HK1127360B - Cationic oligonucleotides, automated methods for preparing same and their uses - Google Patents

Description

Cationic oligonucleotides, automated methods of preparing cationic oligonucleotides, and uses thereof

The present invention relates to cationic oligonucleotides, i.e. oligonucleotide-oligocationic molecules, also referred to herein as cationic oligonucleotides (regardless of their total charge), which can be synthesized stepwise on an oligonucleotide synthesizer. The invention also relates to their use in molecular biology, diagnostics and therapeutic applications.

Oligonucleotides have a wide range of applications in molecular biology and diagnostics, and are likely to be a highly selective class of drugs for the treatment of many diseases.

Oligonucleotides are polyanions that exert their specific activity upon hybridization to complementary sequences carried on other polyanionic nucleic acids.

As drug candidates they must also be able to cross negatively charged cell membranes.

A slight electrostatic consideration may infer that the addition of cationic groups to the oligonucleotide structure contributes to the hybridization energy and cell binding.

To achieve this, a number of synthetic methods for introducing ammonium and guanidinium groups into oligonucleotides have been explored: phosphate backbone substitutions, ribose or nucleobase modifications, and end-bound polycations. However, considerations of hybridization specificity, nucleic acid-processing enzyme activity and metabolite toxicity all indicate that the block approach (block aproach) is the best solution, in which a polycation is attached to an oligonucleotide that is naturally occurring in nature. Unfortunately, however, stepwise automated synthesis of oligonucleotide-cationic peptide conjugates has not been generally practiced. On the other hand, the chemical reaction of the binding between the preformed large blocks is not simple, and especially the "super" zwitterions in water cause problems of poor solubility, purification and identification. In addition, molecular biology and diagnostic applications require rapid and easy synthesis of any given base sequence attached to any organic cation length.

The present inventors have found that on-line computer-driven synthesis of oligonucleotide-oligocations can be achieved by an oligonucleotide synthesizer that inserts vials containing the appropriately activated and protected oligocation derivative into vials containing the four natural bases.

It is therefore an object of the present invention to provide novel cationic oligonucleotides.

It is another object of the invention to provide high-yield automated synthesis of said cationic oligonucleotides.

A further object of the invention relates to the use of said cationic oligonucleotides, in particular in molecular biology, diagnostics and therapeutics.

The present invention thus relates to mixed oligonucleotide oligocation molecules that can be synthesized by automated phosphoramidite chemistry, i.e., phosphodiester.

More specifically, the cationic oligonucleotide A of the invention_iB_jH has an oligonucleotide moiety Ai and an oligocation moiety Bj, wherein

.A_iIs an oligonucleotide residue of an i-mer, i ═ 5 to 50, having natural or unnatural nucleobases and/or pentofuranosyl and/or natural phosphodiester bonds.

Bj is an organic oligocationic moiety of a j-mer, j ═ 1 to 50, where B is selected from the group comprising:

-HPO₃-R¹-(X-R² _n)_n1-X-R³-O-wherein R¹、R² _nAnd R³Are identical or different lower alkylene, X is NH or NC (NH)₂)₂，n1＝2-20，

-HPO₃-R⁴-CH(R⁵X¹)-R⁶-O-wherein R⁴Is lower alkylene, R⁵And R⁶Are identical or different lower alkylene radicals, X¹Is putrescine, spermidine or spermine residue,

-HPO₃-R⁷-(aa)_n2-R⁸-O-wherein R⁷Is lowAlkylene radical, R⁸Is lower alkylene, serine, amino alcohol obtained by reduction of natural amino acids, (aa)_n2Is a peptide containing natural amino acids with cationic side chains such as arginine, lysine, ornithine, histidine, diaminopropionic acid, n2 ═ 2-20.

As used herein the specification and claims, "lower alkyl" and "lower alkylene" preferably refer to optionally substituted C1-C5 straight or branched chain alkyl or alkylene, respectively.

For example a is selected from the group comprising deoxyribonucleotides, ribonucleotides, Locked (LNA) nucleotides and chemical modifications or substitutions thereof such as phosphorothioates (also known as phosphorothioates), 2 '-fluoro groups, 2' -O-alkyl groups or labelling groups such as fluorescers.

The mixed oligonucleotide-oligocation molecule of the invention has^3’A^5’-a B sequence.

Other molecules of the invention have B-^3’A^5’And (4) sequencing.

Other molecules of the invention have B-^3’A^5’-B or^3’A^5’-B-^3’A^5’And (4) sequencing.

This sequence is exemplified in the examples by an oligonucleotide-spermine molecule having the following structure:

wherein A, i and j are as defined above.

Molecules having a as a phosphorothioate nucleotide are particularly advantageous in view of their biological applications, since phosphorothioate oligonucleotides are not hydrolyzed in biological fluids.

The cationic oligonucleotides defined above and their complementary sequences form fast and stable complexes in the context of strand displacement and even in the context of plasmid strand invasion, as exemplified in the examples.

Due to the terminal conjugation, the sequence selectivity is as high as for the native nucleotide.

Thus, the cationic oligonucleotides of the invention are well suited for molecular biology, research reagents and diagnostic applications, such as PCR, real-time PCR, genotyping, in situ hybridization and DNA chips.

These applications are also encompassed by the present invention, and also include the use of oligonucleotide-oligocation molecules as defined above.

Unlike anionic oligonucleotides, the examples show that cationic oligonucleotides of the invention can spontaneously enter the cytoplasm and nucleus of living cells.

In view of their enhanced hybridization and cell penetration properties, they can also be used in therapeutic methods, such as those mediated by antisense and siRNA degradation of messenger RNA, by exon skipping during the maturation of messenger RNA, by triple helix formation with chromatin, by chromatin strand invasion (gene correction).

The invention therefore also relates to a pharmaceutical composition comprising an effective amount of an oligonucleotide-oligocation as defined above and a pharmaceutically acceptable carrier.

The invention therefore also relates to a method of treatment comprising the use of an effective amount of an oligonucleotide-oligocation as defined above, together with a pharmaceutically acceptable carrier.

The mixed oligonucleotide-oligocation molecules defined above are advantageously synthesized stepwise on an oligonucleotide synthesizer by the phosphoramidite pathway according to a method comprising

-inserting the vial containing the activated and protected oligocation B into an oligonucleotide synthesizer of the vial to which the oligonucleotide A as defined above is added, or reversing the order,

stopping the synthesis when the desired length is reached,

cleaving the oligomer from the solid support, and

-removing the protecting group.

