Mechanism of the extremely high duplex-forming ability of oligonucleotides modified with N-tert-butylguanidine- or N-tert-butyl-N′- methylguanidine-bridged nucleic acids

Abstract Antisense oligonucleotides (ASOs) are becoming a promising class of drugs for treating various diseases. Over the past few decades, many modified nucleic acids have been developed for application to ASOs, aiming to enhance their duplex-forming ability toward cognate mRNA and improve their stability against enzymatic degradations. Modulating the sugar conformation of nucleic acids by substituting an electron-withdrawing group at the 2′-position or incorporating a 2′,4′-bridging structure is a common approach for enhancing duplex-forming ability. Here, we report on incorporating an N-tert-butylguanidinium group at the 2′,4′-bridging structure, which greatly enhances duplex-forming ability because of its interactions with the minor groove. Our results indicated that hydrophobic substituents fitting the grooves of duplexes also have great potential to increase duplex-forming ability.

We previously reported that oligonucleotides modified with guanidine-or N -methylguanidine-bridged nucleic acid (GuNA [H] and GuNA[Me], respecti v ely; Figure 2 ) exhibited higher stability against 3 -exonuclease than those modified with 2 ,4 -BN A / LN A (30)(31)(32). Howe v er, the GuNA[H]-and GuNA[Me]-modified oligonucleotides, despite their cationic characters, had almost the same RNA-binding affinity ( T m = +5 • C / mod.) with the 2 ,4 -BN A / LN A-modified ones. We recently demonstra ted tha t GuNA[H]-modified ASOs are well tolerated and have altered organ-specificity, as well as a pr efer ence for skeletal muscle, compared with their 2 ,4 -BN A / LN Amodified counterparts ( 33 ). Moreover, GuNA[H]-modified anti-miRNA oligonucleotides were shown to have high acti vity, possib ly due to the additional interactions between GuNA [H] and miRISC ( 34 ). Here, we report the synthesis and properties of oligonucleotides bearing other GuN A analo gs, w hich have one or two alkyl groups at the guanidine moiety ( Figure 2 ). We found tha t incorpora tion of GuN A [R] or GuN A[R,R] into the oligonucleotide significantly increased the RNA-binding affinity as the N -alkyl group became larger [ tert -butyl

Synthesis and purification of GuNA[R]-modified oligonucleotides (ODN1-ODN3, ODN8-ODN10)
Synthesis of oligonucleotides modified with GuNA[R] was performed using the nS-8 oligonucleotide synthesizer (GeneDesign, Inc.) and the standard phosphoramidite protocol. Custom Primer Support ™ T 40s (GE Healthcare) was used as solid support in a 0.2 mol scale. The amidite was dehydrated and solved in acetonitrile at 0.1 M. The standard synthesis cycle was used to assemble the reagents, except that the coupling time was extended from 25 sec to 16 min for GuNA [R]. We used 0.25 M 5-ethylthio-1 Htetrazole (ETT) in acetonitrile as an activator. The synthesis was carried out in trityl-on mode. The elongated oligonucleotides were treated with a 1:1 mixture of 7 N ammonia solution in methanol and 40% aq. methylamine at room temperature for 4 h to remove the solid support, and then the mixture was hea ted a t 60 • C for 10 h. After deprotection, oligonucleotides were purified using Sep-Pak ® Plus C18 Cartridge, and 5 -dimethoxytrityl group was removed with 2% aq. trifluoroacetic acid on the cartridge. Subsequently, desir ed oligonucleotides wer e further purified using re v erse-phase HPLC with Waters XBridge ™ C18 (4.6 × 50 mm analytical and 10 mm × 50 mm preparati v e) columns, with a linear gradient of acetonitrile in 0.1 M triethylammonium aceta te buf fer (pH 7.0). The purity and structure of the oligonucleotides were confirmed by HPLC and MALDI-TOF mass spectroscopy, respecti v ely.

