Expanding the Scope of 2′-SCF3 Modified RNA

The 2′-trifluoromethylthio (2′-SCF3) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2′-SCF3 adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc-protecting group strategy, are reported. RNA solid-phase synthesis provided site-specifically 2′-SCF3-modified oligoribonucleotides that were investigated intensively. Their excellent behavior in 19F NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule–RNA interaction. Moreover, comparably to the 2′-SCF3 pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off-target effects and their performance in silencing of the BASP1 gene was demonstrated.


Results and Discussion
Synthesis of 2'-SCF 3
We point out that the Bocp rotection concept proved very convenient and was the key for the high efficiencyo ft he synthesis.T herefore, we currently plan to integratet he Boc approach for the syntheses of other 2'-modified (e.g.,2 '-SeCH 3 or 2'-N 3 ) [26,27] guanosine building blocks as well.

Synthesis of 2'-SCF 3 -containing RNA
We used the 2'-O-TOM approach for the solid-phase synthesis of RNA with site-specific 2'-SCF 3 adenosine andg uanosine Scheme2.Synthesis of 2'-SCF 3 guanosine phosphoramidite G7.Starting compound G1 was obtained according to ref. [27].R eactionconditions: a) i. 1.5 equiv F 3 CSO 2 Cl, 3.0 equiv DMAP, in CH 2 Cl 2 ,08C, 20 min;i i. 1.5 equiv CH 3 COS À K + ,1.5 equiv 18-crown-6, 1.5 equiv EtN(iPr) 2 in toluene, 16   www.chemeurj.org modifications. [31,32] Coupling yields of the two novel building blocks were higher than 98 %a ccording to the trityl assay.T he oligoribonucleotides were cleaved from the solid support and deprotected using CH 3 NH 2 in ethanol/H 2 O, followed by treatment with tetrabutylammonium fluoride( TBAF) in tetrahydrofuran (THF). Subsequents ize-exclusion chromatography on aS ephadex G25 column removed salts. The RNA sequences were purified by anion-exchange chromatography under strong denaturating conditions (6 m urea, 80 8C). The molecular weights of the purified RNAsw ere confirmed by liquid chromatography (LC) electrospray ionization (ESI) mass spectrometry (MS). As election of 2'-SCF 3 RNA sequences is listed in the Supporting Information, Ta ble S1. Noteworthy,2 '-SCF 3 guanosine (such as the previously investigated 2'-SCF 3 uridine and2 '-SCF 3 cytidine) [10,11] appeared completely stable under the repetitive oxidative conditions (20 mm aqueous iodine solution) required duringR NA solid-phase synthesis for the transformation of P III into P V ,a nd the subsequentd eprotection ( Figure 1). Unexpectedly,t he 2'-SCF 3 adenosine label turned out to be sensitive duringt he standard deprotection procedure. Only when we added millimolar amountso fthreo-1,4-dimercapto-2,3-butandiol( DTT), ah igh quality of the crude deprotected RNA was achieveda sa nalyzed from the corresponding ion-exchange HPLC traces ( Figure 1B)a nd mass spectrometric experiments. We hypothesize that possible oxidation products (such as sulfoxides, 2'-SOCF 3 )o ft he protected RNA were reduced by this additive,a nd hence, follow-up side-productst hat otherwise dominated during RNA deprotection at high pH values (as ar esult of sulfoxide elimination and successive strand cleavage) could not form any more. This observation is reminiscent of the chemical synthesis of 2'-SeCH 3 RNA that we investigated severaly ears ago; [33] for 2'-SeCH 3 guanosine-modified RNA, the corresponding oxidation products were analyzed in detail by mass spectrometry,a nd additionally isolated and quantitatively reduced by DTT. [26] Unfortunately,o ur attempts to isolateo xidized speciesof2'-SCF 3 RNA have failed so far.

