Sequence-Specific Post-Synthetic Oligonucleotide Labeling for Single

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Sequence-Specific Post-Synthetic Oligonucleotide Labeling for Single-Molecule Fluorescence Applications David Egloff,† Igor A. Oleinich,† Meng Zhao, Sebastian L. B. König, Roland K. O. Sigel, and Eva Freisinger* Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland S Supporting Information *

ABSTRACT: The sequence-specific fluorescence labeling of nucleic acids is a prerequisite for various methods including single-molecule Förster resonance energy transfer (smFRET) for the detailed study of nucleic acid folding and function. Such nucleic acid derivatives are commonly obtained by solid-phase methods; however, yields decrease rapidly with increasing length and restrict the practicability of this approach for long strands. Here, we report a new labeling strategy for the postsynthetic incorporation of a bioorthogonal group into single stranded regions of both DNA and RNA of unrestricted length. A 12-alkyne-etheno-adenine modification is sequenceselectively formed using DNA-templated synthesis, followed by conjugation of the fluorophore Cy3 via a copper-catalyzed azide−alkyne cycloaddition (CuAAC). Evaluation of the labeled strands in smFRET measurements shows that the strategy developed here has the potential to be used for the study of long functional nucleic acids by (single-molecule) fluorescence or other methods. To prove the universal use of the method, its application was successfully extended to the labeling of a short RNA single strand. As a proof-of-concept, also the labeling of a large RNA molecule in form of a 633 nucleotide long construct derived from the Saccharomyces cerevisiae group II intron Sc.ai5γ was performed, and covalent attachment of the Cy3 fluorophore was shown with gel electrophoresis.

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derivatives with suitable functional groups in polymerase chain reactions (PCRs). However, the drawback is the absence of site- or sequence-specificity as multiple modified nucleosides are incorporated throughout the sequence.5,6 Long RNAs produced by in vitro transcription are classically labeled by hybridizing a fluorophore carrying shorter DNA or PNA to artificial internal single stranded loops.8,9 Obviously, such large alterations of the native sequence often have severe negative effects on structure and function of complex three-dimensional architectures. Accordingly, the site- or sequence-specific labeling of longer nucleic acids can be challenging. One strategy successfully applied for the study of large RNA molecules relies on the synthesis of short (partially) labeled RNA fragments that are subsequently assembled enzymatically using DNA splints and T4 DNA ligase.10−16 However, for the assembly of several RNA sequences via splinted ligation, overall yields decrease, and additionally intramolecular RNA structures can influence the efficiency of the reaction.17 Also, one incident of nontemplated nucleotide incorporation at the ligation site by T4 DNA ligase has been reported, and such additional internal bases can be a severe disadvantage depending on the application.17 Another

he development of novel probes, assays, and instruments has led to a wide range of applications for biological research and has helped to increase our knowledge about the underlying mechanisms of living systems.1 One of these techniques is single-molecule Förster resonance energy transfer (smFRET) that permits the study of folding processes and catalysis of single functional nucleic acids in real time.2 Fluorescence studies of nucleic acids usually require the fluorophore(s) to be introduced at a defined position. Shorter nucleic acids are typically obtained using phosphoramiditebased solid-phase synthesis of the strand. This allows the direct site-specific incorporation of either a fluorophore or a functional group that can be used postsynthetically for conjugation with a wide variety of commercially available fluorophore derivatives. Such functional groups include thiol and amine groups, which can be reacted with the respective succinimidyl or maleimide fluorophore derivatives,3 as well as bioorthogonal alkine groups that allow the highly selective and efficient coupling to azide-modified fluorophores or markers via a copper-catalyzed azide−alkyne cycloaddition (CuAAC).4−6 The drawback of solid-phase synthesis is rapidly decreasing synthetic yields with increasing chain length. Accordingly, the preparation of sequences longer than ∼100 nucleotides, which is clearly shorter than the majority of functional nucleic acids, becomes very costly and time-consuming.7 Longer modified DNA strands can be produced by using nucleoside triphosphate © 2016 American Chemical Society

