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctenebased dienophiles (with rate constants up to 380 000 M−1 s−1). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry. abeling of oligonucleotides with fluorophores or affinity tags is of utmost importance in nanotechnology and modern life sciences. In particular, the site-specific introduction of multiple, spectrally different fluorophores is crucial for structure−function studies of biopolymers by bulk or singlemolecule fluorescence spectroscopy and also for the development of DNA probes for imaging purposes.1 While a number of non-nucleosidic modifications can be easily introduced during solid-phase DNA synthesis by phosphoramidite chemistry,2,3 the harsh reaction conditions restrict the variety of modifications that can be inserted. Therefore, postsynthetic labeling strategies have received considerable attention, as they work under milder conditions and furthermore allow the screening over a range of labels in a short time as one phosphoramidite serves as precursor for all different labels that need to be introduced. Nevertheless, conventional postsynthetic labeling protocols suffer from low yield, prolonged reaction time and often require a high concentration of the biomolecule in combination with a huge excess of the coupling partner.4 In contrast, bioorthogonal click reactions are very attractive for this purpose, as they work in aqueous solution under mild conditions and often with quantitative yields.5−9 Among these reactions, the copper-catalyzed (CuAAC)10−13 and strain-promoted azide−alkyne click reactions (SPAAC)14,15 found widest application in the past few years. As azides are rather unstable under the conditions of phosphoramidite solidphase synthesis, the chemical incorporation of “clickable” moieties into oligonucleotides has been focused on alkynecarrying phosphoramidites, followed by postsynthetic labeling with azide-derivatives.16,17 Recently, the successful synthesis of azide-modified RNA-oligonucleotides was reported by using

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© 2012 American Chemical Society

either phosphotriester chemistry18,19 or two-step procedures where a precursor is postsynthetically converted to the azide.20 Another recently upcoming bioorthogonal labeling approach is based on the inverse electron-demand Diels−Alder (DAinv) reaction.21−23 We have previously exploited this strategy for the site-specific labeling of DNA24 and RNA.25 This cycloaddition proceeds rapidly, without transition metals, and allows efficient functionalization of oligonucleotides at room temperature. The selective coupling of two (or more) different labels to specific positions of an oligonucleotide is even more challenging than introducing a single modification. Using cycloaddition chemistry, this goal has been achieved on different routes: in stepwise processes, in which two different conjugation reactions are carried out successively with intervening purification,26 or in which protecting groups must be removed between successive coupling steps in order to direct the reactivity.27 Another approach is the ligation of individually synthesized singly modified fragments.28 These sequential approaches, however, are time-consuming as well as laborious, and often result in low overall yields. Ideally, sitespecific double-modification would be performed concurrently in a one-pot reaction without protection or intervening purification. This goal, however, requires the two different coupling chemistries not only to be bioorthogonal (no side reactions with the functional groups present in the oligonucleotide), but also mutually orthogonal so that no cross-reactivity Received: April 3, 2012 Revised: May 30, 2012 Published: June 19, 2012 1382

dx.doi.org/10.1021/bc300181n | Bioconjugate Chem. 2012, 23, 1382−1386

Bioconjugate Chemistry

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occurs.29,30 To our knowledge, no such systems are known for DNA and RNA modification. Herein, we report the successful combination of CuAAC and DAinv for simultaneous site-specific double-labeling of DNA oligonucleotides in a true one-pot reaction (Figure 1). Both

Figure 1. Double modification of a DNA oligonucleotide by simultaneous DAinv and CuAAC reaction.