The present invention is directed to phosphoramidite reagents for the automated synthesis of building oligocationic repeat blocks B. The following phosphoramidite reagents can be used for this purpose

P(OR⁹)(N(R¹⁰)₂)-O-R¹-(X-R² _n)_n1-X-R³-O-Prot, wherein R¹、R²、R³And n1 is as defined above, X is suitably protected NH or NC (NH)₂)₂，R⁹is-CH₂CH₂CN or lower alkyl, R¹⁰Is lower alkyl, or-N (R)¹⁰)₂Is pyrrolidinyl (pyrrolidinyl group), piperidinyl (piperidinyl group) or morpholinyl (morpholino group), Prot is a protecting group used for oligonucleotide synthesis, e.g. DMT, MMT;

P(OR⁹)(N(R¹⁰)₂)-O-R⁴-CH(R⁵X1)-R⁶-O-Prot, wherein R⁴、R⁵、R⁶Is lower alkylene, X¹Is suitably protected putrescine, spermidine or spermine, R⁹And R¹⁰The definition of (1) is as above;

P(OR⁹)(N(R¹⁰)₂)-O-R⁷-(aa)_n2-R⁸-O-Prot, wherein R⁷、R⁸、R⁹、R¹⁰N2 and Prot are as defined above (aa)_n2Is a peptide containing a natural amino acid with a suitably protected cationic side chain, such as arginine, lysine, ornithine, histidine, diaminopropionic acid, n2 ═ 2-20.

Suitably protected NH or NC (NH)₂)₂Meaning that the protecting group is located on the amino or guanidino group, respectively, such that its functionality is towards the agent in questionThe chemical reaction conditions of (a) are inert.

Such protecting groups are, for example, Phthalimide (PHTH), trifluoroacetic acid, allyloxycarbonyl (Alloc), benzyloxycarbonyl (CBZ), chlorobenzyloxycarbonyl, t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc) and isonicotinyloxy (i-Noc).

According to one embodiment of the invention, the stepwise synthesis of the nucleotide sequence is followed by a stepwise synthesis of the oligocation moiety to obtain a peptide having the sequence (³′A⁵' -B).

According to another embodiment, a reverse step is performed, the stepwise synthesis of the oligocation moiety being followed by a stepwise synthesis of the oligonucleotide sequence to obtain (B-³’A⁵') sequence.

According to yet another embodiment, a mixed sequence is synthesized.

In particular, oligonucleotide sequences capped at both ends (B-³’A⁵' -B) sequences which are resistant to exonucleases in biological fluids, interrupted by cations ((B)³’A⁵’-B-³’A⁵') allows targeting of adjacent nucleic acid sequences.

By using natural amines (e.g. spermine) or peptides (e.g. oligoarginine), the potential toxicity of the metabolites can be avoided. Spermine is indeed present in the cell in millimolar concentrations, and its terminal alkylation is harmless. Furthermore, many nucleoproteins have basic peptide sequences.

It is advantageous to obtain the activated and protected oligocation B by protection of the amino group of the polyamine and subsequent alpha, omega-dihydroxyalkylation to form a diol suitable for oligonucleotide synthesis.

Classical DMT and phosphoramidite extension chemistry works advantageously with base labile TFA protecting groups.

Chemically protected diols are novel products and are within the scope of the present invention.

The present invention particularly relates to intermediates selected from the group comprising:

P(OR⁹)(N(R¹⁰)₂)-O-R¹-(X-R² _n)_n1-X-R³-O-Prot, wherein R¹、R²、R³And n1 is as defined above, X is suitably protected NH or NC (NH)₂)₂，R⁹is-CH₂CH₂CN or lower alkyl, R¹⁰Is lower alkyl, or-N (R)¹⁰)₂Is pyrrolidinyl, piperidinyl or morpholinyl, Prot is a protecting group for oligonucleotide synthesis, e.g., DMT, MMT;

P(OR⁹)(N(R¹⁰)₂)-O-R⁴-CH(R⁵X1)-R⁶-O-Prot, wherein R⁴、R⁵、R⁶Is lower alkylene, X¹Is suitably protected putrescine, spermidine or spermine, R⁹And R¹⁰The definition of (1) is as above;

P(OR⁹)(N(R¹⁰)₂)-O-R⁷-(aa)_n2-R⁸-O-Prot, wherein R⁷、R⁸、R⁹、R¹⁰N2 and Prot are as defined above (aa)_n2Is a peptide containing a natural amino acid with a suitably protected cationic side chain, such as arginine, lysine, ornithine, histidine, diaminopropionic acid, n2 ═ 2-20.

Additional features and advantages of the invention are set forth below. Specifically, a decameric oligonucleotide sequence (A) having spermine (S)₁₀) (hereinafter referred to as A)₁₀S_n) The synthesis of (a) will be given by way of illustration and not by way of limitation. In an embodiment, reference will be made to fig. 1 to 14, respectively, which represent:

FIG. 1, cationic oligonucleotide N₁₀S_n(n-1-2) HPLC analysis on reverse phase column,

-a view of FIG. 2,purification of oligonucleotide N₁₀S_n(n-1-6) HPLC analysis on an anion exchange column,

FIG. 3, analysis of N by polyacrylamide gel electrophoresis₁₀S_n(n-1-6) electrophoretic mobility,

FIG. 4, N₁₀And N₁₀·C₁₀The spontaneous exchange at various temperatures is carried out,

FIG. 5, revealing of N by polyacrylamide gel electrophoresis₁₀And N₁₀S_nThe chain exchange between the two groups of the chain exchange,

FIG. 6, N₁₀S_n·C₁₀The melting temperature of the duplex (where C is the nucleotide complementary to N),

-figure 7: n is a radical of₁₀S_n(n-0-6) and^5’GTGGCATCGC^3’and with^5’GTGGCGTCGC^3’As a result of comparison of the melting temperatures of the duplexes formed therebetween,

FIG. 8, purification of N₁₀S_n(n-1-6) ES-MS analysis of the oligonucleotide,

FIG. 9, phosphorothioate oligonucleotide N₁₂S₁₁F (9A) and N₁₂S₂An HPLC chromatogram of F (9B),

FIG. 10, N₁₂S₂F (10A) and N₁₂S₁₁MALDI-TOF MS mass spectrum of F (10B),

FIG. 11, N₁₄S₄F (11A) and N₂₀S₅An HPLC chromatogram of F (11B),

FIG. 12, N₁₄S₄F (12A) and N₂₀S₅MALDI-TOF MS mass spectrum of F (12B),

FIG. 13, N₁₄S_nF (13A) and N₂₀S_nChain invasion of F (13B) into pGL2 and pGL3 plasmids.