Synthesis and purification of GuNA[R,R]-modified oligonucleotides (ODN4-ODN7, ODN11-ODN18 and ODN24-ODN33)
Synthesis of oligonucleotides modified with GuNA[R,R] was performed using the nS-8 oligonucleotide synthesizer (GeneDesign, Inc.) and the standard phosphoramidite protocol. Primer Support ™ 5G T 350 (GE Healthcare) was used as solid support in a 1.0 mol scale. The amidite was dehydrated and solved in dichloromethane at 0.125 M for GuNA[R,R]-T, or 0.1 M for GuNA[R,R]-A, -G and m C. The standard synthesis cycle was used to assemble the reagents, except that the coupling time was extended from 25 sec to 20 min for GuNA[R,R]. We used 0.4 M 5-benzylthio-1 H -tetrazole (BTT) in acetonitrile as an activator, and 50 g / l tert -butylphenoxy acetic anhy dride (Tac 2 O) in tetr ahydrofur an was used as a capping reagent. Oxidizing steps were performed in an iodide solution for 10 s ( ODN4 -ODN7 , ODN11 , ODN12 , ODN24 -ODN29 ) or 0.5 M (1 S -+-10-camphorsulf on yl)-oxaziridine (CSO) in tetr ahydrofur an for 3 min ( ODN13 -ODN18 , ODN30 -ODN33 ). The synthesis was carried out in the trityl-on mode. For ODN4 -ODN7 , ODN11 -ODN18 and ODN24 -ODN27 , the elongated oligonucleotides were treated with a 1:1 mixture of 38 wt.% methylamine solution in ethanol and 40% aq. methylamine at room temperature for 20 min to remove the solid support, and then the mixture was heated at 60 • C for 4 h. For ODN28 , ODN30 and ODN32 , the oligonucleotides were treated with 0.4 M sodium hydroxide solution in a 4:1 mixture of methanol and water at room temperature for 17 h, followed by 5% aq. acetic acid to stop the reaction. For ODN29 , ODN31 and ODN33 , the oligonucleotides were treated with a 1:1 mixture of 7 M methylamine solution in methanol and 28% aq. ammonium hydroxide at room temperature for 4 h to remove the solid support, and then the mixture was heated at 60 • C for 8 h. After deprotection, crude oligonucleotides were purified using Sep-Pak ® Plus C18 Cartridge, and the 5 -dimethoxytrityl group was removed with 2% aq. trifluoroacetic acid on the cartridge. Subsequently, desired oligonucleotides were further purified using re v erse-phase HPLC with Waters XBridge ™ C18 (4.6 × 50 mm analytical and 10 mm × 50 mm preparati v e) columns, with a linear gradient of acetonitrile in 0.1 M triethylammonium acetate buffer (pH 7.0). The purity and structure of the oligonucleotides were confirmed by HPLC and MALDI-TOF mass spectroscop y, r especti v ely. The following oligonucleotides were synthesized by GeneDesign, Inc.: ODN19 , ODN21 -ODN23 and ODN34 . The following oligonucleotides were pr epar ed pr eviousl y by the authors of this pa per: ODN20 , ODN35 and ODN36 .

UV melting experiments
The UV melting experiments were carried out using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers equipped with a T m analysis accessory. Equimolecular amounts of the target ssRNAs or ssDNAs and oligonucleotides were dissolved in 10 mM sodium phospha te buf fer (pH 7.2) containing 100 mM NaCl to achie v e a final strand concentration of 4 M each. The samples were annealed by heating at 100 • C followed by slow cooling to 5 • C. The melting profile was recorded at 260 nm from 5 to 90 • C at a scan rate of 0.5 • C / min. T m values were taken as the temperatures at which the formed duplexes were half dissociated, determined by the sigmoidal melting curves.

Thermodynamic analysis
The UV melting experiments were carried out using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers equipped with a T m analysis accessory. Equimolecular amounts of the target ssRNAs or ssDNAs and oligonucleotides were dissolved in 10 mM sodium phospha te buf fer (pH 7.2) containing 100 mM NaCl to achie v e final strand concentrations of 0.86, 1.47, 2.43, 4.00, 6.60 and 10.9 M. The samples were annealed by heating at 100 • C followed by slow cooling to room temperature. The melting profile was recorded using the method described in 'UV melting experiments'. The van't Hoff plots were prepared using the T m values in each concentration. For the calculation of H • , S • and G • , the temperature was assumed to be 25 • C (298.15 K).