Received: April 19, 2016 Accepted: July 13, 2016 Published: July 13, 2016 2558

DOI: 10.1021/acschembio.6b00343 ACS Chem. Biol. 2016, 11, 2558−2567

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ACS Chemical Biology strategy is the postsynthetic end-labeling of nucleic acids bringing about the obvious limitation of placing the dyes at the termini of the molecule.18,19 Successful examples include endlabeling of an RNA sequence either at the 5′-end by priming the in vitro transcription reaction with a 5′-azido-5′deoxyguanosine derivative or at the 3′-end using the standard protocol for 3′-end radiolabeling with poly(A) polymerase but in the presence of 3′-N3dATP instead of [α-32P]-dATP.20−22 Both modifications can be subsequently reacted successfully with an alkyne labeled dye in a click reaction under modified conditions.21 Another example is the reaction of RNA with periodate that leads to the oxidative opening of the 3′-sugar ring and the formation of two aldehyde groups that can be coupled to amino-modified molecules.22 Additional enzymes that have been applied to add labels and reactive tags to the ends of RNA are T4 polynucleotide kinase, T4 RNA ligase, and terminal transferase.22 Internal labeling of long nucleic acids has been achieved using S-adenosyl-L-methionine derivatives in conjunction with DNA or RNA methylases,23−25 hybridization of fluorophore-containing nucleotides to nonphysiological hairpin regions,26−28 by targeting the 2′-OH position of RNA using a four-way junction,29 by DNAzyme-mediated strand ligation,30 by DNA-templated S-functionalized 6-thioguanosine-mediated pyrene fluorophore transfer onto G and C bases,31,32 as well as by Tb3+-dependent DNAzyme catalyzed addition of a 5′-GMP linker to an adenine 2′-OH position.33 Taken together, several different techniques for the site-specific labeling of long nucleic acids are presently known. However, there are also certain drawbacks such as low yields, nonspecificity, inflexibility, sequence restriction, high variability with regard to labeling efficiency, considerable alteration of the native sequence, and/or the addition of larger linkers, thus often leading to alterations of the three-dimensional structures and dysfunctions. 26 Accordingly, the demand for new alternative methods is high. The approach used in the present study is based on a DNAtemplated system. Such systems promote chemical reactions by generating a high effective concentration of reaction partners upon interstrand hybridization. We have previously used this technique for the postsynthetic conversion of an internal adenosine into 1,N6-etheno-adenosine (εA) under mild reaction conditions using DNA target strands of varying length, i.e., 16-, 40-, and 123mers.34 To achieve sequence specificity, we designed a reactive group that mimics the action of ethenoforming chloroacetaldehyde and attached it to a short complementary oligonucleotide.34 The reactive aldehyde functionality required for etheno-bridge formation was hereby masked by a 1,2-diol group to prevent premature reaction before strand annealing. Conversion of the vicinal diol group to the reactive aldehyde was performed on demand by oxidative cleavage with NaIO4 in situ. Here, we describe the further development of the method to allow the postsynthetic sequence-specific incorporation of a bioorthogonal alkyne group into an oligonucleotide sequence of any length (Figure 1). This alkyne group can be subsequently used to conjugate a fluorophore in a CuAAC reaction (click chemistry).35,36 The direct attachment of the fluorophore to a nucleotide poses one of the smallest possible alterations of the nucleic acid’s molecular structure. Importantly, as we demonstrate here, nucleobase labeling is the same for DNA and RNA, thus irrespective of, e.g., the presence of the ribose 2′-OH. The basic practicability of this labeling method for single-molecule fluorescence applications is demonstrated using a double-

Figure 1. General approach for the modification of specific sites in target strands (black) using a reactive strand (RS) consisting of a reactive group (gray circle and arrowhead) and a complementary recognition sequence (gray). Upon annealing of the reactive and target strand (step 1), the reactive group is placed in the correct position to induce the formation of the modification, i.e., in this case an alkynesubstituted εA base, in a sequence-selective manner (step 2). Subsequent attachment of a dye to this alkyne modified site yields a sequence-specifically labeled target strand (step 3).

labeled DNA duplex. In addition, single Cy3-labeling of the 633-nucleotide-long D135−L14 group II intron ribozyme construct was attempted. The D135 ribozyme is a truncated form of the group II intron Sc.ai5γ from Saccharomyces cerevisiae and contains the three domains, D1, D3, and D5, that are required for catalysis.37 Two loops were previously introduced into D1 and truncated D4 that allow hybridization with two fluorophore-carrying DNA-oligonucleotides for smFRET analyses.27 As each of the two loops in this D135−L14 construct features a 21-nt-long single-stranded region, these loops are ideally suited to test the presented new modification method directly on a large, biologically relevant RNA molecule.

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RESULTS AND DISCUSSION Sequence-Specific Formation of Alkyne Functionalized εA in DNA Target Strands. The reactive group required for the modification of an adenine base to its 12propargyl-ethenoA (12-propargyl-εA) derivative was prepared in its activated N-hydroxysuccinimide (NHS) ester form (9) in a multistep synthesis (Figure 2a). Compound 9 was subsequently coupled to a complementary amino-modified DNA strand (Supporting Information Figure 1) to yield the reactive sequences RS1 (12 nt) or RS2 (18 nt). RS1 was used for most of the εA-formation procedures described in this work (Figure 2b, Table 1, Supporting Information Figure 2). RS2 was applied for the modification of the group II intron ribozyme (Figure 7a, Table 1). A trade-off for the high reactivity of the reactive group in etheno-forming reactions is the hydrolytic sensitivity of the group at the sulfonate functionality at elevated temperatures (Supporting Information Figures 3, 4). Hence, all functionalization reactions described in the following were performed at RT. The sequence-specific generation of alkyne functionalized εA nucleobases with RS1 was first assessed using short complementary 16nt DNA strands with target A nucleobases in positions (n+1) to (n+4) 2559


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ACS Chemical Biology

Figure 2. (a) Synthesis of the reactive group 9 as NHS ester. The Barbier type reaction to form 4 results in two stereochemical isomers (erythro and threo). Consequently, the final product 9 also represents a mixture of diastereomers. DMAP, 4-dimethylaminopyridine; DCC, N,N′dicyclohexylcarbodiimid; DMF, dimethylformamide; NHS, N-hydroxysuccinimide; THF, tetrahydrofuran. (b) Synthetic scheme for the formation of reactive strand RS1 starting from a 3′-phophorylated oligonucleotide (for the DNA sequence, see Table 1). CTAB, cetyltrimethylammonium bromide; DMSO, dimethyl sulfoxide; K2HPO4, 100 mM K2HPO4 at pH 7.5; PDS, 2,2′-dipyridyl disulfide; PPh3, triphenylphosphine.

