Advances in RNA Labeling with Trifluoromethyl Groups

Abstract Fluorine labeling of ribonucleic acids (RNA) in conjunction with 19F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA‐ligand interactions. This study presents the first syntheses of 2′‐OCF3 guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid‐phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2′‐OCF3 modification is associated with preferential C2′‐endo conformation of the U and G ribose in single‐stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2′‐OCF3 label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2′‐OCF3 modification has potential for applications in therapeutic oligonucleotides. To this end, three 2′‐OCF3 modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2′‐OCF3 nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2′‐OCF3 group without any restrictions in placing the modification into the RNA target sequence.


Introduction
Among the multiple chemical and biophysical approaches to gain insights into RNA structural dynamics and RNA interactions with proteins, other nucleic acids, or small molecules, the use of fluorine labeled RNA combined with 19 F NMR spectroscopy has attracted significant interest in recent years.  This  due to the exceptional properties of fluorine which include its 100 % natural abundance and consequent high NMR sensitivity.Moreover, fluorine exhibits a significant chemical shift dispersion, rendering it highly responsive to conformational and environmental changes.Fluorine atoms are hardly encountered in native biomolecular systems which is advantageous to monitor the 19 F NMR signal in complex substance mixtures, for example in cellular extracts or in small-molecule ligand libraries.However, on the other hand, the lack of fluorine in biomolecules is a drawback because labeling of the biomolecule with a 19 F handle is required and this is particularly challenging for RNA.
Recently, we have reported on 2'-O-trifluoromethyl cytidine and -adenosine modified RNA as a remarkable labeling concept for NMR spectroscopic applications. [45]The ribose 2'-OCF 3 group has the advantage over the widely used 2'-SCF 3 label [28][29][30] in that it is less thermodynamically destabilizing when residing in a double helix.This conforms with access to more diverse labeling patterns allowing to address a broader scope of research questions.In the previous study we demonstrated that 2'-OCF 3 cytidine and adenosine phosphoramidites are readily incorporated into RNA by solid-phase RNA synthesis with yields that are similar to phosphoramidites of the four standard nucleosides (A, C, G, U). [45] Likewise, deprotection follows the standard protocol.Both facts make 2'-OCF 3 labeled RNA accessible with lengths up to 65 and more nucleotides. [45]he introduction of the CF 3 label at the 2'-OH group of the nucleoside is more challenging and admittedly remains an unsolved problem with respect to a high-yielding synthesis of the building blocks. [46,47]Accepting the foreseeable low yields for the actual 2'-OH into 2'-OCF 3 transformation, we set out to expand the set of building blocks toward 2'-OCF 3 guanosine and uridine phosphoramidites.This expansion is urgently needed because thus far, the outstanding performance of the OCF 3 label in NMR spectroscopic approaches has been restricted to adenosine and cytidine labeling patterns in target RNA. [45]Here, we demonstrate how to overcome these restrictions by generating access to the complete set of 2'-OCF 3 nucleosides for RNA labeling.Consequently, any RNA sequence with site-specific 2'-OCF 3 modifications can be furnished by