CuAAC16,17,31 and DAinv24 reactions have been previously described to be bioorthogonal to DNA. We assumed that they could also be mutually orthogonal as alkynes that are reactive in CuAAC reactions should have low reactivity in DAinv cycloadditions due to their low HOMO energy.32 To explore this possibility, we first evaluated three different dienophiles for their use in DAinv modification on DNA by determination of rate constants, followed by investigation of possible cross- and side-reactivity, in combination with CuAAC using different techniques ranging from tandem mass spectrometry (MS/MS) for shorter oligonucleotides to restriction digestion and polyacrylamide gel electrophoretic analysis on longer oligonucleotides. Finally, we carried out one-pot dual labeling on doubly modified oligonucleotides of different sizes and analyzed the reaction mixtures, whereupon all experiments indicated DAinv and CuAAC to be mutually orthogonal. While our previous work used norbornene dienophiles, their moderate reactivity in DAinv reactions (second-order rate constant k2 = 20 ± 2 M−1 s−1) required prolonged reaction times (1 h) and caused nonquantitative reaction yields.24,25 Meanwhile, trans-cyclooctene (TCO)-based dienophiles had been reported to react much more rapidly.33 We therefore synthesized three different TCO-based alcohols as dienophilic moieties, namely, two diastereomers of (E)-cyclooct-4-enol and the cyclopropyl-appendent compound endo-(E)-bicyclo [6.1.0]non-4-en-9-yl methanol,21,33 and converted them into phosphoramidites PA1-PA3 using standard procedures. These phosphoramidites were then used to incorporate a single dienophile at the 5′-terminus of oligonucleotides ODN1a−3a (Figure 2). This required the use of tert-butylhydroperoxide as an oxidizer, as the standard aqueous iodine oxidizer led to quantitative degradation of the TCO moieties. The second-order rate constants of the DAinv reactions with dansyl-tetrazine were determined in a stopped-flow apparatus exploiting the fluorogenic property of these cycloadditions. The values obtained for oligonucleotide ODN1a (k2 = 26 000 ± 600 M−1 s−1), ODN2a (k2 = 83 000 ± 3500 M−1 s−1), and ODN3a (k2 = 380 000 ± 10 000 M−1 s−1) indicate the high reactivity of TCO (factor 1200−19 000, compared to norbornene), which is in agreement with reported data for reaction of tetrazines with trans-cyclooctenols.33,34 The same analysis was applied to an alkyne-only modified oligonucleotide, ODN4, where no reaction with dansyl-tetrazine was observed (Supporting Information, Figure S3−S5). Even after incubation of ODN4 with dansyl-tetrazine for 48 h at room temperature and subsequent LC-MS analysis, no DAinv product of the alkyne was detectable. These data suggest a difference in reactivity of

Figure 2. Dienophile-modified phosphoramidites PA1−PA3 and synthesized oligonucleotides ODN1a−5 with corresponding rate constants of ODN1a−3a in the DAinv reaction.

at least 8 orders of magnitude between TCO-based dienophiles and terminal alkynes. Encouraged by these results, doubly modified oligonucleotides carrying both an alkyne and a dienophile, ODN2b and ODN3b, were synthesized in which one of the two most reactive dienophiles 2 and 3 was introduced at the 5′-end, while the alkynyl residue was attached to an internal position (Figure 2). The possibility to carry out the two cycloaddition reactions concurrently was examined by MS and LC-MS/MS analysis of crude one-pot concurrent DAinv/CuAAC reaction mixtures. High-resolution mass spectrometry served to identify the doubly labeled products, while orthogonality of the two cycloadditions was analyzed by fragmentation of the product signals using tandem mass spectrometry (MS/MS). The fragmentation schemes were then compared with the ones obtained for the nonreacted oligonucleotide ODN2b (Figure 3a), and with those from individual DAinv or CuAAC reactions (Figure 3b,c). When only the DAinv reaction was performed (by reacting the DNA with dansyl-tetrazine, Figure 3b), the dienophile-containing signals (green circles) disappeared, new signals with masses of DAinv products appeared (green stars), while the alkyne-only fragments (red circles) remained unchanged. On the other hand, when only the CuAAC was carried out (by incubation with Alexafluor-594 azide, CuSO4, THPTA, and sodium ascorbate), the fragmentation scheme (Figure 3c) showed the disappearance of the alkyne signals (red circles) and the appearance of CuAAC product peaks (red stars), while the dienophile-related signals (green circles) 1383



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ments, and the appearance of new product peaks (Supporting Information Figure S20). The mass spectrum of the combined product fractions confirmed the formation of the DAinvfunctionalized 5mer and the CuAAC-functionalized 14mer, while there were no traces of the two hypothetical products, namely, CuAAC-functionalized 5mer and DAinv-functionalized 14mer, resulting from cross-reactivity of the two dienophiles (Figure 4).

