Antisense Gapmers with LNA-Wings and (S)-5′-C-Aminopropyl-2′-arabinofluoro-nucleosides Could Efficiently Suppress the Expression of KNTC2

Previously reported (S)-5′-C-aminopropyl-2′-arabinofluoro-thymidine (5ara-T) and newly synthesized (S)-5′-C-aminopropyl-2′-arabinofluoro-5-methyl-cytidine (5ara-MeC) analogs were incorporated into a series of antisense gapmers containing multiple phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in their wing regions. The functional properties of the gapmers were further evaluated in vitro. Compared with the positive control, for the LNA-wing full PS gapmer without 5ara modification, it was revealed that each gapmer could have a high affinity and be thermally stable under biological conditions. Although the cleavage pattern was obviously changed; gapmers with 5ara modification could still efficiently activate E. coli RNase H1. In addition, incorporating one 5ara modification into the two phosphodiester linkages could reverse the destabilization in enzymatic hydrolysis caused by fewer PS linkages. In vitro cellular experiments were also performed, and the Lipofectamine® 2000 (LFA)+ group showed relatively higher antisense activity than the LFA-free group. KN5ara-10, which contains fewer PS linkages, showed similar or slightly better antisense activity than the corresponding full PS-modified KN5ara-3. Hence, KN5ara-10 may be the most promising candidate for KNTC2-targeted cancer therapy.


Introduction
Antisense oligonucleotides (ASOs), composed of approximately 20-bp DNA-like nucleotides, are classified as a kind of mRNA-targeted oligonucleotide therapeutics [1]. Since the first ASO therapeutic, fomivirsen, was approved by the U.S. Food and Drug Administration (FDA) in 1998, nine ASO therapeutics have been approved [2][3][4][5][6][7][8][9][10]. There is no doubt that further research on ASOs will be developed. ASOs can be divided into the following two types depending on the antisense mechanism: the ribonuclease H (RNase H)dependent type and the splice-switching type [11][12][13]. Both types of ASOs need to be taken up into cytoplasm or nucleoplasm for binding with the targeted mRNAs or pre-mRNAs, which would raise several challenges for the clinical application of ASOs. For example, nucleotides experience difficulty passing through cellular or nuclear membranes because of the negative charges on their phosphodiester (PO) linkages. Moreover, natural DNA strands are quickly degraded via nuclease-mediated hydrolysis inside the plasm, resulting in low pharmacological effects. To overcome these challenges, chemically modified nucleosides have been developed and utilized in approved ASOs.
Phosphorothioate (PS) linkages, which contain sulphur substitutions of non-bridging oxygens at each PO linkage, are the most popular chemical modification to improve the

Oligonucleotide Synthesis
The synthesis of phosphoramidite corresponding to 5ara-Me C is shown in Scheme S1 in the Supporting Information. The modified nucleoside analogs 5ara-T and 5ara-Me C were incorporated into a series of LNA-wing antisense gapmers utilizing a DNA/RNA synthesizer via the solid-phase phosphoramidite method. After synthesis, to prevent the additional reaction of acrylonitrile with 5ʹ-C-aminopropyl groups, the controlled-pore glass (CPG) beads were treated with 10% dimethylamine in MeCN at room temperature for 5 min, followed by rinsing with MeCN to selectively remove cyanoethyl groups. The gapmers were then cleaved from CPG beads and deprotected by treatment with a concentrated NH3 solution for 12 h at 55 °C. The corresponding RNA oligomers used in this study were prepared with a DNA/RNA synthesizer. After synthesis, in contrast to antisense gapmers, the RNA oligomers were cleaved from CPG beads and deprotected by treatment with concentrated NH3 solution/40% methylamine (1:1, v/v) for 10 min at 65 °C. Then, 2ʹ-O-TBDMS groups in RNA oligomers were removed using Et3N•3HF (125 µL) in DMSO (100 µL) for 1.5 h at 65 °C. The reaction was quenched with a 0.1 M TEAA buffer (pH 7.0) and the mixture was desalted using a Sep-Pak C18 cartridge. The modified antisense gapmers and RNA oligomers were finally purified by 20% denaturing polyacrylamide gel electrophoresis (PAGE) containing 7 M urea. The sequences of the oligonucleotides used in this study are shown in Tables 1 and S1 (Supplementary Material).

