Saccharide-modified nucleic acid molecules

Abstract

The present invention refers to the transfection of cells using a conjugate comprising at least one saccharide residue at least one nucleosidic component selected from nucleic acids, nucleosides and nucleotides. This conjugate is suitable for the transfection of prokaryotic and eukaryotic cells such as plant cells or mammalian cells including human cells with high efficacy. Thus, a new delivery vehicle for therapeutic molecules including antisense molecules, sRNA molecules, miRNA molecules, antagomirs or precursors of such molecules, as well as the therapeutic nucleosides or nucleotides, is provided. Further, a convenient strategy for developing new lines of plants that exhibit particular traits is provided.

Claims

1. A method of transfecting a cell in vitro, comprising exposing a cell to a conjugate comprising at least one saccharide residue and at least one nucleosidic component, wherein the nucleosidic component is a nucleic acid molecule comprising 5-100 building blocks and optionally comprising at least one modified building block; wherein the saccharide residue is covalently bound to a nucleobase of the at least one nucleosidic component via a linker; and wherein the linker comprises a cyclic group formed by a Click reaction; and wherein the cell is not a human germ cell.

2. The method of claim 1, wherein the saccharide residue is selected from the group consisting of monosaccharides, disaccharides, and linear, branched and circular oligosaccharides.

3. The method of claim 1, wherein the conjugate comprises (i) one saccharide residue and one nucleosidic component, (ii) multiple saccharide groups and one nucleosidic component, (iii) one saccharide residue and multiple nucleosidic components, or (iv) multiple saccharide residues and multiple nucleosidic components.

4. The method of claim 1, wherein the conjugate comprises a saccharide residue, covalently bound to the 3- or 5-terminus of an RNA-transcript.

5. The method of claim 1 for the transfection of plant cells.

6. A method for the down-regulation of genes comprising transfecting a cell in vitro with a conjugate comprising at least one saccharide residue and at least one nucleosidic component, wherein the nucleosidic component is an inhibitory RNA molecule comprising 5-100 building blocks and optionally comprising at least one modified building block; wherein the saccharide residue is covalently bound to a nucleobase of the at least one nucleosidic component via a linker; and wherein the linker comprises a cyclic group formed by a Click reaction; and, wherein the cell is not a human germ cell.

7. The method of claim 6, wherein the saccharide residue is selected from the group consisting of monosaccharides, disaccharides, and linear, branched and circular oligosaccharides.

8. The method of claim 6, wherein the conjugate comprises (i) one saccharide residue and one nucleosidic component, (ii) multiple saccharide groups and one nucleosidic component, (iii) one saccharide residue and multiple nucleosidic components, or (iv) multiple saccharide residues and multiple nucleosidic components.

9. The method of claim 1, wherein the nucleosidic component is a nucleic acid molecule comprising 10-50 building blocks and optionally comprising at least one modified building block.

10. The method of claim 1, wherein the nucleosidic component is a nucleic acid molecule comprising 15-25 building blocksand optionally comprising at least one modified building block.

11. The method of claim 6, wherein the nucleosidic component is an inhibitory RNA molecule comprising 10-50 building blocks and optionally comprising at least one modified building block.

12. The method of claim 6, wherein the nucleosidic component is an inhibitory RNA molecule comprising 15-25 building blocks and optionally comprising at least one modified building block.

Description

FIGURE LEGENDS

(1) FIG. 1 Chemical structure and sequence of saccharide modified siRNA. As a comparative example, anandamide modified siRNA is shown.

(2) FIG. 2 Delivery of glucose modified siRNA to plant cells. Glucose modified siRNA added onto the roots of Arabidopsis thaliana are taken up by the plant and transported along the roots (Alexa=Alexa Fluor 647, Life Technologies).

(3) FIG. 3 Synthesis of saccharide-modified reagents. The depicted reaction scheme shows the preparation of glucose azide (A) and triglucoseazide (B).

