Melting temperatures of 4 μM duplexes formed between 12-base unmodified (F12) or 2’,4’-BNANC-modified (F12-1, F12-31, F12-32, F12-6) oligonucleotides and each of complementary single-stranded RNA (R12R) and single-stranded DNA (R12D)in 10 mM sodium phosphate buffer (pH 7.2) and 100 mM NaCl. The increase in melting temperatureper 2’,4’-BNANC-modification (∆
1. Introduction
Artificial regulation of gene expression is quite important for basic study to analyze unknown biological functions of target genes. Comparison of phenotypes with and without knockdown of expression level of the target genes may be helpful to reveal unknown biological functions of the target genes. Artificial regulation of gene expression is also important for therapeutic applications to reduce expression level of mutated target genes. Knockdown of expression level of the mutated target genes may be useful to avoid undesirable effects produced by the mutated target genes. Antisense and antigene technologies are powerful tools to artificially regulate target gene expression. In antisense technology, a single-stranded oligonucleotide added from outside may bind with target mRNA to form oligonucleotide-RNA duplex[1,2]. The formed duplex may inhibit ribosome-mediated translation of target mRNA due to its steric hindrance, or RNaseH may cleave target mRNA in the formed duplex, which may result in reduction of expression level of target mRNA in both cases[1,2]. In antigene technology, a single-stranded homopyrimidine triplex-forming oligonucleotide (TFO) added from outside may bind with homopurine-homopyrimidine stretch in target duplex DNA by Hoogsteen hydrogen bonding to form pyrimidine motif triplex, where T•A:T and C+•G:C base triplets are formed[3,4]. The formed triplex inhibits RNA polymerase and transcription factors-mediated transcription of target gene due to its steric hindrance, which may result in downregulation of expression level of target gene[3,4].
Serious difficulties, such as poor binding ability of added oligonucleotides with target mRNA or target duplex DNA[5-7], and low stability of added oligonucleotides against nuclease degradation[8], may limit practical applications of the antisense and antigene technologies
In this chapter, we describe the excellent properties of 2’,4’-BNANC-modified oligonucleotides for higher ability to form duplex and triplex and for higher nuclease resistance[11-13]. We also show the biological application of 2’,4’-BNANC-modified oligonucleotides to reduce expression level of target mRNA in mammalian cells[14]. PCSK9 is a serine protease involved in the degradation of LDL receptor [15-17]. Suppression of PCSK9 by reducing expression level of PCSK9 mRNA may cause an increase in the amount of the LDL receptor, resulting in the reduction of serum LDL cholesterol level. Thus, the PCSK9 mRNA has the potential to be an antisense target for the treatment of hypercholesterolemia [15-17]. We present the excellent antisense effect of 2’,4’-BNANC modified antisense oligonucleotides to reduce the expression level of the PCSK9 mRNA.
2. Methods to prepare and characterize oligonucleotides
2.1. Preparation of oligonucleotides
We synthesized complementary oligonucleotides for duplex DNA and unmodified homopyrimidine TFO (Figure 1b, 1c) on a DNA synthesizer using the solid-phase cyanoethyl phosphoramidite method, and purified them with a reverse-phase high performance liquid chromatography on a Wakosil DNA or Waters X-Terra column. 2’,4’-BNANC-modified oligonucleotides (Figure 1b, 1c, 1d) were synthesized and purified as described previously[10,11]. 5’-Biotinylated oligonucleotides were prepared from biotin phosphoramidite for kinetic analyses by Biacore described below. The concentration of all oligonucleotides was determined by UV absorbance. The reported extinction coefficient for poly (dT) [ε265 = 8700 cm-1 (mol of base/liter) -1][18] was used for unmodified and 2’,4’-BNANC-modified homopyrimidine TFO. Complementary strands for duplex DNA were annealed by heating at up to 90 ˚C, followed by a gradual cooling to room temperature. When the removal of unpaired single strands is necessary, the annealed sample was applied on a hydroxyapatite column (BioRad). The concentration of duplex DNA was determined by UV absorbance, considering DNA concentration ratio of 1 OD = 50 µg/ml.