Oligonucleotide Synthesis
The synthesis of phosphoramidite corresponding to 5ara-Me C is shown in Scheme S1 in the Supporting Information. The modified nucleoside analogs 5ara-T and 5ara-Me C were incorporated into a series of LNA-wing antisense gapmers utilizing a DNA/RNA synthesizer via the solid-phase phosphoramidite method. After synthesis, to prevent the additional reaction of acrylonitrile with 5 -C-aminopropyl groups, the controlled-pore glass (CPG) beads were treated with 10% dimethylamine in MeCN at room temperature for 5 min, followed by rinsing with MeCN to selectively remove cyanoethyl groups. The gapmers were then cleaved from CPG beads and deprotected by treatment with a concentrated NH 3 solution for 12 h at 55 • C. The corresponding RNA oligomers used in this study were prepared with a DNA/RNA synthesizer. After synthesis, in contrast to antisense gapmers, the RNA oligomers were cleaved from CPG beads and deprotected by treatment with concentrated NH 3 solution/40% methylamine (1:1, v/v) for 10 min at 65 • C. Then, 2 -O-TBDMS groups in RNA oligomers were removed using Et 3 N·3HF (125 µL) in DMSO (100 µL) for 1.5 h at 65 • C. The reaction was quenched with a 0.1 M TEAA buffer (pH 7.0) and the mixture was desalted using a Sep-Pak C18 cartridge. The modified antisense gapmers and RNA oligomers were finally purified by 20% denaturing polyacrylamide gel electrophoresis (PAGE) containing 7 M urea. The sequences of the oligonucleotides used in this study are shown in Table 1 and Table S1 (Supplementary Material).

RNA-Binding Affinity
The accurate binding of ASOs to target mRNA is necessary for RNase H recognition and the following antisense mechanism. As reported before, although PS linkages negatively affected thermal stability, LNAs could significantly increase RNA binding affinity and a single (S)-5 -C-Aminopropyl-2 -arabinofluoro modification caused few changes to the 50% melting temperature (T m ) values of ASO/RNA duplexes [27][28][29]. Therefore, we hypothesized that the series of KN5ara gapmers would have sufficient RNA binding affinity for therapeutic application. In this study, each KN5ara gapmer was mixed in the same volume of cRNA-1, and then annealed to form ASO/RNA duplexes. Temperature-induced melting was measured by ultraviolet (UV) spectroscopy in a 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl, and T m values were obtained from melting curves using the standard method. Each ∆T m was calculated from [T m (duplex containing each KN5ara gapmers) − T m (duplex containing KN-pos)]. Table 1. Sequence of each gapmer and T m values of duplexes containing these gapmers.

Abbreviation of Gapmers
As shown in Table 1, all KN5ara gapmers maintained a stable duplex structure with complementary RNA strands at 37 • C. The incorporation of 5ara-modified analogs in the gap region showed moderate effects on T m values, while the replacement of LNAs in the wing region with 5ara-T or 5ara-Me C resulted in thermal destabilization. These results are consistent with previous studies showing that the thermal stability of 5ara modification is comparable to that of natural DNA but lower than that of LNA [27]. Furthermore, the T m value of KN5ara-9 was similar to the addition of KN5ara-7 and KN5ara-8, indicating that the continuous introduction of modified analogs might not have an additional impact on thermal stability. The gapmers with fewer PS linkages (KN5ara-10 and KN5ara-11) were more stable than the corresponding full PS gapmers (KN5ara-3 and KN5ara-6).