Results and Discussion
Traditionally, trifluoromethyl ethers are synthesized de novo under harsh reaction conditions using difficult-to-handle chemicals, and requiring pre-functionalized compounds.These methods are limited in practicality/user friendliness and scope. [48]Conceptually, direct OCF 3 formation via electrophilic trifluoromethylation of alcohols is the most practically straightforward approach.It is also considered to be more tolerant to diverse functional groups, but unfortunately, it is the least explored approach, with only few reagents known in the literature that are capable of this transformation. [48]For instance, a O-(trifluoromethyl) dibenzofuranium salt was successfully employed for the formation of aryl and alkyl trifluoromethyl ethers, [49] however, the preparation of the reagent is challenging.Later, the use of a hypervalent iodine compound for the trifluoromethylation of primary and secondary alcohols using zinc triflimide was reported. [50]A drawback, however, was the requirement for a large excess of the alcohol component.Further developments recently led to an electrophilic trifluoromethylating reagent that combines the hypervalent iodine motif with a sulfoximine ligand (HYPISUL), [51] allowing for a broader substrate scope for trifluoromethylation of a variety of secondary and biorelevant alcohols featuring various functional groups.This reagent seems promising but broad applicability remains to be demonstrated.
Since the above mentioned approaches for trifluoromethylation did not work out in our hands on nucleosides, we decided to focus on the transformation of ribonucleoside 2'-O methyl xanthates to the corresponding 2'-OCF 3 modified counterparts albeit this reaction gives generally low yields. [47,48]or guanosine, this path was expected to require nucleobase protection to prevent unintended alkylation during methylation of the xanthogenate using methyliodide.Therefore, O 6 -(4nitrophenyl)ethyl (NPE) together with N 2 -acyl or N 2 -amidine protection was envisaged which additionally promised sufficient solubility in organic solvent required for practicable workup and isolation of the trifluoromethylated nucleoside derivative with free 5' and 3'-OH groups.In this respect, the xanthogenate approach might also be problematic for uridine, however, if so, we were confident that access to 2'-OCF 3 U should be feasible by transformation of 2'-OCF 3 cytidine into the corresponding uridine.

Synthesis of 2'-OCF 3 guanosine
The synthetic route to building block G9 (Scheme 1) started from guanosine G1, which was acetylated at its ribose hydroxyls and the exocyclic NH 2 functionality, providing compound G2.After introduction of the O 6 -(4-nitrophenyl)ethyl group under Mitsunobu conditions furnishing G3 in high yields, selective removal of hydroxylic acetyl groups was achieved in aqueous methanol-triethylamine solution.Treatment of G4 with 1,3dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl 2 ) selectively installed the Markiewicz protecting group at 3'-O and 5'-O and left the 2'-OH available for conversion into the 2'-O-(meth-ylthio)thiocarbonyl functionalized compound G6 by the use of tert-butyl lithium, carbon disulfide and iodomethane.Transformation to the 2'-O-trifluoromethyl derivative G7 was accomplished by treatment with N-bromosuccinimide in hydrofluoric pyridine solution and dichloromethane.Tritylation of the 5'-OH group proceeded in the presence of 4,4'-dimethoxytrityl chloride (DMT-Cl) and dimethylaminopyridine (DMAP) to yield compound G8, which was converted to the corresponding phosphoramidite G9 by reaction with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.This pathway provides compound G9 in eight steps with eight chromatographic purifications in 4 % overall yield; in total, 0.7 g of G9 was obtained in the course of this study.

Synthesis of 2'-OCF 3 uridine
Our diverse and intensive attempts to synthesize 2'-OCF 3 modified uridine directly from uridine unfortunately failed.We therefore conceived a path that includes a pyrimidine nucleobase transformation.The synthetic route to building block U3 (Scheme 2) starts from cytidine C1 which was transformed into N 4 -benzoylated 2'-OCF 3 cytidine C2 in four steps in 9.5 % overall yield, following our previously published protocol. [45]Treatment of C2 with aqueous ammonia in methanol gave the unprotected 2'-OCF 3 cytidine which -after evaporation of the solvents -was directly used for the diazotization reaction with sodium nitrate and acetic acid in aqueous solution to yield 2'-OCF 3 uridine U1.Tritylation of 5'-OH group was achieved applying 4,4'-dimethoxytrityl chloride and dimethylaminopyridine to give compound U2, which was further converted into the corresponding phosphoramidite U3 by reaction with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.This pathway provides compound U3 in eight steps with eight chromatographic purifications in 6 % overall yield (starting from cytidine); in total, 1.0 g of U3 was obtained in the course of this study.
Likewise, RNA containing 2'-OCF 3 uridine were slightly destabilized.This time, we reduced the NaCl concentration from 150 to 100 mM NaCl under otherwise same buffer conditions.Melting profile analysis of the 5'-GAAGG-GCAA-CC(2'-OCF 3 -U)UCG hairpin RNA (Figure 2A) resulted in a 4.6  Taken together, the UV melting study demonstrated that 2'-OCF 3 modified RNA is significantly less thermodynamically destabilizing in comparison to the previously reported 2'-SCF 3 RNAs (Table 1), [29] It otherwise retains all the advantages for 19 F NMR spectroscopy attributed to the CF 3 group, and therefore, a much broader range of applications is foreseeable for 2'-OCF 3 RNA.