2.2. UV melting
Heating of duplex results in monophasic strand dissociation based on the transition between the two states, duplex→2 single strands. Heating of triplex also leads to biphasic strand dissociation according to the transitions between the three states, triplex→ duplex + single strand→3 single strands. Base stacking interactions in the free strands are weaker than those in the bound strands, resulting in a hyperchromic increase in UV absorbance upon heating. UV melting monitors the process of duplex and triplex melting by the temperature dependent change in UV absorbance. First derivative plot of UV absorbance (d
UV melting experiments for duplex and triplex study were carried out on a DU-650 and DU-640 spectrophotometer (Beckman Inc.), respectively, equipped with a Peltier type cell holder. The cell path length was 1 cm. UV melting profiles for duplex study were measured in 10 mM sodium phosphate buffer at pH 7.2 containing 100 mM NaCl. UV melting profiles for triplex study were measured in 10 mM sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2. UV melting profiles were recorded at a scan rate of 0.5 °C/min with detection at 260 nm. The first derivative was calculated from the UV melting profile. The peak temperatures in the derivative curve were designated as the melting temperature,
2.3. CD spectroscopy
CD spectroscopy is sensitive to interactions of nearby bases vertically stacked in strands. Stacking interactions depend on the conformational details of nucleic acid structure. CD spectra provide a certain basis for suggesting the overall conformation of strands in duplex and triplex. The appearance of an intense negative band at the short wavelength range (210-220 nm) in CD spectra indicates the formation of triplex.
CD spectra of the triplex at 20 oC were recorded in 10 mM sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2 on a JASCO J-720 spectropolarimeter interfaced with a microcomputer. The cell path length was 1 cm. The triplex nucleic acid concentration used was 1 µM.
2.4. Electrophoretic mobility shift assay (EMSA)
The 32P-radiolabelled band of triplex migrates slower than that of duplex in native polyacrylamide gel electrophoresis. The formation of triplex results in appearance of a novel radiolabelled band shifted to a new position corresponding to triplex. The percentage of the formed triplex was calculated using the following equation:
where
EMSA experiments for the triplex formation were performed essentially as described previously by a 15% native polyacrylamide gel electrophoresis[12,13,19-27]. In a 9 µl of reaction mixture, 32P-labeled Pur23A•Pyr23T duplex (~1 nM) (Figure 1c) was mixed with increasing concentrations of the specific TFO (Pyr15TM, Pyr15NC7-1, Pyr15NC7-2, Pyr15NC5-1, or Pyr15NC5-2) (Figure 1c) and the nonspecific oligonucleotide (Pyr15xlinkM) (Figure 1c) in buffer [50 mM Tris-acetate (pH 7.0), 100 mM NaCl, and 10 mM MgCl2]. Pyr15xlinkM was added to achieve equimolar concentrations of TFO in each lane as well as to minimize adhesion of the DNA (duplex and TFO) to plastic surfaces during incubation and subsequent losses during processing. After 6 h incubation at 37 oC, 2 µl of 50 % glycerol solution containing bromophenol blue was added without changing the pH and salt concentrations of the reaction mixtures. Samples were then directly loaded onto a 15 % native polyacrylamide gel prepared in buffer [50 mM Tris-acetate (pH 7.0) and 10 mM MgCl2] and electrophoresis was performed at 8 V/cm for 16 h at 4 oC.