(T11−T14, Table 1) with n denoting the base in the respective target strand positioned opposite the 3′-cytosine base of RS1. First, duplex stability was evaluated by thermal melting studies showing a Tm value of 52.6 °C for the duplex formed between T12 and the phosphorylated reactive sequence precursor RS1OPO32−, which corresponds precisely to the calculated value assuming unmodified strands (52.6 °C). Due to its thermal instability, no melting studies were performed with RS1; however, the Tm value for the T12/RS1-SO3− pair, and hence the duplex with degraded RS1, is with 49.6 °C only slightly lower (Supporting Information Figure 15). The single steps of the functionalization reaction are depicted in Figure 3. After strand annealing at RT the reactive group was activated with NaIO4 yielding RS1* followed by quenching of excess NaIO4 with ethylene glycol to prevent degradation of the formed εA derivative.34 Formation of etheno-derivatives proceeds via the respective hydroxyethano intermediates,38 the stability of the latter varying with the type of derivative. A heating step accelerates the dehydration of the here formed 12-propargylhydroxyethanoA (T11-εA* in Figure 3) to 12-propargylethenoA (T11-εA). Product formation was followed and controlled with matrix-assisted laser desorption ionization

time-of-flight (MALDI-TOF) mass spectrometry as exemplified for T11, T11-εA*, and T11-εA in Figure 4. As neither the starting compounds nor the hydroxyethano derivatives show fluorescent properties, formation of highly fluorescent εA was monitored in real-time by recording the emission of the solutions at 350−550 nm upon excitation at 275 nm (Supporting Information Figure 7). Emission was largest for T13, followed by T12 and T14, and lowest for T11, and reached a stable plateau after 2−3 days and heating to 60 °C (Supporting Information Figure 8 for T11). Nevertheless, the fluorescence emission of εA derivatives strongly depends on the sequence context, and quenching can occur especially by stacking interactions with neighboring guanine bases. In contrast to strands T12−T14, such a neighboring guanine base is present in template T11. Hence, the site-selectivity of the reaction was further evaluated with polymerase stop assays (PSAs) using Thermus aquaticus (Taq) polymerase, which is effectively inhibited by etheno-adducts.39,40 For the assay, the 40nt-long template sequences T21−T26 with target adenines in the (n+1) to (n+9) positions (Table 1) were modified with RS1 as described above, and PSAs were performed using the 32 P-labeled primer P1 (Table 1). Product formation was 2560


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ACS Chemical Biology Table 1. List of Oligonucleotide Sequences Applied in This Studya

a

Reactive strand RS1 was used for most modification reactions, RS2 for the modification of the group II intron ribozyme. The short templates T11− T14 were used to follow εA formation via fluorescence, the long templates T21−T26 to assess the site-selectivity of εA formation in polymerase stop assays with the 32P-labeled primer P1. The two complementary strands T31 and P31 were applied in the smFRET measurements. The general applicability of the modification reaction to RNA was investigated with template RNA11. Sequences complementary to RS1 are underlined, target A bases highlighted in black. Sequences RS1, P1, and P31 are depicted 3′ to 5′ to facilitate visualization of complementarity.

considered that we previously showed that modification of cytosine residues to εC occurs under the same conditions.34 On the one hand, the reactivity toward both adenine and cytosine clearly enlarges the application range of the method. On the other hand, if site-specificity is an issue, the strict absence of A or C residues other than the target base in the (n+1) to (n+4) range has to be ensured. So far, the applicability of the method was only tested for single stranded structures, and it is clear that the bases around and including the potential modification site in the target strand need to be unpaired to allow annealing with the reactive strand. Broadening of the application range to double stranded sequences would require the melting of the duplex structures while keeping in mind the limited stability of the reactive group at elevated temperatures. A solution can probably be the use of disrupter sequences in a thermal preannealing step,33 followed by annealing of the reactive strand at RT to the now single stranded target region. Bioorthogonal Labeling of 12-Propargyl-εA Containing Strands with Azide-Functionalized Molecules. Labeling of the 12-propargyl-εA modified target strand T11-εA with azide-functionalized molecules via a CuAAC reaction was first tested using 3-azido-1-propylamine as a model compound under anaerobic conditions and in the presence of catalytic amounts of CuSO4, ascorbic acid, and tris(benzyltriazolylmethyl) amine (TBTA). 43 MALDI-TOF analysis reveals a predominant peak at 5048.7 Da corresponding to the expected product T11-εA-NH2 (Figures 3 and 4d;