2'-OCF 3 ribose conformation
[58][59] Assuming a simple two state equilibrium between the two sugar puckers, the percental population can be directly calculated from the scalar coupling of H1' and H2' of the individual ribose unit.For this purpose, we synthesized the short single-stranded RNA 5'-GGCA(2'-OCF 3 -G)AGGC (Figure 3A) and assigned the H2' of the trifluoromethylated guanosine in position 5 in a 19 F/ 1 H NOESY NMR experiment relying on its proximity to the fluorine label.The 3 J coupling constant to H1'(G5) was then obtained from a 2D 1 H/ 1 H double quantum filtered COSY spectrum (Figure 3B); it amounted to 7.09 Hz which conforms to a C2'-endo population of ca.70 %.For 2'-OCF 3 uridine in the single stranded RNA 5'-GCCU(2'-OCF 3 -U)UGCC (Figure 3C), the 3 J coupling constant between H1' and H2' of the 2'-OCF 3 ribose was determined to be 9.1 Hz which conforms to a C2'-endo population of 91 %.We mention that only 58 % population for C2'-endo conformation was measured for 2'-OCF 3 adenosine in the single strand 5'-GGCAG(2'-OCF 3 -A)GGC. [45]Taken together, these observations provide evidence that forcing the G and U label into a C3'-endo ribose pucker, as mandatory for a double helical A-form RNA, results in a higher energetic penalty than for the 2'-OCF3 adenosine.
Intrigued by the significant line broadening effect upon duplex formation, we used a Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiment to detect and quantify a potential dynamic process on the intermediate chemical shift time scale (Figure 4).For this purpose, we prepared a sample with a slight excess (ca.20 %) of the single strand carrying the 2'-OCF 3 -U label and run a RD experiment with CPMG field strengths up to 5 kHz.A non-flat dispersion profile was observed for the duplex CF 3 resonance, whereas for the sharp single stranded resonances no significant dispersion profile was found.The high quality dispersion data could be fit to an intermediate two state exchange process by using the Richard-Carver equation [60] and an in house written MATLAB script.An excited state population of 2.30 � 0.78 %, an exchange rate k ex (= k forward + k backward ) of 15.208 � 1174 s À 1 and a chemical shift difference of 2.32 � 0.26 ppm was found.We can rule out an exchange process between single and double stranded state, as no RD profile was observed for the single strand resonance.The single stranded 2'-OCF 3 -U populates to ca. 90 % the C2'-endo state (Figure 3C and D).Correlating the chemical shift difference of the single/double stranded state (Δω 2.16 ppm) to the  chemical shift difference between ground and excited state from the RD experiment (Δω 2.32 ppm) supports a sugar pucker equilibrium of the 2'-OCF 3 -U in the duplex between the C2'endo (excited state, 2.3 %) and the C3'-endo (ground state, 97.7 %) sugar pucker.