2.5. Thermodynamic analyses by isothermal titration calorimetry (ITC)
Isothermal titration calorimetry (ITC) relies upon the accurate measurement of heat changes caused by the interaction of molecules in solution and possesses the advantage of not requiring labeling or immobilization of the components[28]. ITC provides a great deal of thermodynamic information about the binding process from only a single experiment. This information includes the binding stoichiometry (
Isothermal titration experiments for the triplex formation were carried out on a VP ITC system (Microcal Inc., U.S.A.), essentially as described previously[12,13,19-22,25-27]. The TFO (Figure 1c) and Pur23A•Pyr23T duplex (Figure 1c) solutions were prepared by extensive dialysis against 10 mM sodium cacodylate-cacodylic acid at pH 6.1 or pH 6.8 containing 200 mM NaCl and 20 mM MgCl2. The Pur23A•Pyr23T duplex solution in 10 mM sodium cacodylate-cacodylic acid at pH 6.1 or pH 6.8 containing 200 mM NaCl and 20 mM MgCl2 was injected 20-times in 5-µl increments and 10-min intervals into the TFO solution without changing the reaction conditions. The heat for each injection was subtracted by the heat of dilution of the injectant, which was measured by injecting the Pur23A•Pyr23T duplex solution into the same buffer. Each corrected heat was divided by the moles of the Pur23A•Pyr23T duplex solution injected, and analyzed with Microcal Origin software supplied by the manufacturer.
2.6. Kinetic analyses by Biacore
Biacore is one example of a class of optical biosensors which can be used to determine the kinetic binding parameters of molecular interactions, such as association rate constant (
Kinetic experiments for the triplex formation were performed on a BIACORE J instrument (GE Healthcare, U.S.A.), in which a real-time biomolecular interaction was measured with a laser biosensor, essentially as described previously[12,13,19-25,27]. The layer of a SA sensor tip with immobilized streptavidin was equilibrated with 10 mM sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2 at a flow rate of 30 µl/min. 40 µl of 50 mM NaOH containing 1 M NaCl was injected 3 times at a flow rate of 30 µl/min to reduce electrostatic repulsion from the surface. After equilibrating with 10 mM sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2, 160 µl of 0.2 µM Bt(biotinylated)-Pyr23T•Pur23A duplex (Figure 1c) solution was added at a flow rate of 30 µl/min to bind with the streptavidin on the surface. After extensive washing and equilibrating the Bt-Pyr23T•Pur23A-immobilized surface with 10 mM sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2, 70 µl of the TFO (Figure 1c) solution in 10 mM sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2 was injected over the immobilized Bt-Pyr23T•Pur23A duplex at a flow rate of 30 µl/min, and then the triplex formation was monitored for 2 min. This was followed by washing the sensor tip with 10 mM sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2, and the dissociation of the preformed triplex was monitored for an additional 2.5 min. Finally, 40 µl of 100 mM Tris-HCl (pH 8.0) for Pyr15TM (Figure 1c), or 40 µl of 10 mM NaOH (pH 12) for Pyr15NC7-1, Pyr15NC7-2, Pyr15NC5-1, and Pyr15NC5-2 (Figure 1c) was injected at a flow rate of 30 µl/min to completely break the Hoogsteen hydrogen bonding between the TFO and Pur23A, during which the Bt-Pyr23T•Pur23A duplex may be partially denatured. The Bt-Pyr23T•Pur23A duplex was regenerated by injecting 0.2 μM Pur23A. The resulting sensorgrams were analyzed with the BIA evaluation software supplied by the manufacturer to calculate the kinetic parameters.
2.7. Stability of oligonucleotides in human serum against nuclease degradation
Stability of oligonucleotides in human serum was examined by the following two procedures[12,13,26].
Analyses by native polyacrylamide gel electrophoresis
Oligonucleotide (Figure 1c) was 5′-end labeled with 32P using [γ-32P] ATP and T4 polynucleotide kinase by a standard procedure. 2 pmol 32P-labeled oligonucleotide was incubated at 37 oC in 200 µl of human serum from human male AB plasma (Sigma-Aldrich Co., USA). Aliquots of 5 µl were removed after 10, 20, 40, 60, and 120 min of incubation, and mixed with 5 µl of stop solution (80 % formamide, 50 mM EDTA) to terminate the reactions. The samples were loaded on 15 % native polyacrylamide gels prepared in buffer [50 mM Tris-acetate (pH 7.0), 100 mM MgCl2], and electrophoresis was performed at 8 V/cm and 4 oC. The gels were scanned and analyzed by BAS system.