Crystallization
Pr epar ation of crystals . Crystalliza tion conditions were screened with the Nucleic Acid Mini Screen ™ kit (Hampton Research) using the hanging drop vapor diffusion technique. Droplets consisting of a mixture of 1 mM oligonucleotide solution and the mini-screen buffer (1:1 or 1:2 v / v) were equilibrated against 1 ml of 35% v / v 2-methyl-2,4pentanediol (MPD) aqueous solution. A crystal suitable for the diffraction experiment was obtained from a droplet containing 1 l of 1 mM oligonucleotide and 1 l of buffer #12, #14 and #10 for ODN25 , ODN28 and ODN29 , respecti v ely. The crystal was mounted in a nylon loop and frozen in liquid nitrogen with the reservoir solution as a cryoprotectant.
X-ray data collection and refinement. For ODN25 , the crystal was diffracted in a Rigaku MicroMax-007HF with R-axis IV++ using copper radia tion. The dif fraction da ta were processed with Mosflm and scaled with Aimless. The initial structure was determined by the molecular replacement method using the single-stranded 10 mer nucleotide containing 2 ,4 -BN A / LN A modification (PDB ID: 1I5W) ( 35 ) as a template model. Rotation and translation searches for molecular replacement were performed by PHASER. Model refinement was performed with REFMAC. For ODN28 and ODN29 , X-ray diffraction data were collected at SPring-8 (Hyo go, Ja pan) with a beamline BL44XU equipped with an EIGER X 16M. One of the crystals was scanned to find the one with strong anomalous scattering at the K-edge absorption of bromine. The XDS software was used for data processing. The initial phases were determined with single-wavelength anomalous dispersion using the hkl2map program of SHELX. The atomic models were built using the molecular gra phics pro gram COO T and refined with Phenix. Geometric restraint library files of GuN A[Me, t Bu] and GuN A[Me,Me] monomers were produced with the CCP4 programs Monomer Library Sketcher and Libcheck. The statistics for data collection and refinement are summarized in Supplementary Tables S6-S8.

Nuclease resistance study
The sample solutions wer e pr epar ed by dissolving 0.75 nmol of oligonucleotides in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCl 2 (sample volume: 100 L).
In each sample solution, same amount (0.03 or 0.12 g) of phosphodiesterase I from Crotalus adamanteus venom (svPDE) was added, and the cleavage reaction was carried out a t 37 • C . A portion of each r eaction mixtur e was r emoved at timed intervals and heated to 90 • C for 2 min to deactivate the nuclease. Aliquots of the timed samples were analyzed by RP-HPLC to evaluate the amounts of intact oligonucleotides remaining. The percentage of intact

Scheme 1. Synthesis of GuN A[R]-T and GuN A[R,R]-T phosphoramidites. Reagents and conditions: (a)
oligonucleotides in each sample was calculated and plotted against the digestion time to obtain a degradation curve with time.

Synthesis of GuNA[R]-and GuNA[R,R]-modified oligonucleotides
Using an automated DNA synthesizer (solid-phase synthesis), the gi v en phosphoramidites were incorporated into oligonucleotides in the same manner as the previous method ( 32 ) ( Table 1 ). After elongation reactions, the oligonucleotides were cleaved from the solid support. At the same time, the protecti v e groups at the nucleobases and the phosphate backbone wer e r emoved by treatment with an ammonia / methylamine solution (7 M ammonium solution in methanol / 40% aqueous methylamine solution = 50:50 v / v) at 60 • C for 10 h. This method provided GuNA[Et]-and GuNA[ i Pr]-modified oligonucleotides ( ODN1 and ODN2 ) at 13% and 22%, respecti v ely.
Howe v er, GuNA[ t Bu]-modified ODN3 was isola ted a t a low yield (2%). The remaining acetyl group at the guanidine moiety was observed by LC-MS analysis of the crude material (see Supplementary Figure S1 in Supporting Informa tion), which indica ted tha t the nearby ter t -butyl group sterically inhibited the deprotection of the acetyl group. In   Figure S2 in Supporting Inf ormation). Theref ore, we used 4tert -butylphenoxy acetic anhy dride (Tac 2 O) as a labile capping reagent. This successfully reduced the production of guanidine-acylated oligonucleotides (see Supplementary Figure S3 in Supporting Information). In addition, 5-(ethylthio)-1H -tetrazole (ETT) and iodine, which could be affected by the basic guanidine moiety, were replaced with BTT and CSO (see Supplementary Tables S1 and S2 in Supporting Information). These changes successfully enabled us to obtain enough GuNA[R,R]-modified oligonucleotides for the following evaluations. The isolated yields of ODN4 -ODN7 were 2-24%, as shown in Table 1 .