(4) FIG. 4 Synthesis of glucose-modified siRNA through Copper (I)-catalysed Azide-Alkyne Cycloaddition (CuAAC).

(5) FIG. 5 Relative expression of Renilla-luciferase in RBL-2H3 cells after treatment with glucose-modified siRNA duplex against Renilla-luciferase in comparison to the same siRNA with anandamide-modification (AEA-siRA). The cells were cultured in medium with 11.1 mM glucose (black) or in medium without glucose (green).

(6) FIG. 6 Principle of the in vitro transcription labelling experiment using T7 RNA polymerase and -glucose labelled GTP.

(7) FIG. 7 Regioselective chemo-enzymatic labelling of RNA using T4 RNA ligase.

EXAMPLES

1. Synthesis of RNA-Saccharide Conjugates

(8) The synthesis of the saccharide modified RNA strand was performed as depicted in FIG. 4. The central element of the synthesis is the Cu-catalyzed alkyne-azide click reaction (34-39) between an alkyne modified RNA strand and the corresponding glucose azide 1. In order to compare the glucose modified RNA strands to other systems, the click method was utilized also for the preparation of a triglucose-RNA conjugate using triglucose monoazide 2, and of a -cyclodextrine modified RNA strand with the -cyclodextrine monoazide 3. Further, an anandamide modified RNA strand was prepared as a comparative example by using anandamide monoazide (AEA) 4. The respective saccharide residues as well as anandamide monoazide are shown in FIG. 1. In all cases a short ethylene glycol spacer was introduced between the RNA strand and the respective ligand. The click-technology enabled in all cases efficient ligation of the saccharide or ananamide molecules to RNA. In addition, the method enabled efficient conjugation at the more difficult to access 3-terminus of the siRNA duplex. 3-modified siRNA strands are typically better tolerated by the RNAi machinery (41). To achieve the 3-end attachment a deoxyuridine phosphoramidite with an octadiine handle at C5 was used during RNA synthesis.

(9) The synthesis of glucose azide and triglucose azide is shown in FIG. 3. For preparing the glucose azide 1 in a first step, an ethylene glycol spacer was introduced into a protected glucose derivative using 2-bromoethanol. In a subsequent step, an azide functionality was introduced before the glucose derivative was deprotected to yield glucose azide 1. For the synthesis of the triglucose azide, first a branched linker was prepared from pentaerythrytol and propargyl bromide. Subsequently, after protecting the remaining alcohol functionality, three glucose azides were added in a Click-reaction. Deprotecting the alcohol functionality and introducing another azide functionality finally yielded the triglucose azide 2.

(10) The azides were subsequently clicked with excellent yields to obtain an alkyne-containing RNA sense strand as shown in FIG. 4. After HPLC purification, the saccharide modified RNAs were hybridized to the antisense counterstrand to obtain the siRNA duplexes depicted in FIG. 1.

2. Delivery of RNA-Saccharide Conjugate into Cells

(11) In order to visualize the delivery of the RNA duplexes into living cells the saccharide modified RNA sense strand was initially hybridized to an antisense strand containing a fluorescein label (Alexa=Alexa Fluor 647, Life Technologies).

(12) Uptake of the glucose modified RNA duplex was studied with Arabidopsis thaliana cells. Glucose modified siRNA was added onto roots of Arabidopsis thaliana. The confocal microscopy studies depicted in FIG. 2 show that unmodified siRNA is as expected unable to enter the cells. Glucose modified siRNA, however, was readily detected inside the respective cells proving uptake and transport along the roots. The same result was also observed with modified dsDNA (not shown).