Analyses by anion-exchange HPLC
1 nmol oligonucleotide (Figure 1c) was incubated at 37 oC in 20 µl of 50 % human serum from human male AB plasma (Sigma-Aldrich Co., USA). After incubation for 20, 60 and 120 min, the samples were mixed with 13 µl of formamide to terminate the reactions, and stored at -80 oC until HPLC analyses. The samples were mixed with 400 µl of HPLC buffer [25 mM Tris-HCl (pH 7.0), 0.5 % CH3CN], and analyzed by anion-exchange HPLC on JASCO LC-2000 Plus series with detection at 260 nm using a linear gradient of 0-0.5 M NH4Cl in HPLC buffer over 45 min to resolve the products. The HPLC column used was TSK-GEL DNA-NPR (Tosoh, Japan). Under these conditions, peaks of all proteins from the human serum could be resolved from those of the intact and degraded TFO. Degradation data from the acquired chromatograms were processed using ChromNAV software as supplied by the manufacturer.
2.8. In vitro assay of PCSK9 gene expression in mouse hepatocyte cell line, NMuLi
Mouse hepatocyte cell line NMuLi (4.0 x 105 cells/ml) was cultivated in 6 well plates (2 ml/well) and incubated for 24 hr at 37 oC under 5 % CO2. Antisense oligonucleotide to target a certain region of PCSK9 gene (Figure 1d), Lipofectamine 2000 (Invitrogen), and Opti-MEM (Invitrogen) were mixed. The final concentration of the antisense oligonucleotide in the mixture was adjusted to 1, 3, 10, 30 or 50 nM. After incubation of the mixture for 20 min at room temperature, the mixture was transfected into the cell line. Cell culture medium was exchanged into the new one at 4 hr after the transfection of the antisense oligonucleotide. Cells were collected at 20 hr after the exchange of the cell culture medium. The collected cells were homogenized by ISOGEN (Nippon Gene) to extract total RNA. Concentration of the extracted total RNA was measured by UV absorbance. Length of the extracted total RNA was analyzed by agarose gel electrophoresis. After adjusting the concentration of the total RNA to 4.0 μg/10 μl, we performed reverse transcription reaction using the total RNA to obtain 1st strand cDNA. Then, we carried out real-time PCR using the obtained cDNA to quantitate the expression levels of PCSK9 mRNA and control housekeeping GAPDH mRNA. We normalized the expression level of PCSK9 mRNA by that of control housekeeping GAPDH mRNA, because the antisense oligonucleotides did not affect the expression level of control housekeeping GAPDH mRNA. We examined the effect of the antisense oligonucleotides on the expression level of PCSK9 mRNA.