analyzed by denaturing polyacrylamide gel electrophoresis (PAGE; Figure 5) and modification yields were calculated from the ratio between the amounts of fully extended and truncated, i.e., etheno-adduct containing, primer (Supporting Information Table 1). Yields are similar for T21 and T22, i.e., 66 ± 3% and 67 ± 5%, respectively. In contrast to the results from the fluorescence emission experiments, modification of the target adenine in the (n+3) position (T23) is, with 56 ± 3%, equally efficient within the error limits. As expected, yields decrease further with increasing distance from the target position, that is 41 ± 1% for (n+4) (T24), 11 ± 1% for (n+6) (T25), and 10 ± 1% for (n+9) (T26). Noticeable is the presence of double bands with varying intensities for the truncated products in most lanes of the PAGE gel, which most likely arise from a reduced site-specificity of polymerization arrest at the modification site. The inhibition of polymerases by 12propargyl-εA has never been studied before and hence neither the exact position nor the efficiency of inhibition is known, but similar effects were observed previously with other modifications.41,42 Evidently, the presented method is not strictly site-specific, which is due to the size and flexibility of the reactive group including the diamine linker. However, especially for larger nucleic acid constructs and considering that also the attached fluorophore labels display a large flexibility due to the linker length and size of the fluorophore, sequence-specific labeling in the (n+1) to (n+4) range is often sufficient. It should be also 2561


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Figure 3. DNA-templated formation of 12-propargyl-εA and CuAAC reaction of azide-containing compounds. After activation of RS1 with NaIO4, the active form RS1* with an electrophilic aldehyde moiety is formed that subsequently reacts with the target adenine, e.g. in target strand T11. The heating step after 3 days accelerates the conversion of the hydroxyethano intermediate T11-εA* to the 12-propargyl-εA structure T11-εA. In a last step, T11-εA is coupled to the model compound 3-azido-1-propanamine or the fluorophore derivative azide-Cy3 to yield T11-εA-NH2 or T11-εACy3, respectively. DMSO, dimethyl sulfoxide; NaOAc buffer, 0.1 M sodium acetate at pH = 5.6; TBTA, Tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl]amine. The numbering of atoms as in the purine ring was retained in all structures for simplicity and convenience and is indicated for T11 and T11-εA.

Figure 4. Reaction control with MALDI-TOF for the formation of T11 and RNA11 derivatives. (a) Unmodified target strand T11 (Mcalc, 4890.2 Da). (b) Modification reaction with RS1 yields a mixture of T11, T11-εA* (Mcalc, 4970.2 Da), and T11-εA (Mcalc, 4952.2 Da). (c) T11-εA* is completely transformed into T11-εA after heat treatment. (d) Reaction of T11-εA with 3-azido-1-propanamine yields T11-εA-NH2 (Mcalc, 5049.9 Da), while reaction with azide-Cy3 gives the fluorophore labeled form T11-εA-Cy3 (Mcalc, 5492.2 Da) in pure form after EtOH precipitation and purification with RP-HPLC (e). (f) The analogous modification of an RNA strand gives the final product RNA11-εA-Cy3 (Mcalc, 5480.7 Da), in which the 3′ nucleoside was lost due to β-elimination at the oxidized ribose moiety.

azide-functionalized Cy3 dye, a fluorophore that is frequently employed in nucleic acid single molecule fluorescence assays

Mcalc, 5049.9 Da). To perform bioorthogonal labeling with a fluorescent dye, T11-εA was incubated with 20 equiv of the 2562


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approximately 5.0 nm. After labeling with Cy3, the reaction mixture was passed over a NAP-5 column to remove excess Cy3 and the product analyzed with RP-HPLC and UV/vis spectroscopy (Supporting Information Figures 9 and 10). Native PAGE experiments using 1 equiv of T31-εA-Cy3 and 20 equiv of P31 clearly demonstrated the formation of an annealed duplex DNA with reduced mobility and emission in both the Cy3 and Cy5 channels (Supporting Information Figure 11). A pronounced decrease of the duplex stability (ΔTm ∼ 30 °C) was observed for the T31-εA-Cy3/P31 pair (Tm ∼ 32 °C) compared to T31/P31 (Supporting Information Figure 16). εA-induced duplex destabilization has been reported before and can be explained by inhibition of Watson−Crick base pairing47 and formation of the bulky εA-Cy3 adduct. However, when modifying, for example, a single nucleotide bulge within a long RNA, the overall decrease in Tm is expected to be much smaller. Having established hybridization of the two strands at RT, the suitability of the labeling method for single-molecule fluorescence was assessed using a total internal reflection fluorescence microscope equipped with a CCD camera.8 Single emitting fluorophores were observed in both the Cy3 and the Cy5 detection channel (Figure 6a). At direct Cy3 excitation, the majority of fluorescence trajectories display stable Cy3 emission at low intensities and high levels of Cy5 emission. This finding can be attributed to the occurrence of Förster resonance energy transfer from Cy3 onto Cy5 (Figure 6b). Upon Cy5 photobleaching, Cy5 cannot act as a FRET acceptor anymore, and a simultaneous increase of Cy3 emission intensity is observed (Figure 6b). Single-step photobleaching was also observed for Cy3 in some cases, as well as concomitant disappearance of Cy3 and Cy5 fluorescence (Supporting Information Figure 12). Single-fluorophore emission trajectories were used to calculate apparent FRET efficiencies and fluorophore stoichiometries over time (Figure 6b, Supporting Information). In the presence of an active Cy5 fluorophore, transfer efficiencies are consistently centered on 0.78, while fluorophore stoichiometry fluctuates around 0.5; i.e., Cy3 and Cy5 are present in a 1:1 ratio. The consistency between individual time traces is illustrated in Figure 6c, which shows a 2D FRET-stoichiometry histogram built from ∼100 000 time bins. These data demonstrate that our chemical method faithfully incorporates a single fluorophore into the DNA strand.26 The absence of subpopulations within the resolution