NMR analysis of RNA small molecule binding
RNA with a single 2'-OCF 3 label provides a powerful sensor to monitor RNA folding and RNA interactions with other biomolecules by 19 F NMR spectroscopy.In this work, we exemplarily applied the 7-aminomethyl-deazaguanine (preQ 1 ) sensing class-I riboswitch from Thermoanaerobacter tengcongensis (Tte) [61][62][63][64][65] as model system and tested two positions of guanosine (G11 and G34) for their potential to follow Mg 2 + induced folding of an RNA pseudoknot and binding of a small molecule (preQ 1 ) to this particular aptamer with high (nanomolar) affinity (Figure 5).In aqueous buffer at pH 6.5, the 2'-OCF 3 -G11 labeled Tte RNA displays a rather broad 19 F NMR signal group (Figure 5A, top), indicating multiple RNA loop conformations in the intermediate to slow exchange regime.Only when Mg 2 + is added, a single sharp resonance dominates (Figure 5A, middle) which is consistent with a pre-organized pseudoknot fold in which G11 is base-paired with C30 (in accordance with crystallography 62 and NMR studies [63,66] ).The observed Mg 2 + induced pseudoknot folding is also in line with the observation by other methods such as 2APfold [67,68] or smFRET spectroscopy. [67,69,70]Once the cognate ligand (preQ 1 ) is added, the 19 F signal shifts downfield (Figure 5A, bottom) consistent with Watson-Crick (WC) base pairing of G11-C30 (see also Figure 3D for comparison).The increased line width of the 19 F signal is likely attributed to restricted rotational freedom of the 2'-OCF 3 group in the rigid ligand-RNA complex.
A strength of single label RNA 19 F NMR analysis as outlined above is the high sensitivity for local conformational rearrangements.In this sense, the 2'-OCF 3 -G11 label reflects changes in the RNA loop conformation, and additionally, responds to pseudoknot formation through WC base pairing to C30.The dynamics of pseudoknot formation can also be pursued from a complementary perspective, namely the RNA 3'-tail.Accordingly, the 2'-OCF 3 -G34 labeled RNA displays a sharp 19 F NMR resonance in Mg 2 + free, aqueous buffer at pH 6.5 (Figure 5B, top) which is consistent with a conformationally flexible unpaired single stranded RNA.When Mg 2 + is added the fraction of the stem-loop RNA with dangling 3'-tail is reduced and two more major 19 F NMR signals appear (Figure 5B, middle).One of them is assigned to a conformation which closely resembles the final ligand-bound RNA fold according to the comparable chemical shift values.The other Mg 2 + -induced conformation likely also reflects a closed (pseudoknotted) conformation but it is structurally more distinct from the final ligand-RNA complex, consistent with the distinct chemical shifts (Figure 5B, bottom).In summary, this example demonstrates the power and convenience of singly labeled 2'-OCF 3 labeled RNA for the detection of RNA conformational states (including an estimate of the timescale for their exchange) and for the detection of RNA ligand interactions by 1D 19 F NMR spectroscopy.

Potential of 2'-OCF 3 RNA for RNA interference
As a novel application for the 2'-OCF 3 modification, we tested the potential of this modification for gene silencing by small interfering RNA (siRNA).The structural proximity of 2'-OCF 3 to 2'-OCH 3 makes it a promising candidate for such applications, in particular under the aspect that 2'-OCH 3 represents the most frequently encountered modification in clinically approved oligonucleotide therapeutics. [71]Albeit the 2'-OCF 3 has a slight destabilizing effect on duplex stability which is not necessarily a disadvantage.[74] Most prominent is the unlocked nucleic acid (UNA) missing the covalent bond between C2' and C3' of a ribose. [71]UNA modifications facilitate antisense strand selection as the RISC guide, and UNA inserts to the seed region of the siRNA guide strand can significantly reduce off target effects. [75]ere, we intended to explore the performance of 2'-OCF 3 for siRNA applications.We employed the model system used previously to knock down the brain acid soluble protein 1 (BASP1) encoding gene by transient siRNA nucleofection in the chicken DF-1 cell line. [76]Expression of the BASP1 gene is specifically downregulated by the evolutionary conserved oncoprotein Myc; [77] conversely, the BASP1 protein is an inhibitor of Myc-induced cell transformation. [76]e synthesized three siRNA duplexes for the BASP1 target gene with the sequence organization depicted in Figure 6A (Supporting Information, Supporting Table 2).The modifications were placed in the antisense strands, two of the siRNA contained a single modification (U6 as; U9 as), and the third one contained both modifications (U6/U9 as).
Expression of the BASP1 gene and of its protein product BASP1 were monitored by Northern and qPCR analysis, and by immunoblotting, respectively.The modified siRNAs U9 and U6/ 9 caused comparable gene silencing as observed for the unmodified reference siRNA.The siRNA U6 -with the modification residing inside the seed region -displayed even slightly increased repression compared to the unmodified siRNA duplex (Figure 6B-D).These results point at the potential of 2'-OCF 3 modifications to tailor siRNAs with advanced performance.In particular, our observation for improved repression of the BASP1 gene with an siRNA carrying the 2'-OCF 3 group in the seed region warrants more comprehensive studies along these lines in the future. [72]