3. Stabilization of duplex by 2’,4’-BNANC-modification
Formation of stable duplexes with complementary single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) under physiological condition is essential for antisense and diagnostic applications. Thermal stability of duplexes formed between a 12-mer unmodified (F12; Figure 1b) or 2′,4′-BNANC-modified (F12-1, F12-31, F12-32, F12-6; Figure 1b) oligonucleotide and each of its complementary 12-mer ssRNA (R12R; Figure 1b) and 12-mer ssDNA (R12D; Figure 1b) was examined at pH 7.2 by UV melting (Table 1). The
Oligonucleotide | R12R: 3’-r(CGCAAAAAACGA)-5’ | R12D: 3’-d(CGCAAAAAACGA)-5’ |
F12: 5’-d(GCGTTTTTTGCT)-3’ | 45 | 50 |
F12-1: 5’-d(GCGTTTTTTGCT)-3’ | 51(+6.0) | 51(+1.0) |
F12-31: 5’-d(GCGTTTTTTGCT)-3’ | 64(+6.3) | 55(+1.7) |
F12-32: 5’-d(GCGTTTTTTGCT)-3’ | 61(+5.3) | 57(+2.3) |
F12-6: 5’-d(GCGTTTTTTGCT)-3’ | 83(+6.3) | 73(+3.8) |
Because 2′,4′-BNANC-modified oligonucleotides (F12-1, F12-31, F12-32, F12-6) exhibited high binding affinity with complementary ssRNA (R12R) as described above, their ability to discriminate bases was evaluated using single-mismatched ssRNA strand (R12X; Figure 1b) (Table 2). Any mismatched base in the ssRNA strands (R12U, R12G, R12C) resulted in a substantial decrease in the
Oligonucleotide | R12X: 3’-r(CGCAAXAAACGA)-5’ | |||
X = A (matched) | X = U | X = G | X = C | |
F12: 5’-d(GCGTTTTTTGCT)-3’ | 45 | 33 | 42 | 30 |
F12-1: 5’-d(GCGTTTTTTGCT)-3’ | 51 | 37 | 46 | 34 |
4. Stabilization of pyrimidine motif triplex at neutral pH by 2’,4’-BNANC-modification
Formation of stable triplex with TFO under physiological condition is essential for antigene application. Thermal stability of the pyrimidine motif triplexes formed between a 23-bp target duplex (Pur23A•Pyr23T; Figure 1c) and each of its specific 15-mer 2’,4’-BNANC-unmodified (Pyr15TM; Figure 1c) or 2’,4’-BNANC-modified (Pyr15NC7-1, Pyr15NC7-2, Pyr15NC5-1, or Pyr15NC5-2; Figure 1c) TFO was investigated at pH 6.8 by UV melting (Figure 2 and Table 3). UV melting curves in the both directions (heating and cooling) are almost superimposable in all cases, indicating that the dissociation and association processes are reversible. The triplex involving Pyr15TM showed two-step melting. Upon heating the first transition at lower temperature,
TFO | ||
Pyr15TM | 39.1 ± 0.1 | 72.4 ± 0.4 |
Pyr15BNANC7-1 | 79.4 ± 0.6 | |
Pyr15BNANC7-2 | 85.8 ± 0.1 | |
Pyr15BNANC5-1 | 75.6 ± 0.7 | |
Pyr15BNANC5-2 | 75.6 ± 0.7 |
5. No significant structural change of pyrimidine motif triplex at neutral pH by 2’,4’-BNANC-modification
Circular dichroism (CD) spectra of the pyrimidine motif triplexes between the target duplex (Pur23A•Pyr23T) and each of the 2’,4’-BNANC-unmodified (Pyr15TM) or 2’,4’-BNANC-modified (Pyr15NC7-1, Pyr15NC7-2, Pyr15NC5-1, or Pyr15NC5-2) TFO were measured at 20 oC and pH 6.8 (Figure 3). A significant negative band in the short-wavelength (210-220 nm) region was observed for all the profiles (Figure 3), confirming the triplex formation involving each TFO[31]. The overall shape of the CD spectra was quite similar among all the profiles (Figure 3), suggesting that no significant change may be induced in the higher order structure of the pyrimidine motif triplex by the 2’,4’-BNANC modification.