Duplex-forming ability of GuNA[R]-and GuNA[R,R]modified oligonucleotides
We next evaluated the duplex-forming ability of the GuNA[R]-and GuNA[R,R]-modified oligonucleotides toward the complementary RNA or DNA strand (  ODN7 ). These results indica ted tha t the bulk y alk yl group on the guanidine moiety played an essential role in increasing duplex stability, and the tandem incorporation of a bulky substituent did not reduce the duplex stability. We further evaluated the thermodynamic parameters of the duplexes formed between the GuNA-modified oligonucleotides and the complementary ssRNA (Table 3 , see also Supplementary Figure S4 for van't Hoff plots). Compared to the GuNA[Me]-modified oligonucleotide ( ODN20 ), ODN2 , ODN3 and ODN5 bearing bulkier substituents were found to stabilize the duplex enthalpically, suggesting a positi v e energy gain by increased nucleobase stacking, hydrogen-bond formation, and / or other electrostatic interactions.

X-r ay crystallogr aphic analysis of the duple x es formed by GuNA[R,R]-modified oligonucleotides
Based on the above results, we specula ted tha t the spatial arrangement of the tert -butyl group at the guanidine moiety in the duplex structure is crucial for increased duplex stability. To clarify this, we conducted an Xr ay crystallogr aphic analysis of GuNA[R,R]-modified oligonucleotides. Two self-complementary sequences were selected; (1) a 10 mer oligodeoxynucleotide (5 -GCGT AT ACGC-3 )( 35 ) and (2) an 8 mer oligodeoxynucleotide (5 -GTG Br UA CA C-3 ), wher e Br U r epr esents a a Underlined text in the sequence indicates modified nucleic acid. ( 43 ) (Figures 3 and 4 ). All three crystal structur es wer e f ound to adopt the A-f orm duplex. Intriguingl y, two GuN A[Me, t Bu]s in the duplexed ODN25 formed different structures from each other ( Figure 3 ). The tert -butyl group of one was located at the minor groove side (see the GuNA[Me, t Bu]-T6 •dA15 base pair in Figure 3 A, and C), whereas that of the other one was located outside of the duplex (see the GuNA[Me, t Bu]-T16 •dA5 base pair in Figure 3 A and B). The results could be explained by a steric repulsion of the two tert -butyl groups because the GuNA[Me, t Bu]s were located close together in the duplexed ODN25 . This means that one of the tert -butyl groups at the minor groov e e xposed another tert -butyl group to the outside. On the other hand, two tert -butyl groups of the duplexed ODN29 were located on the minor groove side, and two GuNA[Me, t Bu]s were found to have similar structures (see Figure 4 A and B). Notably, we found that the tert -butyl-substituted guanidine moiety and the 2-carbonyl group of the thymine nucleobase formed a hydrogen bond (3.4 Å for ODN25 in Figure 3 C and 3.2 Å for ODN29 in Figure 4 B), suggesting that this additional hydrogen-bond contact, as well as the hydrophobic interactions between the tert -butyl group and the minor groove, contributed greatly to duplex stability. In contrast, additional hydrogen-bond contacts were not observed in the duplex formed by the GuNA[Me,Me]-modified ODN28 (Figure 4 C and D). In these three X-ray crystal structures, no counter anion was observed around the guanidium part.