(13) To demonstrate that the delivered siRNA molecules exhibit the desired RNAi effect, a commercially available dual-luciferase reporter assay was utilized. A plasmid containing two luciferases (Renilla and Firefly) was transfected into the cells. RNAi was evaluated by targeting the expression of the Renilla luciferase, whereas the Firefly luciferase served as an internal standard. For these studies the glucose modified siRNA without further fluorescein modification was used. Initial control experiments with unmodified RNA duplexes (no glucose, no fluorescein) showed that the Renilla expression was not affected. In contrast, a dose dependent silencing of Renilla expression in presence of ligand modified siRNA was observed in both cell lines (FIG. 5). Most important, even a relatively low amount of siRNA-ligand conjugate showed already a considerable effect.

(14) The silencing efficacy of glucose modified siRNA was next evaluated in comparison to the ananamide-siRNA conjugate. The result of this comparison is depicted in FIG. 5. Surprisingly, the new glucose modified siRNA is constantly significantly more potent than the ananamide system, which establishes glucose and other saccharide residues as a powerful new delivery tool.

3. 5-RNA Labelling Using -Labelled Nucleotides

(15) For labelling RNA-transcripts at the 5-terminus, first at 39 mer DNA template bearing the T7 promoter sequence followed by a short encoded transcript was prepared. This allowed a primer independent RNA polymerisation reaction, which results in 21 mer RNA transcript. Due to the de novo initialisation of the polymerase, the first-used RNA nucleotide remains as a triphosphate in the transcript providing a unique 5-saccharide labelled transcription product. Since the T7 RNA polymerase usually starts on a CC.sub.n-sequence, which generates G-starting transcripts, the experiment was performed with glucose-labelled GTP. Despite the presence of the glucose residue, the T7-RNA polymerase accepted the labelled triphosphate and continued the transcription process to give the expected glucose-labelled product.

(16) The following coding and template strand encoding a T7 promotor sequence and a 21mer transcript were purchased from METABION.

(17) TABLE-US-00001 Coding: (SEQIDNO:1) 5-dATAATACGACTCACTATAGGC Template: (SEQIDNO:2) 3-dTATTATGCTGAGTGATATCCGGAAAGTGATGAGGATGGA-5

(18) Prior to the transcriptions, the strands were annealed in a thermocycler (Mastercycler Personal from EPPENDORF). Therefore, 20 M of the coding strand was annealed to 20 M of the template strand in buffer (100 mM NaCl, 25 mM Tris-HCl, pH=7.6 at 25 C.) applying the following temperature gradient: 95 C. for 4 min followed by cooling with 2 C./min to 4 C.

(19) In vitro transcriptions were carried out in a 0.2 mL PCR tube in a 20 L setup. To 40 pmol of the hybridized DNA template in transcription buffer (40 mM HEPES pH=7.4, 6 mM MgCl.sub.2, 2 mM, 10 mM DTT), 400 M ATP, CTP, UTP, 20-80 M GTP (400 M for the control) and 400 M glucose labelled GTP 7d were added. The reactions were started by the addition of one unit T7 RNA polymerase (NEW ENGLAND BIOLABS), carefully mixed and then incubated at 37 C. After 5 h the transcriptions were stopped by addition of one volume RNA loading dye (47.5% formamide, 0.01% SDS, 0.01% bromophenol blue, 0.005% xylene cyanol, 0.5 mM EDTA) and 20 L of each sample was analyzed on 20% denaturing polyacrylamide gels (7 M urea, 35 mA, 1000 V) and visualized using a LAS-3000 imaging system (RAYTEST). For visualization of RNA transcripts SYBR green II staining was applied.

4. 3-RNA Labelling Using T4 RNA Ligase

(20) T4 RNA ligase catalyzes the transfer of a cytidine 3, 5-bisphosphate to the 3-OH of single-stranded RNA in the presence of ATP.

(21) Thus, a single-stranded RNA molecule may be reacted in the presence of T4 RNA ligase and ATP with a labelled cytidine 3,5-bisphosphate carrying an alkyne moiety at a phosphate group. A subsequent click-reaction with an azide-modified saccharide residue, as those shown in FIG. 1, allows the preparation of a 3-modified single-stranded RNA molecule as shown in FIG. 7.

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