FIGS. 14A and 14B, cationic oligonucleotide F-S₁₈N₁₉Penetrate into Hela cells.

Example 1: synthesis of phosphoramidite spermine synthon

Spermine-linked phosphoramidite 1 was synthesized from spermine according to scheme 1 below:

(Mes ═ 2, 4, 6-trimethylphenyl; TBDMS ═ t-butyldimethylsilyl; TFA ═ CF-₃CO-; DMT ═ 4, 4' -dimethoxytrityl group)

Tetrakis (2, 4, 6-trimethylbenzenesulfonyl) spermine 2 prepared from spermine was dialkylated to 3. After complete deprotection of 3 under acidic conditions, crude bis (C4-OH)) spermine tetrahydrobromide 4 was fully protected with a pyridine solution of trifluoroacetic anhydride and then the two terminal ester groups of 5 were hydrolyzed under neutral conditions to form diol 6. The monotritylation reaction of 5 was performed statistically using 1 molar equivalent of DMTC1 reagent to provide 7 in 43% yield. The unreacted diol 6 and bistrityl compound 8 were recovered and rebalanced under weakly acidic conditions (trifluoroacetic acid in dichloromethane) to afford 7. The phosphitylation of 7 gives the desired phosphoramidite 1.

N¹，N⁴，N⁹，N¹²-tetrakis (2, 4, 6-trimethylbenzenesulfonyl) spermine (2): the compound was prepared according to the reference Bergeron et al J.Med.chem.2001, 44, 232-.

N¹，N¹²-bis [4- (tert-butyldimethylsilyloxy) butyl group]-N¹，N⁴，N⁹，N¹²-tetrakis (2, 4, 6-trimethylbenzenesulfonyl) -spermine (3): sodium chloride (60%, 1.0g, 25mmol) was added portionwise to a solution of 2(9.31g, 10.0 mmol) in DMF (20ml) under N₂Stirring was carried out at 0 ℃. At room temperatureAfter stirring for 30min, tert-butyl (4-iodobutyloxy) dimethylsilane (7.86g, 25mmol) was added in one portion. The mixture was stirred at room temperature overnight, then at H₂O-CH₂Cl₂(100mL/100mL) was partitioned. The organic phase is separated off and CH is used₂Cl₂The aqueous phase was extracted 3 times (50 mL). The combined organic phases are washed with NaHCO₃(1M) solution washing and then MgSO₄And (5) drying. After evaporation, the pasty residue was purified by flash chromatography with 1: 4 AcOEt: cyclohexane as eluent. The 3-containing fraction was evaporated to a paste-like oil, which was further washed with cold pentane to remove fast-moving impurities and then pumped under vacuum to provide 9.97g (76%) of 3 as an oil: TLC (AcOEt/cyclohexane 1: 4): r_f＝0.28.-IR(KRS-5)：2937，1604，1471，1320，1151，1101，838，777，657，578cm^-1.-¹H NMR(300MHz，CDCl₃)：δ＝-0.01(s，12H)，0.85(s，18H)，1.20-1.45(m，12H)，1.62(m，4H)，2.28(s，6H)，2.29(s，6H)，2.53(s，12H)，2.54(s，12 H)，2.90-3.10(m，16H)，3.42(t，J＝6.1Hz，4H)，6.91(s，4H)，6.92(s，4H).-¹³C NMR(75MHz，CDCl₃)：δ＝4.7，18.9，21，6，23.4，23.5，24.1，24.9，25.7，26.6，30.4，43.5，43.6，45.6，45.7，62.9，132.59，132.64，133.8，140.7，143.0，143.1-MS-ESI(MeOH)：m/z＝1325.85[M+Na]⁺，1303.83[M+H]⁺.-C₆₆H₁₁₀N₄O₁₀S₄Si₂(M_w1304.03), C60.79, H8.50, N4.30, S9.84; measurements C60.74, H8.55, N4.21, S9.63.

N¹，N¹²Bis (4-hydroxybutyl) spermine tetrahydrobromide (4): a solution of hydrogen bromide in acetic acid (33% by weight solution, 80mL, 1.4mol) was added dropwise to 3(9.87g, 7.57mmol) and phenol (29.0g, 0.31mol, 40 equivalents) in CH₂Cl₂(80mL) of the solution. The reaction mixture was stirred at room temperature overnight. After cooling in an ice bath, cold water (100mL) was added with stirring. The organic layer was separated and extracted 3 times with water (20 mL). The combined aqueous layers were treated with CH₂Cl₂Washed 5 times (30mL) and then evaporated to dryness. The resulting wet solid residue was suspended in ether and ground with a spatula (spatula) and the ether supernatant layer was discarded. These operations were repeated (5 times) until a solid phase suspension was obtained. After evaporation and drying in vacuo, compound 4(5.32g) was obtained as a solid. The crude material was used without further purification:¹H NMR(300MHz₁ D₂O)：δ＝1.75-2.10(m，12H)，2.27(m，4H)，3.15-3.35(m，16 H)，3.76(t，J＝12.2Hz，4H).-¹³C NMR(75MHz，D₂O)：δ＝22.9，23.2，23.4，29.0，45.0，45.2，47.7，48.3，61.5.-MS-ESI(MeOH)：m/z＝347.39[M+H]⁺.

