The present invention relates to a method for enzymatically synthesizing chemically modified RNA by using RNA-dependent RNA polymerases (RdRp), especially RdRps from viruses of the Caliciviridae family. The method of the present invention is particularly useful for preparing RNA molecules of increased stability especially with respect to RNA degradation, for example for in vivo applications. Further subject matter of the present invention relates to a kit for carrying out the enzymatic synthesis of the chemically modified RNA.
RNA molecules containing chemically modified ribonucleotides are useful for various applications in science and medicine. In particular, it is generally known that especially backbone-modified RNAs possess an increased stability, e.g. with respect to degradation by RNases, which is of importance for in vivo applications, for example in the case of antisense ribonucleotides as well as siRNAs (small interfering RNAs) for RNA interference (RNAi).
Hitherto, such modified RNA is generally prepared by solid-phase chemical synthesis methods. However, such chemical methods have several drawbacks, for example they are expensive and time consuming. Those drawbacks are an important limitation for the broad diffusion of the RNAi-therapy. Indeed, it is predicted that several tons of RNAi-molecules may be needed each year for the treatment of so far untreatable diseases such as cancer, viral infectious diseases and degenerative diseases (i.e. acute macular degeneration). Those so far untreatable diseases may extensively benefit from RNAi-Therapy.
Accordingly, there is a substantial need for alternative methods for the production of chemically modified RNA molecules, in particular for therapeutic applications. It is known from WO-A-2007/012329 that viruses of the Caliciviridae family can be used for amplification and/or labelling of RNA molecules. In this disclosure, the authors also describe that the calicivirus RdRps can make use of fluorescently coupled, biotin-coupled or radioactively-coupled NTPs used for labelling of the RNA. However, concrete examples that the calicivirus RdRps can factually incorporate such coupled NTPs are completely missing. Furthermore, it is clear that the term NTP analogue in WO-A-2007/012329 is exclusively directed to such analogues that can be used for the detection of the labelled RNA by fluorescence emission, radioactive emission and/or by providing a partner for a chemical or biological binding pair such as for example biotin. Thus, the incorporation of NTP-analogues as envisaged in WO-A-2007/012329 is exclusively directed to the detection of coupled NTPs used for detection of RNA molecules labelled with such NTP-analogues.
Furthermore, another group suggested recently the use of chemically modified ribonucleotides for the inhibition of a norovirus RdRp (Zamyatkin et al. (2008) J. Biol. Chem. 283: 7705-7712).
The solution to this technical problem is provided by the embodiments of the present invention as defined in the claims.
In particular, the present invention provides a method for enzymatically synthesizing chemically modified RNA comprising the steps of:
In view of the prior art it is highly surprising that RdRps, especially corresponding enzymes from the calicivirus family (Caliciviridae) can factually incorporate diverse chemically modified ribonucleotides into dsRNA by synthesizing a complementary strand to a single stranded RNA (ssRNA) template.
It is further contemplated according to the present invention that the incorporation of chemically modified ribonucleoside triphosphates into RNA will be used for the large-scale industrial production of such modified RNA. Thus, it is preferred that chemically modified ribonucleoside triphosphates are used in an amplification reaction which comprises the further steps:
Preferably, n is an integer from 10 to 50, more preferred 15 to 40, most preferred 20 to 30.
The strand separation according step (c) may be carried out by application of heat (heat denaturation), by chemical denaturation or enzymatically, preferably by a helicase. Especially in the case of heat denaturation, it is desirable to add further RdRp molecules to the reaction mixture for performing the next step (b).
It is further preferred that the protein having RdRp activity has a “right hand conformation” and that the amino acid sequence of said protein comprises a conserved arrangement of the following sequence motifs:
with the following meanings:
D: aspartate
Y: tyrosine
S: serine
G: glycine
P: proline
L: leucine
F: phenylalanine
R: arginine
X: any amino acid.
The so-called “right hand conformation” as used herein means that the tertiary structure (conformation) of the protein folds like a right hand with finger, palm and thumb, as observed in most template-dependent polymerases
The sequence motif “XXDYS” is the so-called A-motif. The A-motif is responsible for the discrimination between ribonucleosides and deoxyribonucleosides. The motif “GXPSG” is the so-called B-motif. The B-motif is conserved within all representatives of this RdRp family of the corresponding polymerases from the Caliciviridae. The motif “YGDD” (“C-motif) represents the active side of the enzyme. This motif, in particular the first aspartate residue (in bold, YGDD) plays an important role in the coordination of the metal ions during the Mg2+/Mn2+ dependent catalysis. The motif “XXYGL” is the so-called D-motif. The D-motif is a feature of template-dependent polymerases. Finally, the “XXXXFLXRXX” motif (E-motif) is a feature of RNA-dependent RNA polymerases which discriminates them from DNA-dependent RNA polymerases.