The Ability for E. coli RNase H1 Activation
Before the in vitro cell experiments, we established a simple enzymatic reaction system using RNase H1 purified from E. coli to determine whether RNase H-mediated cleavage could be activated by KN5ara gapmers [33,34]. The KN5ara gapmers used in this experiment were mixed with fluorescein-labeled cRNA-2 at 1:5 before annealing. These duplexes were dissolved in a buffer containing 50 mM Tris-HCl (pH 8.0), 75 mM KCl, 3 mM MgCl 2 , and 10 mM dithiothreitol. Diluted E. coli RNase H1 solution (60 unit/L in H 2 O) was then added, and the mixture was incubated at 37 • C for the required time (0, 1, 5, 15, and 30 min and 1, 2, and 4 h). The aliquots were analyzed using 20% denaturing PAGE and then quantified using a luminescent image analyzer LAS-4000 (Fujifilm). As shown in Figure 2, even though the initial reaction velocity showed a slight change, it was confirmed that the complete cRNA strand was almost cleaved in KN5ara gapmers as well as in the positive control KN-pos after a 5 min reaction. Despite differences in each cleavage pattern, a single 5ara modification moderately affected the cleavage mechanism of E. coli RNase H1, which was consistent with previous reports, because of the remaining recognition portions [27].
Molecules 2022, 27, 7384 5 of 12 differences for KN5ara-6 and -7, KN5ara-4 and -5 were found to be similar to the others . The antisense activity of all KN5ara gapmers is directly evaluated in the following section. ASOs containing multiple 5ara modifications showed extremely high enzyme tolerance in a 3% bovine serum (BS) experiment system [27]. LNAs and PS linkages are also expected to improve the stability of KN5ara gapmers during enzyme-mediated hydrolysis. However, PS linkages may lead to undesirable apoptosis because of their high affinity with plasma proteins, such as paraspeckle proteins [15]. We hypothesized that the application of the 5ara modification could reduce the number of PS linkages while maintaining sufficient nuclease resistance. In this research, a series of fluorescein-labeled KN5ara gapmers were synthesized (Table S1), including positive control (KN-pos-F), and gapmers with full-PS (KN5ara-6-F) or with less PS linkages (KN5ara-10/11-F). For comparison, the Notably, not all KN5ara gapmers were evaluated in this assay. Since the recognition of E. coli RNase H1 would begin with a few nucleotides from the 5 -terminal of ASO, KN5ara-1 and -2, in which the 5ara modification was inserted into 5 -gap region, it might activate E. coli RNase H1 similarly with KN-pos. Meanwhile, as there are no significant differences for KN5ara-6 and -7, KN5ara-4 and -5 were found to be similar to the others. The antisense activity of all KN5ara gapmers is directly evaluated in the following section.
ASOs containing multiple 5ara modifications showed extremely high enzyme tolerance in a 3% bovine serum (BS) experiment system [27]. LNAs and PS linkages are also expected to improve the stability of KN5ara gapmers during enzyme-mediated hydrolysis. However, PS linkages may lead to undesirable apoptosis because of their high affinity with plasma proteins, such as paraspeckle proteins [15]. We hypothesized that the application of the 5ara modification could reduce the number of PS linkages while maintaining sufficient nuclease resistance. In this research, a series of fluorescein-labeled KN5ara gapmers were synthesized (Table S1), including positive control (KN-pos-F), and gapmers with full-PS (KN5ara-6-F) or with less PS linkages (KN5ara-10/11-F). For comparison, the gapmer without any 5ara modification corresponding to KN5ara-11-F was prepared as well. The gapmers described above were dissolved in OPTI-MEM and incubated with 50% BS at 37 • C. During incubation, aliquots from the reactions were taken for the required time (0, 1, 3, 6, 12, 24 and 48 h), then analyzed with 20% PAGE containing 7 M urea and quantified using the Luminescent Image analyzer LAS-4000 (Fujifilm). The results are shown in Figure 3. gapmer without any 5ara modification corresponding to KN5ara-11-F was prepared as well. The gapmers described above were dissolved in OPTI-MEM and incubated with 50% BS at 37 °C. During incubation, aliquots from the reactions were taken for the required time (0, 1, 3, 6, 12, 24 and 48 h), then analyzed with 20% PAGE containing 7 M urea and quantified using the Luminescent Image analyzer LAS-4000 (Fujifilm). The results are shown in Figure 3.
After 48 h incubation, approximately 44% of the KN-pos-F strands remained, while only 26% of KN5ara-12-F strands persisted, apparently owing to the decrease in the two PS linkages. A comparison of KN-pos-F and KN5ara-6-F showed that 5ara modification could further increase the nuclease resistance of gapmers, resulting in a half-life of >48 h with 50% BS treatment. Meanwhile, 44% and 42% of the complete strands of KN5ara-10-F and KN5ara-11-F remained, respectively, indicating the same nuclease resistance as KNpos-F. It is presumed that incorporating one 5ara modification into the two PO linkages could reverse destabilization in enzymatic hydrolysis, which is caused by fewer PS linkages.