Conclusions
Numerous 19 F labels for NMR spectroscopy of nucleic acids have been developed previously.[30] Furthermore, a nine-fluorine-atom label in the form of 5-[4,4,4-trifluoro-3,3-bis(trifluoromethyl)but-1-ynyl] 2'deoxyuridine has also been utilized. [4]Among these options, the ribose trifluoromethyl labels stand out because they meet several important criteria for the practicability of 19 F NMR approaches at the same time.These are their relatively small size, no need for proton decoupling, high sensitivity, large chemical shift dispersion, and equivalent labeling position for all of the four standard nucleosides.45] In the present study, we extend the 2'-OCF 3 labeling concept towards guanosine and -uridine and hence ensure utmost flexibility for labeling any nucleotide within an RNA target sequence.Additionally, we show first biochemical applications.targeting siRNA duplex used in this study; nucleosides in red indicate positions for the modification tested.(B) Biological activities of 2'-OCF3 modified siRNAs, directed against BASP1 mRNA.Chicken DF-1 cells grown on 60 mm dishes were transiently nucleofected with 0.24 nmol (~3.0 μg) aliquots of the individual siRNAs.An equal aliquot of siRNA with a shuffled (random) nucleotide sequence was used as a control.Total RNAs were isolated 2 days after siRNA delivery, and 5 μg of aliquots were analyzed by Northern hybridization using a digoxigenin-labeled DNA probe specific for the chicken BASP1 gene, and subsequently with a digoxigenin-labeled probe specific for the housekeeping chicken GAPDH gene.Sizes for the mRNAs are: BASP1, 2.0 kb; GAPDH, 1.

Experimental Section
For the syntheses and characterization data of compounds G2 to G9 and U1 to U3 see the Supporting Information.

Deprotection of 2'-OCF 3 modified RNA
Solid support was treated with methylamine/ethanol (33 %, 0.7 mL) and methylamine/H 2 O (40 %, 0.7 mL) for 6 h at 37 °C.Supernatant was removed and solid support was washed thrice with tetrahydrofuran/H 2 O (1/1).Combined supernatant and washings were evaporated to dryness and the residue was dissolved in a solution of tetrabutylammonium fluoride in tetrahydrofuran (1.0 M, 1.5 mL) and incubated for 16 h at 37 °C for removal of 2'-O-silyl protecting groups.The reaction was quenched by addition of tetraethylammonium acetate/H 2 O (1.0 M, 1.5 mL, pH 7.4).The solution was reduced to one third of the original volume and desalted with size-exclusion column chromatography (GE Healthcare, HiPrep TM 26/10 Desalting; Sephadex G25) eluting with H 2 O; collected fractions were evaporated and the RNA dissolved in H 2 O (1 mL) for immediate use or storage at À 20 °C.1D 19 F NMR spectra were typically acquired using the following parameters: spectral width 10 ppm, o1p À 60 ppm, 32k complex data points.128 scans were collected with a recycling delay of 1 s resulting in an experimental time of 4 min.