Figure 5. Position of 12-propargyl-εA formation was determined by PSA and denaturing PAGE (12%). Only radioactively labeled products, i.e., P1 and extended products of primer P1, are visible. Target strands without modifications lead to full length products, whereas modified target strands inhibit polymerization at or around the modification site resulting in truncated products of the respective length. Lane 1, P1; lane 2, PSA with unmodified target strand T21; lanes 3−8, PSA with modified target strands T21−T26 (Table 1). The double bands observed for most truncated products most likely arise from a reduced site-specificity of polymerization arrest by 12propargyl-ethenoA and do not indicate εA modification at different sites in the target strand.

(Figure 3).44 After purification (Supporting Information), analysis with MALDI-TOF showed a single peak at 5494.3 Da corresponding to the Cy3-labeled strand (Mcalc, 5492.2 Da; Figure 4e). The overall yield of the HPLC purified fluorophore labeled target strand T11-εA-Cy3 was approximately 25% based on absorption measurements at 260 nm. A certain reduction of the yield can also be attributed to self-reaction of the reactive group within RS1 (Supporting Information Figures 5 and 6). Application of Labeled 12-Propargyl-εA Containing Strands in Single-Molecule Biophysics. The basic suitability of the new labeling strategy for smFRET experiments was assessed using a pair of fluorophore-labeled DNA strands. P31, a 48nt-long strand that carries the FRET acceptor dye Cy5 at its 5′-end and a biotin moiety at its 3′-end for surface immobilization, was obtained commercially.45,46 The 40nt-long strand T31 is complementary to nucleotides 7−46 of P31 and was also obtained commercially in its nonmodified form. The adenine base 10 (bold in Table 1) was postsynthetically functionalized and subsequently labeled with the FRET donor dye Cy3 using the here described protocol. After strand annealing, the two dyes are separated by 11 base pairs, corresponding to approximately 3.7 nm, which should result in a high FRET efficiency given a Förster radius of the two dyes of

Figure 6. Single-molecule imaging of surface-immobilized, postsynthetically labeled DNA. (a) Averaged fluorescence images of the surface-tethered P31/T31-εA-Cy3 duplex with pseudocolors (Cy3, green; Cy5, red). (b) Representative fluorophore emission, apparent FRET, and fluorophore stoichiometry time traces. Single-step photobleaching of Cy5 results in the disappearance of FRET and a change in the Cy3:Cy5 stoichiometry from 1:1 to 1:0. (c) 2D FRET, fluorophore stoichiometry histogram built from 910 000 data points. Consistent FRET and stoichiometry values are observed throughout the data set. 2563


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Figure 7. Direct labeling of the D135−L14 group II intron ribozyme construct. (a) Primary and secondary structure of the D135−L14 ribozyme.27 The target adenine A803 within the D4 loop is highlighted in green. The complementary 18-nt-long reactive sequence RS2 is indicated in blue; the reactive group is attached to the 5′-end adenine marked in magenta. (b) Denaturing PAGE (8%) to evaluate the direct labeling of D135−L14. The left side shows the ethidium bromide (EB) fluorescence under UV light; the right side shows the gel in the Cy3 channel. Lane 1 contains the free Cy3 dye (1.0 μM, 1.0 μL); lane 2, D135−L14 (1.3 μM, 5 μL); lane 3, a mixture of the free Cy3 dye (1.0 μM, 1.0 μL) and D135−L14 (1.3 μM, 5 μL); and lane 4, the D135−L14_A308(Cy3) sample after EtOH precipitation (concentration not determined). The RNA containing band in lane 4 is faint due to low concentration after the labeling and purification procedure. Broad intensive bands above and below the D135−L14_A308(Cy3) construct are due to dye derivatives originating from the CuAAC coupling reaction and an excess of free Cy3 dye.