N¹，N¹²-bis [4- (trifluoroacetoxy) butyl) -N¹，N⁴，N⁹，N¹²-tetrakis (trifluoroacetyl) -spermine (5) (from 4 using TFA₂O/NEt₃): add 4(5.3g, 7.6mmol) of CH in one portion₂Cl₂To the suspension (50mL) was added triethylamine (11.5g, 114mmol, 15 equiv.). The mixture was cooled in an ice bath and trifluoroacetic anhydride (19.1g, 90.9mmol, 12 equivalents) was added dropwise while under N₂Stirring the mixture. The mixture was stirred at room temperature for 3.5 h. After cooling in an ice bath, the resulting solution was washed 3 times with cold water (20mL) and MgSO₄Drying and evaporation gave an oily residue (11.7g) containing the minor product of the reaction (TFA)₂C＝CH-NEt₂(see Schreber, S.L., Tetrahedron Lett 1980, 21, 1027). By two successive flash chromatographies (eluent 1: 1-60: 40 AcOEt: cyclohexane, then 5-10% Et₂O/CH₂Cl₂) This minor product was removed to give 5 as an oil (5.59g, 81%): TLC (AcOEt/cyclohexane 1: 1): r_f＝0.25.-IR(KRS-5)：2955，1789，1690，1467，1352，1197，1147，759，731，692cm^-1.-¹H NMR(300 MHz，CDCl₃)：δ＝1.52-2.06(m，16H)，3.33-3.49(m，16 H)，3.38(m，4 H).-¹³C NMR(75MHz，CDCl₃): the rotamers of the four amine groups make the spectrum more complex. Only the high intensity resonance signal is as followsThe following description is made: δ 23.3, 23.9, 24.1, 24.8, 25.3, 25.6, 26.0, 26.55, 26.61, 44.4, 44.8, 45.7, 46.1, 46.4, 47.3, 48.0, 56.6, 67.3, 67.5, 116.6(q, J288 Hz), 156.9, 157.4, 157.8, 158.6.

N¹，N¹²-bis (4-hydroxybutyl) -N¹，N⁴，N⁹，N¹²-tetrakis (trifluoroacetyl) spermine (6): to a solution of 5(5.39g, 5.84mmol) in MeOH (50mL) was added NaHCO in one portion₃(0.1g, solid), the resulting suspension was stirred at room temperature for 2 h. After evaporation, the oily residue was dissolved in CH₂Cl₂(some fibrous NaHCO is provided)₃Suspension) and in 5-10% MeOH/CH₂Cl₂Elution was performed and purification by flash chromatography was performed to provide 3.61g (85%) of 6: TLC (MeOH 5%/CH)₂Cl₂)：R_f＝0.14.(MeOH 10％/CH₂Cl₂)：R_f＝0.45.-¹H NMR(300MHz，CDCl₃)：δ＝1.51-2.02(m，18H)，3.33-3.51(m，16 H)，3.68(m，4H).-MS-ESI(MeOH)：m/z＝753.33[M+Na]⁺.-C₂₆H₃₈F₁₂N₄O₆·H₂O(M_w748.60), C41.72, H5.39, N7.48, S30.45; measurements C41.97, H5.26, N7.37, S30.14.

From 4 (first using TFA)₂O/pyridine, then NaHCO₃) Preparation 6: add 4(15.3g, 22.8mmol) of CH₂Cl₂(100mL) and pyridine (44mL, 0.54mol) suspension trifluoroacetic anhydride (46mL, 0.33mol) was added dropwise, cooled in an ice bath and concentrated under N₂Stirring was carried out. The mixture was stirred at room temperature for 3 h. Cold water (100mL) was added to decompose excess trifluoroacetic anhydride, cooled in an ice bath, and then CH-quenched₂Cl₂The resulting solution was extracted (4 times, 100mL +50mL +25 mL. times.2). The combined extracts were washed with cold water (50 mL. times.3) and MgSO₄Dried and evaporated to give crude 5 as an oil (19.4g, 92%). The oil was dissolved in MeOH (100 mL). Adding NaHCO₃(solid, 0.1g) and the suspension was stirred overnight. After evaporation of the solventUsing 5-7% MeOH: CH₂Cl₂The residue was purified by flash chromatography as eluent to yield 10.1g (61%) of 6 as an oil.

N¹- [4- (dimethoxytrityloxy) butyl]-N¹²- (4-hydroxybutyl) -N¹，N⁴，N⁹，N¹²-tetrakis (trifluoro-acetyl) -spermine (7): add DMTCl (757mg, 2.23mmol) to a solution of 6(1.46g, 2.00mmol) in pyridine (3mL) and rinse with 1mL of pyridine. The mixture is at room temperature N₂Stirring was continued for 4h and then pyridine was removed by repeated co-evaporation with toluene. The residue was subjected to two successive flash chromatographies (eluent 2-5% MeOH/CH)₂Cl₂Then 10-15% acetone/CH₂Cl₂) Purification to yield 7(879mg, 43%) and bis-DMT derivative 8(648mg, 24%) as a foam. The starting diol 6(350mg, 24%) was also recovered. Data of 7: TLC (acetone/CH)₂Cl₂1∶9)：Rf＝0.20.-¹H NMR(300MHz，CDCl₃)：δ＝1.51-2.03(m，17H)，3.11(m，2H)，3.32-3.51(m，16H)，3.71(m，2H)，3.81(s，6H)，6.84(m，4H)，7.19-7.46(m，9H).-MS-ESI(MeOH)：m/z＝1055.52[M+Na]⁺.-C₄₇H₅₆F₁₂N₄O₈Calcd (Mw 1032.95) C54.65, H5.46, N5.42, F22.07; measurement C54.46, H5.58, N5.37, F21.63.

Compound (7) derived from diol (6) and bis-DMT derivative (8): add 6(1.4g, 1.9mmol) and 8(2.5g, 1.9mmol) of CH₂Cl₂Trifluoroacetic acid (50. mu.L, 0.6mmol) was added to the solution, and the mixture was stirred at room temperature for 30 min. The solution is mixed with 1M Na₂CO₃The solution was washed 3 times with MgSO₄Dried and evaporated. Using a continuous 5% AcOEt/CH₂Cl₂(750mL)、33％AcOEt/CH₂Cl₂(500mL)、7％MeOH/CH₂Cl₂(500mL) and 10% MeOH/CH₂Cl₂(500mL) the residue was separated by flash chromatography (column diameter: 50mm, SiO₂Height: 15cm) to yield 8(1.1g), 7(1.2g) and 6(1.3 g).

