Article pubs.acs.org/bc
Site-Specific Difunctionalization of Structured RNAs Yields Probes for microRNA Maturation Ugo Pradère and Jonathan Hall* Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland S Supporting Information *
ABSTRACT: Modified oligonucleotides bearing multiple functional labels are valuable tools in RNA biology. Efficient synthetic access to singly modified short DNAs and RNAs has been developed in the past years and paved the way to a first generation of oligonucleotide tools. Here, we describe an efficient procedure for the site-specific hetero bis-labeling of long RNAs. We exemplified the method with the preparation of Cy3/Cy5 pre-microRNAs labeled at selected internal sites as probes for microRNA maturation.
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for long structured RNAs. The Carell group was the first to report a click-based method for the heterolabeling of DNAs in which trimethylsilyl- and triisopropylsilyl-protected alkyne groups were selectively unmasked under acidic and fluoridic conditions, respectively, prior to conjugation of the labels.20 Although this works well on small DNAs, it is hardly applicable to long RNAs because acid treatment also causes loss of the terminal dimethoxytrityl (DMT) group, which is needed for the separation of full-length RNA product from shorter failure sequences. Furthermore, some degree of cross-labeling (conjugation of the
INTRODUCTION The site-specific modification of oligonucleotides using azide/ alkyne copper(I) catalyzed cyclo-addition (click) reactions has become the method of choice in recent years for a variety of applications including cross-linking,1−6 strand-ligation,7−10 or labeling with, for example, dyes.11 Thus, procedures are wellestablished for a single labeling or a multiple homolabeling of oligonucleotides using click (alkyne-bearing oligonucleotide)12−16 or reverse (or inverse)-click (azide-bearing oligonucleotide)17−19 reactions. Although some examples of heterolabeling of DNA and RNAs have been reported, significant drawbacks limit their use
Scheme 2. Approach Used to Evaluate Conditions for Azidation of CPG-Bound Oligoribonucleotides: Azidation of Pre-miR-21 at its 3′-End
Scheme 1. Synthetic Strategy for Preparation of Cy3/Cy5 Bis-Labeled pre-miRNAs Using 2′-O-Propargyl/2′-OMethylenetriazolobutylbromide Modified Nucleosides in a Click/Reverse-Click Procedure
Received: December 9, 2015 Revised: January 18, 2016
© XXXX American Chemical Society
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DOI: 10.1021/acs.bioconjchem.5b00661 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry Scheme 3. Approach Employed to Evaluate Click Conditions in Solutiona
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(a) Structure of Cy3 alkyne 2, Cy5 alkyne 3, and Cy3 azide 4. (b) Time course of Cy5-labeling of pre-miR-21at its 5′-end.
pre-miRNAs (Scheme 1), which were subsequently used to sense for distinct steps of miRNA maturation in an in vitro assay.
label to an undesired alkyne site) is inevitably observed on long RNA structures because click reactions are rarely quantitative.21 Using an approach similar to that reported by Wengel,22 we described recently bis-heterolabeling of long RNAs in which solid phase synthesis was interrupted for selective coupling of the first label on solid support.21 Although this afforded the desired modified RNAs, cross-labeling produced complex mixtures in some cases which were hardly separable. Brown1 and Micura23 both reported bis-heterolabeling of RNA using a combination of click or reverse-click and activated-acid-amine coupling reactions. Although this procedure circumvents cross-labeling, the use of the azido-modified nucleoside is restricted to the 3′-end of RNA due to the incompatibility of azides and the phosphoramidite method.24 The group of Morvan was the first to combine click and reverse-click methods on oligonucleotides using an alkyne- and a bromide-bearing phosphoramidite at the terminal positions of short DNAs.25 The absence of cross-labeling and the mild reaction conditions appeared to be suitable for the bis-heterolabeling of long RNAs. Here we report the development of a practical, site-specific heterobis-labeling of long RNAs using a click/reverse-click procedure. A single, readily available nucleoside precursor served as the source for both sites of conjugation. We exemplified the approach with preparation of several Cy3/Cy5 bis-labeled
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RESULTS AND DISCUSSION We investigated 2′-O-haloalkyl nucleoside phosphoramidites as a means to introduce azido-modified ribonucleosides. We synthesized 2′-O-methylenetriazolobutyl bromide cytidine phosphoramidite 1 in two steps from the DMT-protected 2′-O-propargyl-cytidine intermediate by click reaction with 1-azido-4-bromobutane, followed by phosphitylation (Supporting Information). The ready accessibility of 2′-O-propargylmodified ribonucleosides contributes important flexibility to our overall approach. As long RNAs are less reactive and generally more difficult to functionalize than short RNAs on solid support21particularly close to their 3′-endswe optimized each step of the synthesis, beginning with azidation of bromo-containing ribonucleosides on solid support. Pre-miR21 modified at its 3′-end (ORN1; Supporting Information) was used as a model substrate. Morvan et al. described quantitative azidation of short DNAs using 200 equiv of NaN3 and NaI for 1.5 h at 65 °C.25 We used a milder temperature of 45 °C in order to help ensure RNA stability. Controlled-pore glass (CPG) bearing a protected oligoribonucleotide was distributed equally B