6. Promotion of pyrimidine motif triplex formation at neutral pH by 2’,4’-BNANC-modification
The pyrimidine motif triplex formation between the target duplex (Pur23A•Pyr23T) and each of the 2’,4’-BNANC-unmodified (Pyr15TM) or 2’,4’-BNANC-modified (Pyr15NC7-1, Pyr15NC7-2, Pyr15NC5-1, or Pyr15NC5-2) TFO was examined at pH 7.0 by EMSA (Figure 4). Total oligonucleotide concentration ([specific TFO (Pyr15TM or 2’,4’-BNANC-modified TFO)] + [nonspecific oligonucleotide (Pyr15xlinkM)]) was kept constant at 1000 nMto minimize loss of DNA during processing and to assess sequence specificity. While incubation with 1000 nM Pyr15xlinkM alone did not cause a shift in electrophoretic migration of the target duplex (see
7. Thermodynamic analyses of pyrimidine motif triplex formation involving 2’,4’-BNANC-modified TFO at neutral pH
We examined the thermodynamic parameters of the pyrimidine motif triplex formation between the target duplex (Pur23A•Pyr23T) and each of the 2’,4’-BNANC-unmodified (Pyr15TM) or 2’,4’-BNANC-modified TFO at 25 °C and pH 6.8 by ITC. To investigate the pH dependence of the pyrimidine motif triplex formation, the thermodynamic parameters of the triplex formation between Pur23A•Pyr23T and Pyr15TM were also analyzed at 25 °C and pH 6.1 by ITC. Figure 5a shows a typical ITC profile for the triplex formation between Pyr15NC7-1 and Pur23A•Pyr23T at 25 °C and pH 6.8. An exothermic heat pulse was observed after each injection of Pur23A•Pyr23T into Pyr15NC7-1. The magnitude of each peak decreased gradually with each new injection, and a small peak was still observed at a molar ratio of [Pur23A•Pyr23T]/[Pyr15NC7-1]=2. The area of the small peak was equal to the heat of dilution measured in a separate experiment by injecting Pur23A•Pyr23T into the same buffer. The area under each peak was integrated, and the heat of dilution of Pur23A•Pyr23T was subtracted from the integrated values. The corrected heat was divided by the moles of injected solution, and the resulting values were plotted as a function of a molar ratio of [Pur23A•Pyr23T]/[Pyr15NC7-1], as shown in Figure 5b. The resultant titration plot was fitted to a sigmoidal curve by a nonlinear least-squares method. The binding constant,
Table 4 summarizes the thermodynamic parameters for the pyrimidine motif triplex formation with each of Pyr15TM and the 2’,4’-BNANC-modified TFOs at 25 ˚C and pH 6.8, and those with Pyr15TM at 25 ˚C and pH 6.1, obtained from ITC. The signs of both ∆
TFO | pH | ∆ | ∆ | ∆ | ||
Pyr15TM | 6.1 | (5.81 ± 0.99) x 106 | 13.9 | -9.23 ± 0.11 | -92.0 ± 1.5 | -278 ± 5 |
Pyr15TM | 6.8 | (4.19 ± 2.0) x 105 | 1.0 | -7.67 ± 0.38 | -38.5 ± 7.5 | -103 ± 26 |
Pyr15BNANC7-1 | 6.8 | (5.30 ± 0.45) x 106 | 12.6 | -9.17 ± 0.05 | -56.1 ± 1.0 | -157 ± 4 |
Pyr15BNANC7-2 | 6.8 | (4.10 ± 0.69) x 106 | 9.8 | -9.02 ± 0.11 | -62.1 ± 2.4 | -178 ± 8 |
Pyr15BNANC5-1 | 6.8 | (3.73 ± 0.43) x 106 | 8.9 | -8.96 ± 0.07 | -54.3 ± 1.5 | -152 ± 5 |
Pyr15BNANC5-2 | 6.8 | (3.82 ± 0.29) x 106 | 9.1 | -8.98 ± 0.05 | -53.8 ± 1.1 | -150 ± 4 |
8. Kinetic analyses of pyrimidine motif triplex formation involving 2’,4’-BNANC-modified TFO at neutral pH
To examine the putative mechanism involved in the increase in
TFO | ||||||
Pyr15TM | (2.01 ± 0.11) x 102 | 1.0 | (1.05 ± 0.29) x 10-3 | 1.0 | (1.91 ± 0.88) x 105 | 1.0 |
Pyr15BNANC7-1 | (3.81 ± 0.60) x 102 | 1.9 | (6.96 ± 1.14) x 10-5 | 0.066 | (5.47 ± 2.10) x 106 | 28.6 |
Pyr15BNANC7-2 | (4.61 ± 0.29) x 102 | 2.3 | (7.99 ± 0.83) x 10-5 | 0.076 | (5.77 ± 1.07) x 106 | 30.2 |