Typical representatives of the above types of RdRps are the corresponding enzymes of the calicivirus family (Caliciviridae). The RdRps of the calicivirus family are capable of synthesizing complementary strands using as a template any ssRNA template in vitro, including heterologous viral, eukaryotic and prokaryotic templates. The ssRNA template may be positive stranded or negative stranded.
The above-defined protein having RdRp activity is capable of synthesizing a complementary on a strand both by elongation of a corresponding primer and by novo synthesis of a complementary strand in the absence of a primer. The primer, if desired, may be a sequence specific primer or may be a random primer or may be an oligo-T-primer or an oligo-U-Primer. More details of the characteristic features of the calicivirus RdRp can be found in WO-A-2007/012329
Preferably, the RNA-dependent RNA-polymerase is an RdRp of a human and/or non-human pathogenic calicivirus. Especially preferred is an RdRp of a norovirus, sapovirus, vesivirus or lagovirus, for example the RdRP of the norovirus strain HuCV/NL/Dresden174/1997/GE (GenBank acc. No AY741811) or of the sapovirus strain pJG-Sap01 (GenBank acc. No AY694184) or an RNA-dependent RNA polymerase of the vesivirus strain FCV/Dresden/2006/GE (GeBank acc. No DQ424892).
According to especially preferred embodiments of the invention the RdRp is a protein having an amino acid sequence according SEQ ID NO: 1 (norovirus-RdRP), SEQ ID NO: 2 (sapovirus-RdRP) or SEQ ID NO: 3 (vesivirus-RdRP). The person skilled in the art is readily capable of preparing such RdRP, for example by recombinant expression using suitable expression vectors and host organisms (cf. WO-A-2007/012329). To facilitate purification of the RdRp in recombinant expression, it is preferred that the RdRp is expressed with a suitable tag (for example GST or (His)-6-tag) at the N- or C-terminus of the corresponding sequence. For example, a histidine tag allows the purification of the protein by affinity chromatography over a nickel or cobalt column in a known fashion. Examples of embodiments of RdRps fused to a histidine tag are the proteins having an amino acid sequence according to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. SEQ ID NO: 4 corresponds to a norovirus-RdRP having a histidine tag. SEQ ID NO: 5 corresponds to the amino acid sequence of a sapovirus-RdRP having a histidine tag. SEQ ID NO: 6 corresponds to the amino acid sequence of vesirius-RdRP having a histidine tag.
The method of the present invention is particularly useful for providing short RNA molecules for gene silencing applications, either by antisense technology or RNA interference.
Therefore, the ssRNA template to be used in the method of the present invention has preferably a length of 8 to 45 nucleotides, preferably of 15 to 30 nucleotides, preferably of 21 to 28 nucleotides, more preferably of 21 to 23 nucleotides. The molecules of the latter length are particularly useful for siRNA applications. The chemically modified RNA products of the methods of the present invention preferably have an increased stability as compared to the non-modified dsRNA analogues.
Especially for this purpose, the chemical modification of the at least one modified ribonucleoside triphosphate to be incorporated by the RdRp activity into the complementary strand can have a chemical modification(s) at the ribose, phosphate and/or base moiety. With respect to molecules having an increased stability, especially with respect to RNA degrading enzymes, modifications at the backbone, i.e. the ribose and/or phosphate moieties, are especially preferred.
Preferred examples of ribose-modified ribonucleoside triphosphates are analogues wherein the 2′-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN with R being C1-C6 alkyl, alkenyl or alkynyl and halo being F, CI, Br or I. It is clear in the context of the present invention, that the term “modified ribonucleoside triphosphate” or “modified ribonucleotide” also includes 2′-deoxy derivatives which may at several instances also be termed “deoxynucleotides”.
Typical examples of such ribonucleotide analogues with a modified ribose at the 2′ position include 2′-O-methyl-cytidine-5′-triphosphate, 2′-amino-2′-deoxy-uridine, 2′-azido-2′-deoxy-uridine-5′-triphosphate, 2′-fluoro-2′-deoxy-guanosine-5′-triphosphate and 2′-O-methyl-5-methyl-uridine-5′-triphosphate.
Examples of ribonucleoside triphosphates leading to a phosphate backbone modification in the desired dsRNA product are phosphothioate analogues.