DOI: 10.1021/acs.bioconjchem.5b00661 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
conjugation efficiency, and the second label by reverse-click reaction in solution (azide modified RNA). We validated this protocol with the conjugation of Cy3 and Cy5 fluorescent dyes at various internal and terminal positions of pre-miR-21 (ORN3−6, Table 1). Thus, the CPG-bound
into different vials to ensure identical starting materials for azidation reactions carried out over 1, 2, 4, and 8 h. Oligonucleotide deprotection with aqueous methylamine/ammonia and HF.Et3N treatment, followed by reverse-phase HPLC, showed two separated peaks in the chromatogram, one of which was the desired azidoproduct (Peak B, ORN1-N3). Analysis of the faster-eluting peak (Peak A) by mass spectrometry suggested that it contained unreacted starting material and products formed from substitution and elimination of the bromide by methylamine (Scheme 2). After 4 h, a maximum 80% conversion of bromide to azide had occurred (Supporting Information 3.1). In order to increase the conversion, harsher conditions were evaluated including: a 5-fold increase in NaN3 and NaI concentrations and equivalents; replacement of NaN3 and NaI by the corresponding lithium salts; and an increase in reaction temperature to 65 °C (Supporting Information 3.2). However, none of these conditions raised the conversion above ∼85%. We then investigated the reverse-click reaction between an azide-modified RNA and an alkyne-modified label on solid support and in solution. We examined the reverse-click conjugation of Cy3-alkyne 2 (Scheme 3a, Supporting Information 7.2) to ORN2-N3 on solid support using our previously reported conditions for click labeling.21 Although the desired Cy3labeled product (ORN2-Cy3) was isolated, RNA degradation (>40%) and loss of the DMT group (>65%) occurred (Supporting Information 5) to a much greater degree than previously observed.21 We attempted to resolve this with modifications to the protocol including a 5-fold reduction of the catalyst system; a 5-fold increase of tris(benzyltriazolylmethyl)amine (TBTA) ligand; use of the alternative ligand (tris(hydroxypropyltriazolylmethyl)amine (THPTA); exchange of water for PBS buffer; and the addition of TRIS base. None of these conditions reduced the RNA degradation (Supporting Information 5), though the addition of TRIS base and PBS buffer avoided loss of the DMT group. Solution phase reverse-click reactions were evaluated with Cy5-alkyne 3 (Scheme 3a, Supporting Information 7.3) and purified 5′-N3-pre-miR-21 (ORN2-N3), which was previously prepared by azidation of 5′-Br-pre-miR-21. It is necessary to perform conjugation reactions in solution when labels (e.g., Cy5) are unstable to the harsh basic conditions of oligonucleotide deprotection. Following the conditions of Finn,26 ORN2-N3 was reacted at 25 °C for 1 h with 3 in a 1:1 mixture of water/PBS buffer containing CuSO4·5H2O, sodium ascorbate and THPTA ligand. 5′-Cy5-pre-miR-21 (ORN2-Cy5) was obtained in 26% conversion (Scheme 3, Supporting Information 4). Extending the reaction time to 3 h increased the conversion moderately (49%). However, increasing the temperature to 45 °C enhanced the reaction conversions to 86% and 95% after 1 and 2 h, respectively. Under these conditions RNA degradation accounted for roughly 10−12%, similar to that seen at 25 °C. When we applied these optimized conditions to pre-miR-21, this time modified at its 3′-end, we obtained a quantitative yield of the Cy5 labeled product (ORN1-Cy5, Supporting Information 4). This solved the previously reported limitation of low labeling efficiency close to the 3′-end of the RNA on solid support, probably due to steric hindrance with the CPG.21 Taken together, it seemed clear that the best procedure for the bis-labeling of long RNAs would be the introduction of the first label by click reaction (propargyl modified RNA) on solid support at the position closest to the 5′-end (i.e., furthest from the 3′-end) to minimize any influence of the CPG on the
Table 1. Post-Synthetic Site Specific Bis-Labeling of Pre-miRNAs at Different Positions with Cy5 Alkyne 3 Cy3/Cy5 PremiRNA
position modif.a
ORN3 (miR-21) ORN4 (miR-21) ORN5 (miR-21) ORN6 (miR-21) ORN-7 (miR-106a) ORN-8 (miR-106a) ORN9 (miR-124) ORN10 (miR-124) ORN11 (miR-20b) ORN12 (miR-20b) ORN13 (miR-122) ORN14 (miR-122)
1/60 32/60 13/45 13/49 8/52 8/35 7/49 32/49 8/52 8/30 11/50 11/31
conv. clickb
conv. azid.c
conv. reverse clickd
> > > > > > > > > > > >
72% 68% 66% 65%
> > > > 66% 59% 62% 63% 72% 63% 70% 71%
90% 90% 90% 90% 90% 90% 90% 90% 90% 90% 90% 90%
95% 95% 95% 95%
overall conv. 62% 58% 56% 56% 63% 53% 59% 60% 65% 57% 63% 64%
a Nucleotide number of the modification (alkyne-/bromo-) from the 5′-terminus. bClick performed on solid support: estimated conversions in the click reactions were calculated from the peak integrals of products and starting materials. cAzidation performed on solid support: estimated conversions in the azidation step were calculated from the peak integrals of products and starting materials. dReverseclick reaction performed on solution phase: estimated conversions in the click reaction were calculated from the peak integrals of products and starting materials.