Antisense Activity
In addition to the physical experiments and simple in vitro enzymatic assays, in vitro cellular experiments were performed to knock down KNTC2 in A549tGFP cells to evaluate the antisense activity of each KN5ara gapmer. The pre-cultured A549tGFP cells were treated with KN5ara gapmers at a final concentration of 4.0 nM or 2.5 µM, and were transfected with 0.3% Lipofectamine ® 2000 (LFA) or not, respectively. After incubation, total After 48 h incubation, approximately 44% of the KN-pos-F strands remained, while only 26% of KN5ara-12-F strands persisted, apparently owing to the decrease in the two PS linkages. A comparison of KN-pos-F and KN5ara-6-F showed that 5ara modification could further increase the nuclease resistance of gapmers, resulting in a half-life of >48 h with 50% BS treatment. Meanwhile, 44% and 42% of the complete strands of KN5ara-10-F and KN5ara-11-F remained, respectively, indicating the same nuclease resistance as KN-pos-F. It is presumed that incorporating one 5ara modification into the two PO linkages could reverse destabilization in enzymatic hydrolysis, which is caused by fewer PS linkages.

Antisense Activity
In addition to the physical experiments and simple in vitro enzymatic assays, in vitro cellular experiments were performed to knock down KNTC2 in A549tGFP cells to evaluate the antisense activity of each KN5ara gapmer. The pre-cultured A549tGFP cells were treated with KN5ara gapmers at a final concentration of 4.0 nM or 2.5 µM, and were transfected with 0.3% Lipofectamine ® 2000 (LFA) or not, respectively. After incubation, total mRNA inside the cells was extracted, followed by reverse transcription of the targeted KNTC2 mRNA. A quantitative real-time polymerase chain reaction (qRT-PCR) was performed in duplicate, and relative KNTC2 mRNA levels were calculated, as shown in Figure 4.
In the LFA-free group, KN-pos showed antisense activity comparable to that in the LFA+ condition. All KN5ara gapmers were clearly less active, indicating that cellular uptake might be reduced, although the extent of the reduction varied greatly depending on the site of the 5ara modification. Meanwhile, KN5ara-4 efficiently knocked down KNTC2 mRNA, and the decrease in antisense activity of KN5ara-8 was also reversed by continuous 5ara modifications (KN5ara-9). Notably, significant property degradation was observed in KN5ara-11, as compared to KN5ara-6, whereas KN5ara-10 showed similar or slightly better antisense activity than KN5ara-3.
In more detail, it is obvious that KN5ara-4 showed the most effective antisense activity in both methods, when used with lipofection or not. The relative KNTC2 mRNA level tended to increase according to the shift of 5ara modification to the 3ʹ-terminal, which suggests 5ara modification might be accepted well in the center of the gap region. Further investigation is need, however, of the synthesis of 5ara modified adenosine and guanosine analogs. Moreover, according to previous studies, the incorporation of a single 2ʹ-OMe modification at gap position 2 could reduce the PS-derived toxicity while maintaining enough antisense activity [16]. In this study, KN5ara-3 and -10 obtained a 5ara modification at gap position 2; additionally, KN5ara-10 retained even less antisense activity for PS linkages. Therefore, KN5ara-10 might be the most promising candidate for reducing PSderived cytotoxicity. Further experiments studying the cytotoxicity are planned.  LFA-free groups were incubated with the gapmers for 48 h, and further incubated for 24 h after exchanging medium. LFA+ groups were incubated with the gapmers for 24 h, and further incubated for 24 h after exchanging medium. Treatment without gapmers was used as a control (unTF). The total mRNA inside cells were extracted, followed by the reverse transcription of the targeted KNTC2 mRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in duplicate, and the relative KNTC2 mRNA levels were calculated via the ddCT method.