Figure 3. (a) MS/MS spectrum of nonreacted heptamer oligonucleotide ODN2b, (b) MS/MS spectrum of DAinv product of ODN2b, (c) MS/MS spectrum of CuAAC product of ODN2b, (d) MS/MS spectrum of simultaneous DAinv/CuAAC reaction on ODN2b.

remained unchanged. Importantly, neither the primary mass spectra nor the MS/MS spectra indicated the presence of multiply or wrongly functionalized fragments that would indicate cross-reactivity. These results demonstrate the selectivity of the two cycloaddition reactions for their intended functional groups. When both reactions were run simultaneously (by incubation of the oligonucleotide with Alexafluor594 azide, CuSO4, THPTA, sodium ascorbate, and dansyltetrazine), the mass signals of the dienophile- (green circles) as well as alkynyl-containing fragments (red circles) disappeared, while the DAinv (green star) and CuAAC product (red star) peaks appeared (Figure 3d). An identical MS/MS analysis was conducted for DAinv, CuAAC, and simultaneous DAinv/ CuAAC on ODN3b where similar fragmentation schemes were obtained. (For MS spectra, LC traces, and analysis of MS and MS/MS spectra, see Supporting Information). These results demonstrate thateven when run simultaneously in a one-pot reactionboth reactions are orthogonal to each other, whereas the reactivity of the DAinv can be tuned by the choice of the dienophile. To further analyze the labeling efficiency and orthogonality of the two reactions, we applied the one-pot double labeling reaction to a 19mer oligonucleotide carrying dienophile 2 and an alkyne moiety, ODN2c (Figure 2), bearing a restriction cleavage site in between the alkyne and TCO substitutions. After labeling, the oligonucleotide was hybridized to its complementary strand and restriction digested to yield a 5mer that was expected to contain exclusively the DAinv product and a 14mer that should be CuAAC product only. HPLC analysis in comparison with a restriction digest of the nonreacted oligonucleotide ODN2c showed the quantitative disappearance of the two unmodified oligonucleotide frag-

Figure 4. Calculated (dotted line) and measured (straight line) MS spectra of DAinv and CuAAC products of doubly labeled ODN2b after restriction digestion (DdeI restriction endonuclease).

After having demonstrated the orthogonality and efficiency of the labeling protocol, we in a next step applied this protocol to the preparative synthesis of a doubly labeled oligonucleotide. Varying amounts of ODN2c (162 pmole to 1.62 nmole) were subjected to the concurrent DAinv/CuAAC reaction, and the crude reaction mixture was purified on a denaturing polyacrylamide gel, followed by isopropanol precipitation. This protocol afforded the pure doubly labeled oligonucleotide in isolated yields of ∼33% and amounts sufficient for typical biophysical experiments (Supporting Information Figure S21). This approach opens up the possibility to label long, biologically relevant oligonucleotides simultaneously with different fluorophores as required for structural studies, e.g., by FRET spectroscopy. As proof-of-principle for the applicability to longer oligonucleotides, a TCO- and alkyne-modified 233mer oligonucleotide was amplified by PCR using singly modified primers ODN1a−3a (for DAinv, forward primer) and ODN5 (for CuAAC, reverse primer). The PCR product was then subjected to either individual DAinv and CuAAC reactions, or to a simultaneous one-pot DAinv/CuAAC reaction using TAMRA-tetrazine and Cy5 azide. After polyacrylamide gel electrophoretic separation of the reaction mixtures, the gels were subjected to fluorescence imaging. The TAMRA scan (excitation 532 nm) revealed all products of a 1384