HPLC analysis and quantification of 2'-OCF 3 modified RNA
For the 2D 19 F- 13 C HMQC experiments at natural 13 C abundance the following parameters were used: spectral width in the indirect 13 C dimension was set to 10 ppm, and the spectral width in the direct 19 F dimension was set to 10 ppm.A total of 64 complex points was collected in the indirect 13 C dimension (acquisition time = 21 ms) and 1024 complex points were collected in the direct 19 F dimension (acquisition time = 91 ms).768 scans were collected with a recycling delay of 1 s resulting in an experimental time of 16 h.The carrier frequency was placed at À 58 ppm in the 19 F dimension and in the 13 C dimension at 120 ppm.The 1 J CF coupling constant was set to 270 Hz.
For experimental details concerning RNA interference and analysis of gene silencing see the Supporting Information.
[a] Buffer conditions: 10 mM Na 2 HPO 4 , 150 mM NaCl, pH 7.0.ΔH and ΔS values were obtained by van't Hoff analysis according to references 54 and 55.Errors for ΔH and ΔS, arising from non-infinite cooperativity of two-state transitions and from the assumption of a temperature-independent enthalpy, are typically 10-15 %.Additional error is introduced when free energies are extrapolated far from melting transitions; errors for ΔG are typically 3-5 %. [b] Data reproduced from Ref. [29].[c] Buffer: Same as [a] but 100 mM NaCl.

Figure 4 . 19 F
Figure 4. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiment to detect and quantify a potential dynamic process of the 2'-OCF 3 modified ribose in single-stranded vs duplex RNA.(A) RNA sequences and 19 F NMR spectra of a mixture of duplex and single strand in a ratio of 1.0 to 1.2.(B) 19 F-relaxation dispersion profiles of 2'-OCF 3 U5 recorded at 565 and 659 MHz 19 F-Larmor frequency.The statistics of the two-state exchange process are shown as inset (for discussion see main text).R 2 (transverse relaxation rate), ν CPMG (CPMG field strength).Dots represent experimental data, black crosses repeat experiments and the solid line is the best fit to an intermediate exchange process using the Carver-Richards equation.MC Monte Carlo iterations for error statistics.