5-bromourasil nucleobase
We further evaluated the T m values of the crystalized self-complementary oligonucleotides, ODN25 , ODN28 and ODN29 (Table 5 ) Table 3 , GuNA[ t Bu] and GuNA[Me, t Bu] stabilized the duplex enthalpically. Taken together, the increased duplex stability can be understood by the following three processes: (i) the tert -butyl group of GuNA[ t Bu] or GuNA[Me, t Bu] bound tightly to the minor groove, (ii) an additional hydrogen bond formed, and (iii) the nearby T •A base-pair strengthened. In addition, we compared the X-ray crystal structures of ODN25 and ODN29 , containing GuNA[Me, t Bu], with those of the original sequences (PDB IDs: 1I5W and 8HU5) ( 35 , 43 ) and have confirmed that the overall structures of duplexes containing GuNA[Me, t Bu] are quite similar to those of the original LNA-modified and unmodified DNA duplexes (see Supplementary Figures S5 and S6 in Supporting Information). Although the X-ray crystal structure is a snapshot, the GuNA[Me, t Bu] modification is expected to have little effect on the ov erall duple x structure and the stacking geometries. Using space-filling models, we confirmed tha t the ter t -butyl groups fit tight into the minor groove (see Supplementary Figures S5B and S6B in Supporting Information).  Supplementary Table  S4 in Supporting Information) and their T m values were determined (Table 6 ) Here, 2 ,4 -BN A / LN A-T-modified ODN21 was also used as a control, having the bridging structure. As shown in Figure 3 , GuNA[Me]-T-modified ODN20 exhibited a high misma tch discrimina tion ability compared to the na tural ODN19 ( 44 ) and the 2 ,4 -BN A / LN A-T-modified ODN21 ( 45 ). The strong destabilization of the T •G mismatched wobble base pair by GuNA[Me] was observed. However, as we had expected, the T •G mismatch discrimination ability was much more significant when the substituent groups at the guanidine moiety became larger (see ODN3 and ODN5 in Figure 5 ); here, the steric repulsion between the 2-amino group of the facing guanine nucleobase and the substituent groups at the guanidine moiety (shown as R 1 and R 2 in Figur e 6 ) occurr ed. Among the modified oligonucleotides evaluated in Figure 5

Nuclease stability of GuNA[R]-and GuNA[R,R]-modified oligonucleotides
For application to therapeutic oligonucleotides, the nuclease stability of modified nucleic acids is essential.
Ther efor e, we also evaluated the nuclease stability of GuNA[R]-modified oligonucleotides (Figure 7 ). Poly T oligonucleotides modified at the second position from the 3 -end were synthesized (Supplementary Table S5 in Supporting Information) and subjected to the stability assay against 3 -exonuclease (svPDE: phosphodiesterase I from Crotalus adamanteus venom). As anticipated, oligonucleotides bearing bulkier alkyl groups were found to have higher enzymatic stability ( tert -butyl > isopropyl > ethyl > methyl > hydro gen). Understandabl y, the bulkier alkyl groups blocked the access of nuclease more efficiently. We observed similar trends in the GuNA[R,R]modified oligonucleotides (see Supplementary Figure S7 in Supporting Information), and all GuN A analo gs synthesized here were found to increase nuclease stability more than 2 ,4 -BN A / LN A.

CONCLUSION
In conclusion, we synthesized and evaluated a series of GuNA deri vati v es. We found that the substitution of the guanidine moiety by bulk y alk yl groups, such as isopropyl and ter t -butyl, grea tly increased the stability of the duplex formed by GuNA-modified oligonucleotides and the DN A or RN A complement. We also conducted an Xr ay crystallogr aphic analysis of GuNA[Me, t Bu]-modified oligonucleotides, and the gi v en data showed that the tertbutyl group bound to the minor groove, and an additional hydrogen bond was formed. These are important factors for the significantly increased duplex-forming ability of GuNA[ t Bu]-and GuNA[Me, t Bu]-modified oligonucleotides. We belie v e tha t the da ta shown here have strong implications for de v eloping nov el types of artificial nucleic acids in the future. We also demonstra ted tha t

DA T A A V AILABILITY
All relevant datasets that support the findings of this study, including the procedures for monomer synthesis, as well as the compound characterization data ( 1 H, 13 C and 31 P NMR spectra for all synthesized monomers, the HPLC and MS data for the synthesized oligomers), UV-melting curves and X-r ay crystallogr aphy data (Supplementary Tables S6-S8) are available in the Supplementary Information. Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data bank under accession numbers of 8HIS for duplexed ODN25 , 8I50 for duplexed ODN28 and 8HU5 for duplexed ODN29 .

SUPPLEMENT ARY DA T A
Supplementary Data are available at NAR Online.