Pyr15BNANC5-1 | (4.60 ± 0.18) x 102 | 2.3 | (9.36 ± 1.29) x 10-5 | 0.089 | (4.91 ± 1.01) x 106 | 25.7 |
Pyr15BNANC5-2 | (4.30 ± 0.67) x 102 | 2.1 | (6.61 ± 0.81) x 10-5 | 0.063 | (6.51 ± 2.06) x 106 | 34.1 |
Table 5 summarizes the kinetic parameters for the pyrimidine motif triplex formation with each of Pyr15TM and the 2’,4’-BNANC-modified TFOs at 25 °C and pH 6.8, obtained from BIACORE. The magnitudes of
9. Increased stability of 2’,4’-BNANC-modified oligonucleotides in human serum against nuclease degradation
A major difficulty associated with the use of oligonucleotides as
10. Excellent antisense effect of 2’,4’-BNANC-modified antisense oligonucleotides on the expression level of PCSK9 mRNA
We examined
11. Discussion
The
The
Because the formed triplex structure involving Pyr15TM at pH 6.1 and that involving Pyr15TM at pH 6.8 are the same, the magnitude of ∆
Although the
The increase in the
Kinetic data have demonstrated that the 2’,4’-BNANC modification of TFO considerably decreased the
The nuclease resistance of the 2’,4’-BNANC-modified TFO in human serum was significantly higher than that of the unmodified TFO (Figures 7 and 8). The 2’,4’-BNANC modification increased the nuclease resistance of TFOs in human serum. Previously, the nuclease resistance of the phosphorothioate backbone, in which a nonbridging oxygen of a phosphodiester group was replaced by a sulfur atom, was known to be significantly higher than that of the unmodified backbone[37,38]. However, the
The 2’,4’-BNANC-modified antisense oligonucleotides showed the excellent antisense effect to reduce the expression level of PCSK9 mRNA (Figure 9). As discussed above, the 2′,4′-BNANC-modified oligonucleotides exhibited significantly higher binding affinity with complementary ssRNA than unmodified oligonucleotides (Table 1). Also, the 2’,4’-BNANC modification significantly increased the nuclease resistance of oligonucleotides in human serum (Figures 7 and 8). The significantly higher binding affinity with complementary ssRNA and the significantly higher nuclease resistance of oligonucleotides in human serum may achieve the excellent antisense effect of the 2’,4’-BNANC-modified antisense oligonucleotides. We conclude that the 2’,4’-BNANC-modified antisense oligonucleotides may be useful to reduce the expression level of the target mRNA.
12. Conclusion
The present study has clearly indicated that the 2’,4’-BNANC modification increased the thermal stability of the duplex with complementary ssRNA and ssDNA at neutral pH. It has also clearly demonstrated that the 2’,4’-BNANC modification of TFO increased not only the thermal stability of the pyrimidine motif triplex but also the
We thank Dr. S. M. Abdur Rahman, Dr. Kiyomi Sasaki, Ms. Hiroko Takuma, Ms. Sayori Seki, and Mr. Haruhisa Yoshikawa for their technical assistance. We acknowledge Prof. Satoshi Obika and Prof. Kazuyuki Miyashita for their useful discussions. The present work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (22113519), Grant-in-Aid for Exploratory Research (20655038), Grant-in-Aid for Scientific Research (B) (21350094) and Grant-in-Aid for JSPS Fellows (22-10383) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also supported partly by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), the Program for Precursory Research for Embryonic Science and Technology (PRESTO), and the Creation and Support Program for Start-ups from the Universities of the Japan Science and Technology Agency (JST)
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