Postsynthetic Labeling of a 633-nt-long Group II Intron Ribozyme Construct. The sequence and proposed secondary structure of the construct D135−L14 is depicted in Figure 7a. The chosen target position A803 (marked in green) is located in the D4 loop, and the respective 18-nt-long reactive sequence RS2 (marked in blue) is designed to hybridize to the entire single stranded loop region. The modification and labeling protocol was only slightly adapted (see the Methods section), involving in particular purification of the ribozyme after the εA forming reaction using 8% denaturing PAGE. After Cy3-labeling and EtOH precipitation of the RNA, further analysis was performed using denaturing PAGE (8%). Total RNA was visualized with ethidium bromide (EB) staining, free or bound Cy3 dye by detecting its fluorescence in the Cy3 channel. The RNA obtained after labeling shows the same running behavior and hence comparable molecular mass and charge as the unmodified D135−L14 construct and in addition can be visualized in the Cy3 channel. Mere intercalation of free Cy3 dye can be excluded as the control sample consisting of a mixture of unmodified D135−L14 and free Cy3 dye does not show Cy3 fluorescence at this position in the gel. Accordingly, covalent Cy3 attachment to the large ribozyme construct using the here presented method was successful. No further evaluations concerning yield of the modification and labeling reaction as well as site-specificity of the modification were performed. We could thus demonstrate that DNA as well as RNA can be successfully sequence-specifically modified and labeled with fluorophore dyes suitable for smFRET studies.

limits of the technique confirms sequence-specific Cy3 incorporation into strand T31. Postsynthetic Labeling of RNA. RNAs are versatile molecules that play essential roles in almost every aspect of cellular metabolism.48 As a consequence, they are in the focus of numerous biophysical studies, many of which require their specific labeling with fluorophores. However, RNA is considerably less stable than DNA and, among others, prone to hydrolytic backbone cleavage. Hence, we also attempted, as a proof-of-principle, the sequence-specific introduction of a 12propargyl-εA moiety into a short RNA sequence to evaluate its stability under the reaction conditions applied (Supporting Information Figure 13). For this, we used the RNA equivalent to template T11, i.e., strand RNA11 (Table 1), and the modification protocol described above. RP-HPLC purified RNA11-εA-Cy3 (Supporting Information Figure 14) was obtained in 11% yield. MALDI-TOF analysis shows one peak centered around 5480.9 Da corresponding to the Cy3-labeled RNA strand without the 3′-terminal cytosine (Mcalc, 5480.7 Da; Figure 4f). It is known that NaIO4 oxidizes the 2′-3′-vicinal diol at the terminal ribose moiety of RNA to yield the 2′,3′dialdehyde. While this dialdehyde is prone to β-elimination leading to cleavage of the terminal nucleoside as observed here,49 it can also be used for further (fluorescent) labeling upon reaction with hydrazine derivatives followed by reduction with sodium cyanoborohydrate.19 Importantly, no further modification or degradation due to the reaction conditions was observed. The loss of the 3′-terminal nucleoside can be easily compensated by the addition of an additional and dispensable nucleotide during in vitro transcription and does not pose a drawback of the method. 2564