Spermine-linked phosphoramidite (1): add 7(844mg, 817. mu. mol) and triethylamine (230. mu.l, 1.65mmol, 2 equiv.) to CH₂Cl₂(4mL) solution 2-cyanoethyl- (N, N-diisopropylamino) chlorophosphite acid (205. mu.L, 0.92 mmol, 1.1 equiv.) was added and the mixture was N.sub.t. at room temperature₂Stirring for 40 min. The mixture is passed through NEt₃(1％NEt₃CH (A) of₂Cl₂Cyclohexane 1: 2 solution; 400mL) saturation (125 mL of 1% NEt were used sequentially₃CH (A) of₂Cl₂Cyclohexane 1: 2 solution and 100mL of 1% NEt₃ CH₂Cl₂Cyclohexane 1: 1 solution) of SiO₂Column (diameter: 20mm, height: 15cm) to give 1 as an oil (735mg, 73%):¹HNMR(200MHz，CDCl₃)：δ＝1.13-1.35(m，12H)，1.51-2.06(m，16H)，2.66(t，J＝6.4Hz，2H)，3.11(m，2H)，3.32-3.98(m，20H)，3.81(s，6H)，6.84(m，4H)，7.15-7.51(m，9H).-³¹P NMR(81MHz，CDCl₃): 148.06, 148.13, 148.19, 148.3 (resolved by amine group rotaisomerism).

Example 2: synthesis, purification and characterization of decamer oligonucleotides having the following structural formula

Said oligonucleotide being hereinafter referred to as N₁₀S_n(N₁₀An oligonucleotide moiety; s ═ spermine residue, n ═ 1-6)

Automated synthesis: a series of chemical syntheses of a sequence having the same sequence N using standard solid phase cyanoethyl phosphoramidite chemistry on an Expedite DNA synthesizer according to the following scheme₁₀＝³’CACCGTAGCG⁵' and increasing number of decameric oligonucleotides of spermine residue S:

according to classical oligonucleotide synthesis, the last N moiety is a nucleoside.

Reagents for automated DNA synthesis were purchased from Glen Research (Eurogentec).

For automated synthesis, standard 1. mu. mol coupling cycles were used, except for the coupling of spermine phosphoramidite 1 by extension of the coupling time (15min) and the use of a slightly more concentrated phosphoramidite solution (1mL phosphoramidite) (90mg) in acetonitrile).

The trityl fraction was collected, diluted and analyzed with a spectrophotometer to determine the yield of stepwise couplings.

The coupling yield of the four natural nucleotides is over 97%, while the coupling yield of spermine phosphoramidite under the above coupling conditions is 90-96%.

For purification-identification purposes, a DMT-ON (ON ═ oligonucleotide) pattern was used in all cases, while keeping the 5' -terminal DMT group ON the oligomer uncleaved.

And (3) post-synthesis treatment: following automated synthesis, the oligomer was cleaved from the solid support and deprotected completely using standard conditions (treatment with concentrated ammonia at room temperature for 90min for cleavage followed by overnight deprotection at 55 ℃).

And (3) purification: first two anionic oligonucleotides N₁₀S₁And N₁₀S₂Purification was carried out by standard HPLC methods on a reverse phase nucleosil C-18 column (Macherey-Nagel 10X 250mM) in DMT-on conditions by linear gradient elution with acetonitrile (5-35%, 20min) ammonium acetate solution (pH7, 20 mM). The oligonucleotide was then purified as A_cOH/H₂O-4/1 (500ml) was treated at room temperature for 20min to remove the trityl group. After dilution with water (5mL), DMT-OH was removed by ether extraction (3X 2mL) and concentratedThe aqueous phase is condensed to form oligomers.

FIG. 1 shows the oligonucleotide N₁₀S₁And N₁₀S₂Using a reverse phase nucleosil C-18 column (Macherey-Nagel 4.6X 250mM) with a linear gradient of acetonitrile (5-35%, 20min) ammonium acetate solution (pH7, 20 mM): a) n is a radical of₁₀S₁Coarse, DMT-ON; b) n is a radical of₁₀S₁Purified c) N₁₀S₂Coarse extract, DMT-ON; d) n is a radical of₁₀S₂And (4) purifying.^*A benzamide;^**a truncated sequence.

Poly-Pak II was used according to the manufacturer's instructions, except that the final oligonucleotide elution was performed by acetonitrile/concentrated ammonia/water (20: 4: 80)^TM(Glen Research/Eurogentec) column purification of neutral oligo N₁₀S₃And cationic oligomer N₁₀S₄、N₁₀S₅And N₁₀S₆(with or without DMT group). The fraction containing the oligonucleotide can be visualized using a TLC plate. After collection of the fractions, the solvent was removed by lyophilization. The oligomers collected in this way generally contain a benzamide contamination. The oligomer was dissolved in dilute aqueous ammonia (50mM) and the benzamide was removed by extraction with ether (3 times). The purified oligonucleotide was dissolved in dilute aqueous ammonia (50mM) using the following extinction coefficients (260nm, mol)^-1dm³cm^-1) The concentration of the compound is measured:

ε＝(15.4N_A+11.5N_G+7.4Nc+8.7N_T)×0.9×10³.

FIG. 2 shows HPLC analysis of purified oligonucleotides: a linear gradient elution was carried out on an anion exchange column (DionexPA-1009X 250mM) using NaCl (100-: a) n is a radical of₁₀S₁，b)N₁₀S₂，c)N₁₀S₃，d)N₁₀S₄，e)N₁₀S₅，f)N₁₀S₆。

Due to the use of bondingChemically, each polyamine carries a phosphate group, thus resulting in an additional net positive charge. 7 oligonucleotides (N)₁₀S_n)^3n-9n-0.. 6, which carry a total charge of-9, -6, -3, 0, +3, +6, +9, respectively, upon complete ionization, so that amounts of 80 to 250 nanomolar can be achieved.

Electrophoretic mobility:

they were studied for electric field migration at pH7 using polyacrylamide gel electrophoresis and their migration was visualized by silver-mirror staining. Mu.l of loading buffer (10mM HEPES pH7.4, 150mM NaCl, glycerol) containing compound (0.5nmol) was loaded onto native polyacrylamide gels (15%, TAE pH 7). Electrophoresis was carried out at 5V/cm and 4 ℃ for 17 h. Silver staining was performed according to Rabilloud et al, Electrophoresis, 1987, 9, 288-. The results are given in figure 3. Spermine-free oligonucleotide N₁₀(lane 1) the movement to the anode was the fastest and showed only weak silver staining under the conditions that polyamine-containing oligonucleotides were shown.

N₁₀And N₁₀·C₁₀Spontaneous exchange of

Autofluorescence of N₁₀·C₁₀ ^*Add oligonucleotide C to the duplex solution (50pmol, HEPES 10mM pH7.4, NaCl 150mM)₁₀(wherein C is a nucleotide complementary to N) (50pmol or 500 pmol). The mixture was incubated at 37 deg.C, 20 deg.C or 10 deg.C for 4h, and then loaded onto a non-denaturing polyacrylamide gel (15%, TAE pH 7). Electrophoresis was carried out at 5V/cm and 4 ℃ for 17 h. Gel pair C was scanned using a Typhoon 8600Imager₁₀ ^*And (6) detecting. As shown by the results shown in FIG. 4, N was found at 10 ℃₁₀And N₁₀·C₁₀Spontaneous exchange between them is not obvious.