The results suggested that there was no obvious change in the antisense activity of KN5ara gapmers with a single 5ara modification when LFA was used. KN5ara-4/5 showed much higher KNTC2 knockdown efficiency than the positive control KN-pos, although KN5ara-8 showed a 2-fold decrease. Interestingly, compared to KN5ara-8, KN5ara-9, which has another 5ara modification at the same site as KN5ara-7, maintained antisense activity similar to that of KN5ara-7, suggesting that continuous 5ara modifications may induce further improvement. Contrastingly, KN5ara-3 and KN5ara-10 exhibited similar antisense activity, whereas KN5ara-11 had slightly lower activity than KN5ara-6.
In the LFA-free group, KN-pos showed antisense activity comparable to that in the LFA+ condition. All KN5ara gapmers were clearly less active, indicating that cellular uptake might be reduced, although the extent of the reduction varied greatly depending on the site of the 5ara modification. Meanwhile, KN5ara-4 efficiently knocked down KNTC2 mRNA, and the decrease in antisense activity of KN5ara-8 was also reversed by continuous 5ara modifications (KN5ara-9). Notably, significant property degradation was observed in KN5ara-11, as compared to KN5ara-6, whereas KN5ara-10 showed similar or slightly better antisense activity than KN5ara-3.
In more detail, it is obvious that KN5ara-4 showed the most effective antisense activity in both methods, when used with lipofection or not. The relative KNTC2 mRNA level tended to increase according to the shift of 5ara modification to the 3 -terminal, which suggests 5ara modification might be accepted well in the center of the gap region. Further investigation is need, however, of the synthesis of 5ara modified adenosine and guanosine analogs. Moreover, according to previous studies, the incorporation of a single 2 -OMe modification at gap position 2 could reduce the PS-derived toxicity while maintaining enough antisense activity [16]. In this study, KN5ara-3 and -10 obtained a 5ara modification at gap position 2; additionally, KN5ara-10 retained even less antisense activity for PS linkages. Therefore, KN5ara-10 might be the most promising candidate for reducing PS-derived cytotoxicity. Further experiments studying the cytotoxicity are planned.

Conclusions
In summary, the novel synthesis of (S)-5 -C-Aminopropyl-2 -arabinofluoro-5-methylcytidine (5ara-Me C) was accomplished in this study. The properties of a series of LNA-wing antisense gapmers containing (S)-5 -C-Aminopropyl-2 -arabinofluoro (5ara) modification, named KN5ara gapmers, were evaluated. It was revealed that each KN5ara gapmer could bind to the complementary RNA strand with high affinity and could be thermally stable under biological condition. The ability for E. coli RNase H1 activation was moderately affected by the incorporation of a single 5ara modification, although the cleavage pattern was obviously changed, which is consistent with previous reports. To determine whether the 5ara modification could be an alternative to PS linkage, the nuclease resistance of several KN5ara gapmers was evaluated using a simple enzymatic reaction system. As a result, incorporating one 5ara modification inside the two PO linkages could reverse the destabilization in enzymatic hydrolysis caused by fewer PS linkages. In addition, in vitro cellular experiments were performed to knockdown KNTC2 in A549tGFP cells. The LFA+ group showed a relatively higher antisense activity than the LFA-free group, while KN5ara-4 showed superior antisense activity in both methods. Compared to KN5ara-7, KN5ara-8, and KN5ara-9, continuous 5ara modifications at certain sites might induce further improvement. KN5ara-10, which contained fewer PS linkages, showed similar or slightly better antisense activity than the corresponding KN5ara-3. Hence, given the possibility of lower PS-derived cytotoxicity, KN5ara-10 might be the best candidate for KNTC2-targeted ASO for cancer therapy in this study. However, the further cytotoxicity assay of these KN5ara gapmers should be performed in future studies, and gapmers with fewer PS linkages than KN5ara-10 should also be synthesized and evaluated as well.