Probing of RNA structures by 19 FN MR spectroscopy
To evaluate the applicability, and importantly,t he uniformity of the 2'-SCF 3 labeling concept not only with respect to pyrimidine but also with respectt op urine nucleosides, we demonstrate as ingle-case study for NMR spectroscopicR NA probing here. Figure 4A depicts the secondary structure model for the Thermoanaerobactert engcongensis preQ 1 class-I riboswitch. [37][38][39] This RNA becomes preorganized into ap seudoknot fold when Mg 2 + is presenta tp hysiological concentrations. The distribution between the stem-loop fold (SL) with an unpaired strand overhang and the more compact RNA pseudoknot (P)w as nicely reflected by the two 19 Fr esonances at À40.2 and À40.5 ppm, in 3:7r atio ( Figure 4B,m iddle). Ligand addition in fourfold excessr esulted in ad ominant population of the preQ 1 -bound RNA complex (C), reflected by an ew signala t À40.8 ppm ( Figure 4B,b ottom). Thes implicity of the population analysis based on 19 Fs pectra becomes obvious from ad irect comparison with the corresponding NH imino proton 1 HNMR spectra ( Figure 4C). Note that the imino protons exchange with the solvent and therefore result in much weaker intensities for the two dynamic, ligand-unbound RNA confor- For the 2'-SCF 3 moiety,the 3-bond scalar coupling constants of H1' and H2' ( 3 JH1'-H2')were determined to be 9.7 and 8.0 Hz, respectively.These account for 96 and 80 %C 2 '-endo (South) populationsi nC2'/C3'-endo equilibria. [35,36] Notethat for pyrimidine nucleosidest he C2'-endo (South) population was 100 %. [10,11] Conditions: c RNA = 0.3 mm;25mm sodium cacodylate, pH 7.0, 298 K. mations ( Figure 4C,t op, middle) while signali ntensities are higher for the significantly more stable preQ 1 -aptamer complex ( Figure 4C,b ottom), hence impairing an accurate quantification of populations of the coexisting folds.
As as econd example for 19 FNMR spectroscopic applications of the novel labels, we analyzed duplex formation by titration and in temperature-dependent manner.T he 14 bp RNA contained as ingle 2'-SCF 3 adenosine in the middler egion. Its meltingt emperature was readily obtained. The corresponding set of data is depicted in the Supporting Information, Figure S2.
The NMR analysis presented in Figure 4t ogether with other examples that we demonstrated previously for the pyrimidine series makesu sc onfident that the 2'-SCF 3 label awaits rapid and widespread applications. [10,11] Its performance confirmso ur originale xpectations for facile NMR spectroscopic probingo f RNA structure rearrangements and RNA-ligand interactions.