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propargyl bromide in the presence of elemental zinc yielded the alkyne-substituted glycerol derivative (4) in a mixture of stereoisomers. Coupling to 3-chlorosulfonyl-benzoic acid methyl ester (5), subsequent hydrolysis of the methyl ester moiety, and deprotection of the diol group afforded compound 8. Finally, activation of the carboxyl group in 8 yielded the reactive group as an NHS ester (9), which was directly used for the synthesis of the reactive strand (RS). For this purpose, a commercially obtained 3′-phosphorylated complementary oligonucleotide sequence was modified with 1,2-diaminoethan as described51,52 and reacted with compound 9 (Figure 2b).53 The resulting reactive strands RS1 and RS2 were purified and RS1 characterized by MALDI-TOF (Supporting Information Figure 2). To avoid hydrolysis of the reactive group at the sulfonate functionality that occurs at elevated temperatures, all primer-template annealing procedures were performed at RT (Supporting Information Figures 3 and 4). Selected NMR and ESI-MS spectra for RS1 can be found in the Supporting Information (Figures 17−26). General εA-Formation Procedure with Reactive and Target Strands. In a typical procedure, the reactive strand (0.6 nmol, 5 μL) and the respective target strand (0.3 nmol, 5 μL) were mixed with 60 μL of NaCl (1 M) and 50 μL of NaOAc (20 mM, pH = 5.5) and incubated for 15 min. After the addition of 30 μL of 10 mM NaIO4 in NaOAc (20 mM, pH = 5.5), the mixture was incubated in a thermomixer (25 °C, 500 rpm) for 90 min. NaIO4 was quenched with 30 μL of 15 mM ethylene glycol in NaOAc (20 mM, pH = 5.5), and incubation was continued under the same conditions. After 3 days, the reaction mixture was heated to 60 °C for 2 h to accelerate dehydration of the hydroxyethanoA intermediate. Reaction mixtures were used without dilution or purification for fluorescence measurements and RP-HPLC analyses. Samples were purified with NAP-5 columns (GE Healthcare) or with ZipTip C18 (EMD Millipore) for PSAs (see below) and MALDI-TOF MS measurements. εA-Formation Procedure of the D135−L14 Group II Intron Ribozyme Construct. Hybridization of RS2 and the D135−L14 construct was performed according to the published protocol.27 In detail, D135−L14 (15 μL, 3.5 μM) was heated at 90 °C for 1 min, incubated at 42 °C for 3 min, and then combined with 1 μL of NaCl (5 M) and a solution of RS2 in 15 μL of NaOAc (1.0 M, pH 5.5). The mixture was incubated at RT for 10 min. After the addition of 15 μL of 20 mM NaIO4 in 20 mM NaOAc (pH 5.5), the mixture was incubated in a thermomixer (25 °C, 500 rpm) for 90 min. NaIO4 was quenched with 30 μL of 15 mM ethylene glycol in 20 mM NaOAc (pH 5.5), and incubation was continued under the same conditions. No heating step to 60 °C to accelerate εA formation was performed. The reaction was stopped after 2 days by EtOH precipitation. Precipitates were collected and subjected to purification using denaturing PAGE (8%). The modified D135−L14 was recovered by “crush and soak” in a buffer containing 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS, pH 6), 1 mM EDTA, and 250 mM NaCl followed by precipitation with three volumes of EtOH. Polymerase Stop Assay (PSA). Reaction mixtures obtained from the εA-formation reaction as described above were dried after ZipTip C18 purification and redissolved in ddH2O to obtain a concentration of 40 nM, calculated based on the extinction coefficient of the respective target strand. A total of 5 μL of this solution was combined with ddH2O (7.5 μL), GoTaq buffer (4 μL), and 32P-labeled primer P1 (1 μL). P1 and the target strand were annealed by incubation at 90 °C for 2 min and slowly cooling down. After the addition of dNTPs (0.5 μL, 10 mM) and Taq DNA polymerase (2 μL, 0.2 U), the mixtures were incubated at 55 °C for 30 min. The reactions were quenched with 15 μL of stop solution (82% formamide, 0.16% xylene cyanol, and 0.16% bromophenol blue) and incubated at 90 °C for 5 min prior to analysis by 12% denaturing PAGE. Visualization and quantification was conducted on a Typhoon Scanner FLA 9500 with ImageQuant TL (1D gel analysis, background subtraction Rolling Ball: 500). General Procedure for the Cu(I)-Catalyzed Huisgen 1,3Dipolar Cycloaddition. A total of 50 μL of DMSO, 8 μL of 10 mM ascorbic acid (80 nmol), and either 3-azido-1-propanamine or the azide-Cy3 derivative (80 nmol in 20 μL of DMSO) were added to a

CONCLUSIONS Postsynthetic modification of nucleic acids is an appealing strategy to introduce labels for fluorescence-based biophysical studies, not least because this also allows, e.g., the labeling of native sequences or constructs obtained by in vitro transcription. Applying the principle of templated synthesis, we used a complementary reactive strand to generate 12-propargylεA derivatives within target strands of different lengths with yields of approximately 65%. With a basic smFRET experiment, the specific and stoichiometric subsequent labeling with an azide Cy3 fluorophore via click chemistry was shown. The templated strategy puts no limits to the total length of the target strand as demonstrated with the successful labeling of a 633-nt-long RNA construct, and hence this strategy overcomes a significant obstacle in fluorescence-based biophysics. With the reported basic smFRET experiment, we could further show that the amount of labeled strands obtained is more than sufficient to perform the biophysical measurements. Taken together, our method overcomes many of the existing drawbacks: (i) targeting the adenine nucleobase, both DNA and RNA can be labeled; (ii) the introduced etheno propargyl linker, i.e., “C5H4,” ensures minimal perturbation of the nucleic acid; (iii) the reaction conditions are such that no internal degradation is observed. In addition, great flexibility for labeling is ensured as the postsynthetic incorporation of any azidefunctionalized compound is possible. For application in smFRET studies, a second dye can be introduced either by using a labeled complementary strand as demonstrated here, by additional end-labeling of the modified strand, or by using the here presented approach but with introduction of a functional group different from an alkyne.