Solid-Phase Oligonucleotide Synthesis
The synthesis was carried out with a DNA/RNA synthesizer by the phosphoramidite method. After the synthesis, the RNA oligomers were cleaved from CPG beads and deprotected by treatment with concentrated NH 3 solution/40% methylamine (1:1, v/v) for 10 min at 65 • C, while the DNA-based oligomers were treated with concentrated NH 3 solution for 12 h at 55 • C. Notably, before the treatment of the NH 3 solution, the CPG beads were treated with 10% dimethylamine in MeCN for 5 min followed by rinsing with MeCN to selectively remove cyanoethyl groups, if there are analogs introduced in oligomers. Then, 2 -O-TBDMS groups in RNA oligomers were removed by Et 3 N·3HF (125 µL) in DMSO (100 µL) for 1.5 h at 65 • C. The reaction was quenched with 0.1 M TEAA buffer (pH 7.0), and the mixture was desalted using a Sep-Pak C18 cartridge. The oligomers were purified by 20% PAGE containing 7 M urea to give highly purified oligonucleotides.

MALDI-TOF/MS Analysis of ONs
The spectra were obtained with a time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). A solution of 3-hydroxypicolinic acid (

Thermal Denaturation Study
The solution containing 3.0 µM ASO/RNA duplexes were prepared by mixing the ASOs (600 pmol) with complementary target cRNA-1 (600 pmol) in a buffer of 10 mM sodium phosphate (pH 7.0) containing 100 mM NaCl, and then heated at 90-100 • C, followed by being cooled gradually to room temperature. Thermally induced transitions were monitored at 260 nm with a UV/vis spectrometer fitted with a temperature controller in quartz cuvettes with a path length of 1.0 cm. The sample temperature was increased by 0.5 • C/min.

RNase H Assay
The ASO/RNA duplexes used for RNase H assay were prepared by mixing the ASOs (600 pmol) with fluorescein labeled complementary target cRNA-2 (3000 pmol) in 75 µL of 50 mM Tris-HCl (pH 8.0) containing 75 mM KCl, 3 mM MgCl 2 and 10 mM dithiothreitol, followed by heating at 90-100 • C for 5 min and cooling gradually to room temperature. Then, 70 µL diluted RNase H solution (60 unit/L in H 2 O) was added, and subsequently the mixture was incubated at 37 • C for the required time. Aliquots of 5 µL were diluted with 100% formamide (10 µL). Samples were subjected to electrophoresis in 20% PAGE containing 7M urea and quantified by Luminescent Image analyzer LAS-4000 (Fujifilm).

Nuclease Resistance of Single-Stranded ASO
Fluorescein labeled ASOs (300 pmol) were dissolved in OPTI-MEM (37 µL) and used for the serum stability test. 1.0 µL of the oligomer solution was diluted in 10 µL stop solution (10% formamide in 10 mM EDTA) as the control sample (0 min). Then, 36 µL bovine serum was added to achieve a final concentration of 50% (v/v), and subsequently the mixture was incubated at 37 • C for the required time. Aliquots of 2.0 µL were diluted with 10 µL stop solution. Samples were subjected to electrophoresis in 20% PAGE containing 7M urea and quantified by Luminescent Image analyzer LAS-4000 (Fujifilm).

ASOs In Vitro Activity Assay
A549tGFP cells were established by transfection with pGIPZ (Horizon Discovery) into a human lung cancer cell line A549 (ATCC). For LFA-free (LFA-) group, A549tGFP cells were plated into 96-well plates at 2 × 10 3 cells/well, followed by the cultivation in D-MEM