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DAinv/SPAAC system, we could not observe any side reaction, such as the reaction of the azide with the alkene or the coupling of the tetrazine with the alkyne. To our knowledge, the DAinv reaction with the TCO reactants described here isby several orders of magnitudethe fastest bioorthogonal modification reaction ever reported for nucleic acids. Future efforts will now be directed to increase the rate of the CuAAC reaction, too. We expect that the combination of CuAAC and DAinv will find broad application in biochemistry, polymer science, and nanotechnology. The commercial availability of a wide range of azide derivatives for conjugationfrom dyes, amino acids, sugars, nucleotides to whole peptide tags or solid matrices combined with the current rapid development of conjugatable tetrazine derivatives will facilitate the one-step synthesis of a broad array of double-functionalized biomolecules. While the requirement for copper ions hinders applications inside living cells, this does not present a problem for many other biological applications, from in vitro studies to fixed-cell labeling and cellsurface labeling.36,37

reaction with tetrazine, while all azide-derived products were visualized in the Cy5 scan (excitation 633 nm, Figure 5). For

Figure 5. 15% Denaturing polyacrylamide gel of DAinv/CuAAC labeling reaction on an alkyne/TCO-modified ds233mer PCRproduct: (1) negative control incubating nonmodified PCR product with dansyl-tetrazine, Cy5 azide, THPTA, CuSO4, and sodium ascorbate; (2−4) CuAAC on TCO-modified PCR products; (5) DAinv on alkyne-modified PCR product; (6) CuAAC on doubly modified PCR product of ODN2a; (7) DAinv on doubly modified PCR product of ODN2a; (8−10) concurrent DAinv/CuAAC on doubly modified PCR products of ODN1a−3a. FP and RP are unmodified oligonucleotides having the same nucleotide sequence as ODN1a−3a and ODN5, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details and analysis of MS/MS spectra. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+49) 6221-54 4851; Fax: (+49) 6221-5464 30; E-mail: [email protected].

the PCR product based on ODN3a, no DAinv reaction product was observed (Figure 5, lane 10), likely due to decomposition of the highly reactive bicyclo[6.1.0]nonenyl ring system during multiple heating cycles to 98 °C. In contrast, the cyclooctenolbased dienophiles withstand such treatment without problems and CuAAC as well as DAinv products could be detected (Figure 5, lanes 6−9). For doubly labeled PCR products of primers ODN1a and ODN2a, the bands in both scans overlapped exactly, indicating attachment of both dyes to the same macromolecular species (Figure 5, lanes 8−9). We again checked for cross-reactivity by incubating the TCO-only modified PCR product with a 500-fold excess of Cy5 azide, CuSO4, THPTA, and sodium ascorbate, and also the alkyneonly PCR product with a 10-fold excess of TAMRA-tetrazine. The absolute absence of any faint green or red band in these control experiments even after 1 h of reaction time unambiguously demonstrates the selectivity and orthogonality of these cycloadditions toward oligonucleotide functionalization (Figure 5, lanes 2−5). Very recently, Karver et al. reported the orthogonality of the DAinv reaction and the strain-promoted alkyne−azide cycloaddition (SPAAC), and the application of this methodology to cell-surface labeling.29 As strained alkynes are known to react as dienophiles in DAinv reactions,35 this approach required finetuning of reactivities by the careful choice of reactants, and the authors settled for a less reactive tetrazine in combination with the highly reactive TCO (k2 = 210 M−1 s−1), for which they observed excellent orthogonality to the azide/dibenzocyclooctyne system. In our system, no compromise in tetrazine reactivity was necessary to achieve orthogonality of the cycloadditions, leading to rate constants of up to 380 000 M−1 s−1 for the DAinv reaction. This allows completion of the reaction within seconds even at low micromolar concentration of both biomolecule and labeling reagent. In contrast to the

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Grant # Ja 794/3). The authors thank Dr. Murat Sünbül for providing TAMRA.

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REFERENCES

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Site-Specific One-Pot Dual Labeling of DNA by Orthogonal

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