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METHODS

Synthesis and characterization of chemical compounds are described in detail in the Supporting Information. Outlines of key procedures are detailed in the following. Materials. Unless stated otherwise, starting materials were obtained in the highest commercial grades and used without further purification. Solvents for organic syntheses were distilled prior to use and, if necessary, dried under standard conditions. HPLC solvents were obtained in HPLC grade from Roth AG and Sigma-Aldrich, deuterated solvents were purchased from Armar AG. H2O used for all manipulations with oligonucleotides was purified with a TKA GenPure dispenser and water as well as buffer solutions for enzymatic reactions were sterilized either by autoclaving or sterile filtration. All unmodified oligonucleotide strands were purchased from Microsynth AG with the exception of the biotin and Cy5 labeled strand P31 that was obtained from Lumiprobe GmbH. Oligonucleotides were dissolved in water at a concentration of 100 μM. The azide-Cy3 derivative was prepared as a 0.1% w/v solution in dimethylformamide (DMF) immediately before use. Enzymes for radioactive labeling and PSAs were purchased from Promega AG (Calf Intestinal Alkaline Phosphatase, GoTaq DNA polymerase with 5X Colorless GoTaq Reaction Buffer) and New England Biolabs (T4 Polynucleotide Kinase). [γ-32P]-ATP for radioactive labeling of primer P1 was obtained from PerkinElmer AG at a concentration of 150 mCi mL−1. 1 H and 13C NMR spectra were recorded on a Bruker ARX-300 or AV-400 spectrometer, ESI mass spectra on a Bruker Esquire 6000 spectrometer, and MALDI-TOF spectra on an Autoflex time-of-flight mass spectrometer with a 337 nm nitrogen laser, and fluorescence measurements were carried out on a Varian Cary Eclipse fluorescence spectrophotometer. Synthesis of the Reactive Strand (RS). For the synthesis of the reactive group, D-mannitol (1) was selectively protected with 2,2dimethoxypropane and subsequently oxidized with NaIO4 to afford stereopure protected glyceraldehyde (3, Figure 2a).50 Treatment with 2565


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ACS Chemical Biology Notes

solution of the respective 12-propargyl-εA containing target strand (4 nmol, 150 μL) in 1 M triethylammonium acetate (TEAA) buffer at pH = 7.2. The solution was briefly vortexed and saturated with argon for 30 s before 4 μL of a 10 mM solution containing CuSO4 and the TBTA ligand in a 1:1 ratio (40 nmol) in H2O/DMSO (45:55) was added. The solution was saturated with argon for 1 min and the vial closed. After incubation for 16 h at 20 °C and precipitation with 700 μL (∼3 volumes) of EtOH in the presence of 10 μL of 3 M NaCl, the resulting product was dissolved in deionized water and purified by RPHPLC (Supporting Information Figure 10). smFRET Imaging and Data Processing. Microfluidic channels were built from quartz slides (Finkenbeiner) and passivated with biotinylated BSA.8 The 48nt-long strand P31 was obtained commercially with the acceptor dye Cy5 attached to its 5′-end and a biotin moiety to its 3′-end and was tethered to the surface via a biotin−streptavidin linkage. Subsequently, the postsynthetically functionalized and subsequently Cy3 labeled strand T31-εA-Cy3 was annealed with P31 at RT for 5 min. Excess dye was washed out, and smFRET clips were recorded in 50 mM MOPS, 100 mM KNO3, 1% w/v D-glucose, 1× enzymatic oxygen scavenging solution (165 U/ mL glucose oxidase, 2170 U/mL catalase), and 1 mM 6-hydroxy2,5,7,8-tetramethylchroman-2-cyclooctatetraene (Trolox, pH 6.90). Cy3 and Cy5 emissions were followed for 18 min at a time resolution of 300 ms using a home-built total internal reflection fluorescence microscope at alternating laser excitation as described.8,54−56 Clips were corrected for background noise and cross-talk, followed by spot detection and colocalization of corresponding coordinates. Photon counts (PC) were determined via integration over 3 × 3 pixels around the central coordinates, yielding three emission timedependent PCs: Cy3 emission upon Cy3 excitation, Cy5 emission upon Cy5 excitation, and Cy5 emission upon Cy3 excitation Cy5em Cy5em (PC(t)Cy3em Cy3ex , PC(t)Cy5ex , PC(t)Cy3ex ). Single-fluorophore emission trajectories were used to calculate apparent FRET efficiencies and fluorophore stoichiometries over time using the following two equations:54 FRET (t ) =

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank E. Fiorini for providing the D135−L14 group II intron ribozyme sample. This work was supported by the University of Zurich [Forschungskredit to D.E. and S.L.B.K.], an ERC starting grant [MIRNA No. 259092 to R.K.O.S.], the Swiss National Science Foundation [to R.K.O.S. and E.F.], SystemsX.ch [to R.K.O.S.], and the COST Action CM1105.

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DEDICATION This work is dedicated to Prof. Dr. Bernhard Lippert with the very best wishes on his 70th birthday.

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PC(t )Cy5em Cy3ex Cy5em PC(t )Cy3em Cy3ex + PC(t )Cy3ex

stoichiometry (t ) =

(1)

Cy5em PC(t )Cy5em Cy3ex + PC(t )Cy5ex Cy5em Cy5em PC(t )Cy3em Cy3ex + PC(t )Cy3ex + PC(t )Cy5ex

(2) Data analysis was performed using the MATLAB-based software package MASH (D. Kowerko, M. Hadzic, R. Börner, S. L. B. König, M. Ritter, R. K. O. Sigel, unpublished results).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00343. Experimental procedures including syntheses and analyses of compounds (Figures 1−6, 17−26), investigation of ε-base formation in the target strands (Figures 7 and 8, Table 1), performance of the CuAAC reaction (Figures 9−11), smFRET measurements (Figure 12), analyses of melting temperatures (Figure 15 and 16), and RNA modification (Figures 13 and 14) (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0041-44-635-4621. E-mail: [email protected]. Author Contributions †

Both authors contributed equally 2566


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