N₁₀And N₁₀S_nChain exchange between

Detection of N under physiological salt conditions₁₀S_nFor natural duplex N₁₀·C₁₀Strand displacement ability of (1).

Will be refinedAmine conjugates N₁₀S_n(50 or 500pmol) to fluorescent N₁₀·C₁₀ ^*Duplex solution (50pmol, 10mM HEPES ph7.4, 150mM NaCl). The mixture was incubated at 10 ℃ for 4h and then loaded onto a non-denaturing polyacrylamide gel (15%, TAE pH 7). Electrophoresis was carried out at 5V/cm and 4 ℃ for 17 h. Fluorescence was detected by gel scanning using a Typhoon 8600 Imager.

Spermine conjugation had a significant effect on the strand exchange reaction as shown in figure 5. With competition N₁₀S_nIncreased number of spermine residues, corresponding to N₁₀·C₁₀ ^*Also become weaker, promoting slower moving, less negatively charged N₁₀S_n·C₁₀ ^*And (4) forming a complex. This effect on N₁₀S₃I.e. especially for conjugates that no longer carry formal negative charges. Indeed, spermine forms NH in the minor groove₂ ⁺The interchain network of the bidentate hydrogen bonds cleaves duplex DNA structure, so spermine is more likely to react with N₁₀S_nBut not N₁₀And (4) combining. But when strand exchange occurs at preformed (N)₁₀S_n)^3n-9/(N₁₀·C₁₀)^18-An additional favorable kinetic factor for electrostatic complexes may also play a role, as is the case with n > 3.

N₁₀S_n·C₁₀Melting temperature of the duplex

The stability of the double-stranded nucleic acid is compared by measuring its melting temperature, i.e., the temperature at which the complementary strands separate from each other. Record N₁₀S_n·C₁₀Optical density of the solution at 260nm versus temperature T (o.d.).

The melting temperature T was measured in HEPES 10mM pH7.4 (black line, diamonds) and HEPES 10mM pH7.4 +150mM NaCl (grey line, circles)_m. Using a CARY4000 spectrophotometer equipped with a temperature control unit, the sample was heated gradually (1 ℃/min) while recording the absorbance at 260nm, to obtain all duplexes (3.75nmol, 1ml buffer)Melting curve. Melting of the duplex results in hyperchromic shift, T_mThe temperature at which the first derivative curve do.d./dT ═ f (t) reaches a maximum value. The results are given in fig. 5.

Natural duplexes in HEPES solution (10mM, pH7.4) at T_mMelting was carried out at 30 ℃ (fig. 5). The increase in the number of spermines bound leads to T_mIs remarkably increased. N is a radical of₁₀S₆·C₁₀At T_mMelt at 75.2 ℃ approximately 45 ℃ higher than the natural duplex. T is_mThe curve being S-shaped with the point of inflection corresponding to neutral N₁₀S₃An oligonucleotide.

The melting temperature under physiological salt conditions was also recorded. T is_mThe curve f (N) appears much weaker (damped), and it is noteworthy that it corresponds to N₁₀S₃The previous curves intersect. Thus, when N < 3, N₁₀S_nAnd C₁₀The oligonucleotides are all anionic and repel each other in the duplex; the increase in salt concentration shields the repulsive force, thereby increasing T_m. When N > 3, N₁₀S_nBecome positive ions and attract C₁₀The salt-induced electrostatic shielding then reduces stability.

For neutral N₁₀S₃The stability of the duplex is independent of salt concentration.

N₁₀S_n(n-0-6) and⁵’GTGGCATCGC³' and⁵’GTGGCGTCGC³' comparison of melting temperatures of duplexes formed between them

Single base mismatch discrimination of oligonucleotide-spermine conjugates was detected. At C₁₀ ＝⁵’GTGGCATCGC³Within the sequence context of' the literature recommends the centrally located A to G conversion as the most stringent test.

The melting temperature T was measured in HEPES 10mM pH7.4 + NaCl 150mM_m. Using CARY4000 Spectroscopy light with temperature control UnitThe melting curves of all duplexes (3.75nmol, 1ml buffer) were obtained by gradually heating the sample (1 ℃/min) while recording the absorbance at 260 nm. T is_mIs the temperature at which the first derivative curve do.d./dT ═ f (t) reaches a maximum. These results are given in fig. 7 (diamonds correspond to diamonds)⁵’GTGGCATCGC³', triangle corresponds to⁵’GTGGCGTCGC³’)。

Natural N in 150mM NaCl₁₀·C₁₀The transition temperature of the duplex was reduced from 50.6 ℃ to 42.9 ℃, i.e.DT on occurrence of a mismatch_m7.7 ℃. In principle, the increase in stability due to electrostatic forces of non-specific end binding should not impair the base pair specificity, denoted Δ Δ G. This was true for the observations that complementary and mismatched target oligonucleotides were shown to have an average Δ T_mQuasi-parallel (quasi-parallel) Tm ═ f (n) curve at 7.9 ℃.

Purification of N₁₀S_nES-MS analysis of oligonucleotides

The oligonucleotide is dissolved in a solution containing the final concentration of 5X 10^-5M1% Triethylamine in 50% aqueous acetonitrile (v/v). 100mL aliquots were added to the ion source of an Applied biosystems Mariner 5155 mass spectrometer at a flow rate of 5 mL/min. The results are given in FIG. 8 (mosaic: deconvoluted spectra): a) n is a radical of₁₀S₁，b)N₁₀S₂，c)N₁₀S₃，d)N₁₀S₄，e)N₁₀S₅，f)N₁₀S₆. Neutral and cationic oligomer N₁₀S_3-6Becomes more difficult and it is therefore necessary to accumulate several spectra to obtain an acceptable signal-to-noise ratio.

Example 3: synthesis, purification and characterization of 12-mer phosphorothioate oligonucleotides having the following structural formula

Said oligonucleotide being hereinafter referred to as N₁₂S_nF (N-12 mer phosphorothioate oligonucleotide moiety; S-spermine residue, N-2 or 11; F-fluorescein bound to thymine).