oligoribonucleotides were subjected to click conjugation with the Cy3-azide 4. They were then converted to azides, cleaved from the CPG, deprotected (NH4OH/MeNH2, HF·Et3N) and purified by HPLC. This yielded Cy3-monolabeled intermediates primed with azide for the second conjugation reaction (ORN3−6-Cy3/N3, Scheme 4a, Supporting Information 6). Although the DMT-on HPLC step efficiently removed failure sequences and unreacted alkyne groups, the aforementioned side products resulting from the reaction of methylamine with unreacted bromides were not separated at this stage (Scheme 4b left panel, Supporting Information 6). However, these were easily removed at the final product purification (vide infra). For the second conjugation of ORN3−6 by reverse-click reaction with Cy5-alkyne 3, heating the reaction mixtures at 45 °C for 2 h only partially converted the starting materials. However, increasing the temperature to 65 °C provided a complete conversion without any obvious additional degradation. A final HPLC purification then isolated the desired bislabeled Cy3/Cy5 product (ORN3−6-Cy3/Cy5, Table 1), and separated cleanly the aforementioned side-products from the first labeling reaction (Scheme 4b right panel, Supporting Information 6). This complete procedure worked equally well on other pre-miRNAs including pre-miR-106a, −124, 20b, and −122 (ORN7−14-Cy3/Cy5; Table 1) in overall conversions ranging from 53% to 65% with cyanine dyes positioned either in the miRNA guide and passenger strands or in the guide and the terminal loops (TL). Oligonucleotides bearing fluorescent dyes forming a fluorescence resonance energy transfer (FRET) pair are valuable tools for biology in a wide array of applications.27,28 For example, Cy3 and Cy5 are commonly used dyes, in which C
DOI: 10.1021/acs.bioconjchem.5b00661 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Scheme 4. (a) Optimized Method for the Synthesis of Bis-Labeled Pre-miRNAs. (b) HPLC Spectra with Different Solvent Gradients of, Respectively, Crude ORN6-Cy3/N3 and Crude ORN6-Cy3/Cy5
3′-terminus with the Cy3 donor (Figure 1). This was attributed to the greater distance between the fluorophores of ORN4Cy3/Cy5, although a difference in the orientation of the dipole moment might also have been partly responsible.30 Interestingly, despite the apparent proximity of donor and acceptor at the 5′ and 3′-termini of ORN3-Cy3/Cy5, only a moderate FRETmidway between that of ORN4-Cy3/Cy5 and ORN6Cy3/Cy5was observed. The digestion of pre-miRNAs by RNase A caused a strong drop (>5-fold) of fluorescence emission at 675 nm after excitation of the three bis-labeled probes, consistent with the loss of FRET. A small decrease observed with the controls was in line with reports that the fluorescence of cyanine dyes is enhanced when bound to oligonucleotides (∼2 fold), which is presumably lost after digestion.31 The processing of a pre-miRNA by the DICER enzyme cleaves the TL region and yields a miRNA duplex composed of guide and passenger strands.32 This partially complementary duplex is then bound by Argonaute proteins to form the activated RNA-induced silencing complex (RISC), from which the miRNA passenger strand is ejected. The rate of this multistep process is influenced by several factors including sequence of the pre-miRNA,33 phosphorylation of the
nonradiative energy is transferred from the donor (Cy3) to the acceptor (Cy5) when they are positioned in close proximity (