-modified siRNA
As an ovel application fort he 2'-SCF 3 modification, we tested the potential of this modification for gene silencingb ys mall interfering RNA (siRNA). Nucleosides with destabilizinge ffects on Watson-Crick base pairing are of specific interest for the development of oligonucleotide therapeutics. [2,4,23] Most prominent is the highly flexible unlocked nucleic acid (UNA;o r" seconucleoside") modification. [23] UNA, missingt he covalentC 2 'À C3' bond of ar ibose sugar,i sn ot conformationally restrained, and can be used to influence oligonucleotide flexibility.U NA insertsr educe duplex T m values by 5t o1 08Cp er insert, [23] they facilitateantisense strand selection as the RISC guide, and UNA modifications to the seed region of as iRNA guide strand can significantly reduceo ff target effects. [44] The comparable extento fd estabilization of UNA and2'-SCF 3 modifications prompted us to explore ap otential role of the latter in siRNA approaches. For reasons of comparability,w e employed the same model system used previously to knock down the brain acid soluble protein 1( BASP1) encoding gene by transient siRNA nucleofection in the chickenD F-1 cell line. [45,46] Expression of the BASP1 gene is specifically suppressed by Myc, an evolutionary conserved oncoprotein; [47] conversely,t he BASP1 protein is an efficient inhibitor of Mycinduced cell transformation. [46] We synthesized five siRNA duplexes for the BASP1 target gene with the sequence organization depicted in Figure 5A (see also the Supporting Information, Table S2), two of them with as ingle2 '-SCF 3 adenosine (A15) or guanosine (G8) in the sense strand,t wo of them with as ingle 2'-SCF 3 guanosine modification in the antisense strand,v ery close to (G10) or within (G2) the seed region,a nd another one with two 2'-SCF 3 modifications (G2 and A13) in the antisense strand. We determined the thermodynamic parameters for two of the five modified siRNAs( A15 sa nd G2 as;s eed region)b yU Vm elting profilea nalysisa nd-as expected-found significant destabilization compared to the native siRNA (Supporting Information, Figure S3).
The modified siRNAs caused complete gene silencing as observed for the non-modified reference duplex only when the modification resided in the sense strand ( Figure 5B). Instead, siRNA activity was impaired for the 2'-SCF 3 -G10 antisensemodified siRNA. Not unexpectedly,t he two siRNAs carrying the 2'-SCF 3 modification in the seed region at the critical position2 of the antisense strand [23] were not active. These results indicate that the 2'-SCF 3 modification is apromising tool for the alternative design of siRNAs with reduced off-target effects and warrantsc omprehensive studies in the future along these lines. [44] Conclusion The ribose 2'-trifluoromethylthio group makes ribonucleic acids an attractive reporter for spectroscopic investigationso f . NMR spectroscopic analysis of Mg 2 + -assisted RNA pseudoknot formation, and subsequent stabilization through binding of asmall ligand (Thermoanaerobacter tengcongensis preQ 1 class-Ir iboswitch), using a2 '-SCF 3 guanosinel abel. A) RNA secondary structure model,B)corresponding 19 FNMR spectra,a nd C) imino proton 1 HNMR spectra. Conditions: c RNA = 0.3 mm,2 5mm sodium cacodylate, pH 7.0, 298 K; additions: c Mg2 + = 2.0 mm;f ollowed by c preQ1 = 1.2 mm.The cytosine that forms aW atson-Crick base pair with the preQ 1 ligand is highlighted in grey.
Chem.E ur.J.2015, 21,10400 -10407 www.chemeurj.org RNA structure, structural dynamics, folding, and ligand interactions. So far,o nly RNA with 2'-SCF 3 modified pyrimidine nucleosides hasb een accessible by chemical synthesis. The syntheses of the novel 2'-SCF 3 purine nucleoside phosphoramidites and the corresponding RNAsh as been demonstrated in this work and significantly expands the scope of applications for this modification. Their excellent behavior in 19 FNMR probing of structurep reformation and ligand binding was exemplified for the preQ 1 class-Ir iboswitch and for meltingo fa nR NA duplex. Moreover,a ll 2'-SCF 3 -modified nucleosides cause thermodynamic destabilization when they reside in double helices. Since this property is reminiscent of "unlockedn ucleic acid" (UNA) which is widely used for siRNA technologiest om inimize off-target effects, [23,44] we have highlighted the principal potential of 2'-SCF 3 RNAs for siRNA design as ap romising novel application of this modification.

Deprotection of 2'-SCF 3 -containing RNA
The solid support was transferred into as crew-capped Eppendorf tube and 1.5 mL of a1 :1 mixture of solution of methylamine in ethanol (33 %) ammonia and aqueous methylamine (40 %) was added and the reaction proceeded at room temperature for 4t o 6h with occasional shaking. IMPORTANT:F or deprotection of 2'-SCF 3 adenosine containing RNA, the deprotection solution additionally contained 150 mm of threo-1,4-dimercapto-2,3-butandiol (DTT). [26,33] The suspension was filtered and all volatiles evaporated. The residue was dissolved in 1.0 mL of 1 m tetrabutylammonium fluoride trihydrate in THF and kept at 37 8Cf or 12 h. The reaction was quenched by addition of 1.0 mL of triethylammonium bicar-bonate buffer (1 m,p H7.4) and the organic solvent was evaporated. The solution was subjected to size-exclusion chromatography on an Amersham HiPrep 26/10 desalting column (2.6 10cm; Sephadex G25). The crude RNA was eluted with H 2 O, dried, and redissolved in 1mLH 2 O.

RNA interference and Northern analysis
Lyophilized synthetic siRNA duplexes were dissolved, annealed, and delivered into chicken DF-1 cells by electroporation as described. [45] To tal RNA isolation, and analysis of gene silencing by Northern hybridization using specific 32 P-radiolabelled DNA probes for detection of BASP1 and GAPDH mRNAs were done as described previously. [45,46]