Automated synthesis: synthesis of 12-mer phosphorothioate oligonucleotide sequences N with 2 or 11 spermine residues S Using solid phase Cyanoethylphosphoramide chemistry on an Expedite DNA synthesizer₁₂＝ ³’GCGACTCATGAA⁵'. Super-mild CE phosphoramidites and super-mild vectors (Glen Research/Eurogentec) were used to avoid oligomer cleavage during examination. Phosphorothioate linkages within the 12-mer oligonucleotide moiety were generated using standard sulfuration reagents (Glen Research/Eurogentec). fluorescein-dT phosphoramidite (Glen Research/Eurogentec) was used for 5' -end labeling. Spermine phosphoramidite coupling was performed using the coupling protocol described in example 2.

The trityl fraction was collected, diluted and analyzed on a spectrophotometer to determine the yield of stepwise couplings.

For purification-identification purposes, the DMT-ON mode was used in all cases, while keeping the 5' -terminal DMT group ON the oligomer uncleaved.

And (3) post-synthesis treatment: following automated synthesis, the oligomer is cleaved from the solid support and deprotected completely using concentrated ammonia overnight at room temperature.

And (3) purification: using Poly-Pak II according to the manufacturer's instructions^TMColumn (Glen Research/Eurogentec) purification of DMT-ON Compound N₁₂S₂F and N₁₂S₁₁F。

Purified oligonucleotide N was analyzed on an anion exchange chromatography column (SAX1000-8) using an aqueous alkaline condition (100mM ammonia, pH11) with a NaCl gradient (0.75-2.5M, 20min)₁₂S_nF (n ═ 2, 11). FIG. 9 shows the HPLC spectrum (A: N)₁₂S₁₁F，B：N₁₂S₂F)。

MALDI-TOF MS analysis of purified oligonucleotides

The oligonucleotides were dissolved in 500. mu.L of deionized water. The sample and HPA matrix were mixed on the plate. Once crystallized, the sample was analyzed using a BRUKER Ultraflex MS device. FIG. 10A: n is a radical of₁₂S₂Calculated F5460, measured 5459 (above) and fig. 10B: n is a radical of₁₂S₁₁Calculating the value of F: 9135 measured value: 9125 (lower) shows the results.

Example 4: strand invasion of plasmid DNA by 14-mer and 20-mer fluorescent oligonucleotides

The compounds shown above are hereinafter referred to as N₁₄S_nF (N ═ oligonucleotide moiety; S ═ spermine residue with N ═ 2-4; F ═ fluorescein residue) and N₂₀S_nF (N ═ oligonucleotide moiety; S ═ spermine residue with N ═ 3 to 5; F ═ fluorescein residue).

These fluorescent oligonucleotides were synthesized according to the procedure described in example 2.5 '-Fluorescentphosphoramidite (Glen Research/Eurogentec) was used for 5' -end labeling. FIGS. 11 and 12 show the most substituted N, respectively₁₄S₄F and N₂₀S₅Analytical grade HPLC and MALDI-TOF mass spectra of F compound as purity and structure (N)₁₄S₄F calculated 6470, measured 6478; n is a radical of₂₀S₅F calculated value 8813, measured value 8815).

Selection of the oligonucleotide sequence N located within the luciferase Gene sequence of the pGL3 control plasmid (Promega)₁₄S_nF and N₂₀S_nF. To assess the sequence specificity of strand invasion, pGL2 control plasmid (Promega) was used. The GL2 luciferase sequence was 95% identical to GL3, N₁₄S_nF and N₂₀S_nThe sequences targeted by F contain one and two mismatches, respectively.

Detection of N under physiological salt and temperature conditions₁₄S_nF and N₂₀S_nThe ability of the F strand to invade pGL3 instead of pGL2 plasmid.

To the plasmid solution (1.5. mu.g, 0.43pmol, 10mM HEPES pH7.4, 150mM NaCl) was added the fluorescent conjugate N₁₄S_nF and N₂₀S_nF (8.65 pmol). The mixture was incubated at 37 ℃ for 24h and then loaded onto an agarose gel (1.3%, TAE pH 7.4). Electrophoresis was performed at room temperature for 45min, after which the emitted green fluorescence was detected by gel scanning using a Typhoon 8600 Imager. After incubation of the gel in ethidium bromide solution for 15min, a red fluorescence photograph of the gel was taken using a UV transilluminator. These results are given in fig. 13.

Red and green fluorescence are evidence of double-stranded plasmid DNA and fluorescent oligonucleotide, respectively. Their co-localization with pGL3 but not pGL2 therefore confirmed strand invasion. Compound N₁₄S₃F and N₂₀S_nF, when incubated with pGL3 but not pGL2, showed a faint green fluorescent band associated with the plasmid.

Example 5: cationic oligonucleotides penetrate cells

The day before the experiment, Hela cells cultured in MEM medium containing 10% (v/v) fetal bovine serum were cultured at 50-60X 10³Cells/well were seeded into 4-well borosilicate Lab-Tek dishes. The complete medium was replaced with 0.5ml serum-free MEM medium. Preparation of 5' -end-cationic fluorescein-conjugated oligonucleotide F-S with sterile PBS₁₈N₁₉Formulation (wherein N₁₉TCGAAGTACTCAGCGTAAG). This was added to the cells to a final concentration of 2. mu.M.After 4 hours, the medium was changed to 1ml of fresh serum-containing medium. The first photograph was taken using a zeissaxioviert 25 fluorescence microscope fitted with a FITC filter (fig. 14A, left). All cells carry fluorescence, some of which is localized in intracellular vesicles, and most importantly, also dispersed throughout the cytoplasm and nucleus. After 24h, the medium was replaced with 1ml of phenol red-free MEM medium. Propidium iodide (1mM, water) was added to give a final concentration of 10. mu.M. After 10 minutes, a second photograph was taken showing that most healthy cells without propidium (propidium less) still have fluorescence (fig. 14B, right). Control cells incubated under similar conditions using the F-N19 oligonucleotide showed no fluorescence.

The present invention thus provides a flexible automated synthesis of cationic oligonucleotides that can form fast and stable complexes with their complementary sequences even in the context of strand invasion. Due to the terminal conjugation, the sequence selectivity for the native oligonucleotide was still as high. Moreover, due to its cationic nature, intracellular delivery does not require complexes with cationic carrier molecules. Together, these properties make oligonucleotide-oligocation conjugates an attractive alternative to oligonucleotides for molecular biology, diagnostics, and therapeutic uses.