Figure 6 .
Figure 6.Gene silencing by 2'-OCF 3 modified siRNAs.(A) Sequence of the brain acid soluble protein 1 gene (BASP1)[76] targeting siRNA duplex used in this study; nucleosides in red indicate positions for the modification tested.(B) Biological activities of 2'-OCF3 modified siRNAs, directed against BASP1 mRNA.Chicken DF-1 cells grown on 60 mm dishes were transiently nucleofected with 0.24 nmol (~3.0 μg) aliquots of the individual siRNAs.An equal aliquot of siRNA with a shuffled (random) nucleotide sequence was used as a control.Total RNAs were isolated 2 days after siRNA delivery, and 5 μg of aliquots were analyzed by Northern hybridization using a digoxigenin-labeled DNA probe specific for the chicken BASP1 gene, and subsequently with a digoxigenin-labeled probe specific for the housekeeping chicken GAPDH gene.Sizes for the mRNAs are: BASP1, 2.0 kb; GAPDH, 1.4 kb.The levels (%) of BASP1 expression were determined using the program ImageQuant TL and are depicted as bars in relation to mock transfections (no siRNA, 100 %).Vertical bars show standard deviations (SD) from independent experiments (n = 3).Statistical significance was assessed by using a paired Student t-test (***P < 0.001, ****P < 0.0001).(C) The same as (B) but analyzed by quantitative polymerase chain reaction (qPCR) using each 2.5 ng cDNA template reverse transcribed from total RNA, and primers specific for chicken BASP1 or GAPDH.All siRNAs depicted contain overhangs of 2'-deoxynucleosides (lower case letters).(D) Immunoblot analysis using cell extracts prepared 3 days after siRNA delivery and antibodies specific for the BASP1 or GAPDH proteins.The levels (%) of BASP1 expression were determined using the program ImageQuant TL and are depicted as bars in relation to mock transfections (no siRNA, 100 %).Vertical bars show standard deviations (SD) from independent experiments (n = 4).Statistical significance was assessed by using a paired Student t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 6.Gene silencing by 2'-OCF 3 modified siRNAs.(A) Sequence of the brain acid soluble protein 1 gene (BASP1)[76] targeting siRNA duplex used in this study; nucleosides in red indicate positions for the modification tested.(B) Biological activities of 2'-OCF3 modified siRNAs, directed against BASP1 mRNA.Chicken DF-1 cells grown on 60 mm dishes were transiently nucleofected with 0.24 nmol (~3.0 μg) aliquots of the individual siRNAs.An equal aliquot of siRNA with a shuffled (random) nucleotide sequence was used as a control.Total RNAs were isolated 2 days after siRNA delivery, and 5 μg of aliquots were analyzed by Northern hybridization using a digoxigenin-labeled DNA probe specific for the chicken BASP1 gene, and subsequently with a digoxigenin-labeled probe specific for the housekeeping chicken GAPDH gene.Sizes for the mRNAs are: BASP1, 2.0 kb; GAPDH, 1.4 kb.The levels (%) of BASP1 expression were determined using the program ImageQuant TL and are depicted as bars in relation to mock transfections (no siRNA, 100 %).Vertical bars show standard deviations (SD) from independent experiments (n = 3).Statistical significance was assessed by using a paired Student t-test (***P < 0.001, ****P < 0.0001).(C) The same as (B) but analyzed by quantitative polymerase chain reaction (qPCR) using each 2.5 ng cDNA template reverse transcribed from total RNA, and primers specific for chicken BASP1 or GAPDH.All siRNAs depicted contain overhangs of 2'-deoxynucleosides (lower case letters).(D) Immunoblot analysis using cell extracts prepared 3 days after siRNA delivery and antibodies specific for the BASP1 or GAPDH proteins.The levels (%) of BASP1 expression were determined using the program ImageQuant TL and are depicted as bars in relation to mock transfections (no siRNA, 100 %).Vertical bars show standard deviations (SD) from independent experiments (n = 4).Statistical significance was assessed by using a paired Student t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).
Analysis of crude and purified RNA was performed by anion exchange chromatography on a Dionex DNAPac® PA-100 column (4 mm×250 mm) at 80 °C with flow rate of 1 mL/min.For RNA shorter or equal to 15 nucleotides, a gradient of 0-40 % B in 30 min and for RNA longer than 15 nucleotides a gradient of 0-60 % B was used; Eluent A: 20 mM NaClO 4 and 25 mM Tris-HCl (pH 8.0) in 20 % aqueous acetonitrile; Eluent B: 0.6 M NaClO 4 and 25 mM Tris-HCl (pH 8.0) in 20 % aqueous acetonitrile.HPLC traces were recorded at UV absorption at 260 nm.The RNA was quantified on an Implen P300 Nanophotometer.Mass spectrometry of 2'-OCF 3 modified RNA RNA samples (3 μL) were diluted with 40 mM Na 2 H 2 (EDTA)/H 2 O (5/ 4) for a total volume of 30 μL, injected onto C18 XBridge 2.5 μm (2.1 mm×50 mm) at a flow rate of 0.1 mL/min and eluted with 0-100 % B gradient at 30 °C (Eluent A: 8.6 mM triethylamine, 100 mM 1,1,1,3,3,3-hexafluoroisopropanol in H 2 O; Eluent B: methanol).RNA traces were analyzed on a Finnigan LCQ Advantage Max electrospray ionization mass spectrometer with 4.0 kV spray voltage in negative mode.NMR measurements of 2'-OCF 3 modified RNA RNA samples were lyophilized as triethylammonium salts and dissolved either in 280 μL or 400 μL NMR buffer (15 mM Na-[AsO 2 (CH 3 ) 2 ] • 3H 2 O, 25 mM NaCl, 3 mM NaN 3 , in D 2 O or 9/1 H 2 O/D 2 O, pH 6.5) and transferred into restricted volume Shigemi tubes or standard 5 mm NMR tubes.Sample concentrations varied between 0.1 and 1 mM and experiments were run at 298 K unless otherwise stated.All NMR experiments were conducted on a Bruker 600 MHz Avance II + NMR or a 700 MHz Avance Neo NMR both equipped with a Prodigy TCI probe.