Claims (19)

1. Oligonucleotide-oligocation molecule A_iB_jH, which can be synthesized by automated phosphoramidite chemistry, said molecule having an oligonucleotide moiety A_iAnd an oligocationic moiety B_jWherein

.A_iIs an i-mer oligonucleotide residue, i ═ 5 to 50, an oligomer having natural or unnatural nucleobases and/or pentofuranosyl and/or natural phosphodiester linkages, and chemical modifications or substitutions thereof,

.B_jis an organic oligocationic moiety of a j-mer, j ═ 1 to 50, whereinB is selected from the group consisting of

-HPO₃-R¹-(X-R² _n)_n1-X-R³-O-wherein R¹、R² _nAnd R³Is C1-C5 lower alkylene, X is NH or NC (NH)₂)₂，n1＝2-20，

-HPO₃-R⁴-CH(R⁵X¹)-R⁶-O-wherein R⁴Is C1-C5 lower alkylene, R⁵And R⁶Are identical or different C1-C5 lower alkylene, X¹Is putrescine, spermidine or spermine residue,

-HPO₃-R⁷-(aa)_n2-R⁸-O-wherein R⁷Is C1-C5 lower alkylene, R⁸Is C1-C5 lower alkylene, serine, natural amino alcohol, (aa)_n2Is a peptide containing a natural amino acid having a cationic side chain, n2 ═ 2-20.

2. The molecule of claim 1, wherein the oligonucleotide is selected from the group consisting of deoxyribonucleotides, ribonucleotides, Locked (LNA) nucleotides, and chemical modifications or substitutions thereof.

3. The molecule of claim 2, wherein the modification or substitution is phosphorothioate, 2 '-fluoro, 2' -O-alkyl.

4. The molecule of any one of claims 1 to 3, wherein the labeling group is a fluorescent agent.

5. The molecule of claim 1, wherein the amino acid is arginine, lysine, ornithine, histidine, diaminopropionic acid.

6. The molecule of claim 1, having³′A⁵' -B sequence.

7. The molecule of claim 1 which has B-³′A⁵' sequence.

8. The molecule of claim 1 which has B-³’A⁵' -B or³’A⁵′-B-³’A⁵' sequences and combinations thereof.

9. A method of obtaining the oligonucleotide-oligocation molecule of any one of claims 1 to 8 by using stepwise synthesis on an oligonucleotide synthesizer via the phosphoramidite pathway, the method comprising

An oligonucleotide synthesizer for inserting the vial containing the activated and protected oligocation B into the vial containing the oligonucleotide A, or reversing the order,

stopping the synthesis when the desired length is obtained,

cleaving the oligomer from the solid support, and

-removing the protecting group.

10. The method of claim 9, wherein the phosphoramidite reagent is selected from the group comprising:

P(OR⁹)(N(R¹⁰)₂)-O-R¹-(X-R² _n)_n1-X-R³-O-Prot, wherein R¹、R²、R³And n1 is as defined above, X is suitably protected NH or NC (NH)₂)₂，R⁹is-CH₂CH₂CN or C1-C5 lower alkyl, R¹⁰Is C1-C5 lower alkyl, or-N (R)¹⁰)₂Is pyrrolidinyl, piperidinyl or morpholinyl, Prot is a protecting group for oligonucleotide synthesis;

P(OR⁹)(N(R¹⁰)₂)-O-R⁴-CH(R⁵X1)-R⁶-O-Prot, wherein R⁴、R⁵、R⁶Is C1-C5 lower alkylene, X¹Is suitably protected putrescine, spermidine or spermine, R⁹And R¹⁰The definition of (1) is as above;

P(OR⁹)(N(R¹⁰)₂)-O-R⁷-(aa)_n2-R⁸-O-Prot, wherein R⁷、R⁸、R⁹、R¹⁰、n2、(aa)_n2And Prot are as defined above.

11. The method of claim 9 or 10, wherein the stepwise synthesis of the oligonucleotide sequence is followed by a stepwise synthesis of the oligocation moiety to obtain a peptide having the sequence³′A⁵' -B.

12. The method of claim 9 or 10, wherein the stepwise synthesis of the oligocation moiety is followed by a stepwise synthesis of oligonucleotide sequences to obtain B-³′A⁵' A compound of the sequence.

13. The method of claim 9 or 10, comprising synthesis of a mixed sequence.

14. The method of claim 13, which comprises a double-ended capped oligonucleotide sequence B-³’A⁵' -B or sequences in which the oligonucleotide sequence is interrupted by a cation³’A⁵′-B-³’A⁵' Synthesis of (I).

15. The method of claim 9, wherein the activated and protected oligocation B is obtained by protection of the amino group of a polyamine and subsequent alpha, omega-dihydroxyalkylation to form a diol suitable for oligonucleotide synthesis.

16. Phosphoramidite reagent of the formula:

P(OR⁹)(N(R¹⁰)₂)-O-R¹-(X-R² _n)_n1-X-R³-O-Prot, wherein R¹、R²、R³And n1 is as defined above, X is suitably protected NH or NC (NH)₂)₂，R⁹is-CH₂CH₂CN or C1-C5 lower alkyl, R¹⁰Is C1-C5 lower alkyl, or-N (R)¹⁰)₂Is pyrrolidinyl, piperidinyl or morpholinyl, Prot is a protecting group for oligonucleotide synthesis;

P(OR⁹)(N(R¹⁰)₂)-O-R⁴-CH(R⁵X1)-R⁶-O-Prot, wherein R⁴、R⁵、R⁶Is C1-C5 lower alkylene, X¹Is suitably protected putrescine, spermidine or spermine, R⁹And R¹⁰The definition of (1) is as above;

P(OR⁹)(N(R¹⁰)₂)-O-R⁷-(aa)_n2-R⁸-O-Prot, wherein R⁷、R⁸、R⁹、R¹⁰N2 and Prot are as defined above.

17. Use of the oligonucleotide-oligocation molecule of any one of claims 1 to 8 in the preparation of a composition for use in methods of biology and diagnostics.

18. The use of claim 17, wherein the composition is used for PCR, real-time PCR, genotyping, in situ hybridization, and DNA chip manufacturing.

19. A pharmaceutical composition comprising an effective amount of an oligonucleotide-oligocationic molecule of any one of claims 1 to 8 and a pharmaceutically acceptable carrier.