According to the present invention, the at least one modified ribonucleoside trisphophate may also be selected from analogues having a chemical modification at the base moiety. Examples of such analogues include 5-aminoallyl-uridine-5′-triphosphate, 6-aza-uridine-5′-triphosphate, 8-aza-adenosine-5′-triphosphate, 5-bromo-uridine-5′-triphosphate, 7-deaza-adenosine-5′-triphosphate, 7-deaza-guanosine-5′-triphosphate, N6-methyl-adenosine-5′-triphosphate, 5-methyl-cytidine-5′-triphosphate, pseudo-uridine-5′-triphosphate, and 4-thio-uridine-5′-triphosphate.
The above and other chemically modified ribonucleoside triphosphates are commercially available, for example from Sigma-Aldrich Chemie GmbH, Munich, Germany or Trilink technologies, USA
According to the present invention, the term “conditions sufficient for RNA synthesis” means the conditions, in particular relating to buffer, temperature, salt and metal ion (if applicable) conditions that allow the RdRp to synthesise an RNA strand complementary to a template strand. Appropriate buffer, salt, metal ion, reducing agent (if applicable) and other conditions of RdRps are known to the skilled person. With regard to the RdRPs of caliciviruses, it is referred to WO-A-2007/012329. Thus, the ssRNA template is used in amounts of, e.g. 1 microgram to 4 microgram per 50 microliter reaction volume. The concentration of the ribonucleoside triphosphates (including the modified ribonucleoside trisphosphate (s)) is preferably in the range of from 0.1 micromol/l to 1 micromol/l, for example 0.4 micromol/l. The concentration of the RdRp may be for example 1 micromol/l to 10 micromol/l.
Typical buffer conditions are 10 to 80 mM, more preferred 20 to 50 mM HEPES, pH 7.0-8.0, 1 to 5 mM, for example 3 mM magnesium acetate, magnesium chloride, manganese acetate or manganese chloride and 1 to 5 mM of a reducing agent, for example DTT.
A typical stop solution contains 2 to 10 mM, preferably 4 to 8 mM ammonium acetate, and 50 to 200 mM, for example 150 mM EDTA.
Short RNA templates (e.g. as defined above) are usually prepared by chemical synthesis of two separate strands that are then annealed in vitro using an annealing buffer containing for example Tris-Hcl 50 mM, pH 8.0, NaCl 200 mM, EDTA 1 mM. Other methods for providing the ssRNA template include enzymatic manipulations, for example transcription initiation depending on selected sequences (so called “promotor”) or using a specific primer and RNA synthesis by DNA-dependent RNA polymerases following degradation of the DNA strand.
Preferred reaction volumes range from 20 to 200 microliter, preferably 50 to 100 microliter. Typically, the buffer conditions and other conditions as outlined above are provided by mixing appropriate stock solutions (usually 5× or 10× concentrated), adding the RdRPl and the ssRNA template and double distilled or deionised water to the desired final reaction volume.
A further subject matter of the present invention is a kit for carrying out the inventive method. The kit of the present invention comprises a protein having RdRp activity (preferred examples are defined above), ribonucleotides (rATP, rGTP, rCTP, rUTP, preferably as stock solutions), at least one chemically modified ribonucleoside triphosphate (as defined above and preferably selected from the examples outlined above), an appropriate buffer (preferably as a stock solution as defined above), and, optionally, a stop solution (preferably a stop solution as defined above, more preferred in the form of a 5× or 10× stock solution) and, also optionally, a helicase.
The figures show:
a shows graphical representations of elution profiles resulting in purification of the single-stranded RNA template used for enzymatic synthesis with calicivirus RdRp.
b shows graphical representations of elution profiles resulting in purification of the double-stranded RNA products of the enzymatic synthesis using calicivirus RdRp. The product of a reaction for a template ssRNA was purified from the reaction mixture by ion exchange chromatography and fractionized.
c shows a photographical representation of a polyacrylamid gel electrophoresis of the fractions indicated (marked with a dashed box) in
a shows graphical representations of elution profiles resulting in purification of the double-stranded RNA products of the enzymatic synthesis using calicivirus RdRp. The products of a reaction for a normal dsRNA product (upper panel), a dsRNA product including 2-O—CH3—CMP (2′-O-methyl-cytidine-5′-monophosphate, medium panel) and a dsRNA containing 2-F-dGMP (2′-fluoro-2′-deoxy-guanosine-5′-monophosphate) were purified from the reaction mixture by ion exchange chromatography and fractionized.
b shows a photographical representation of a polyacrylamid gel electrophoresis of the fractions indicated (marked with a dashed box) in
The following non-limiting examples further illustrate the present invention.
The general reaction mixture is as follows:
The reaction mix consists of
The reaction mixture is incubated for 2 h at 30 to 42° C. depending on the RdRp used.
X, Y, Z, or W corresponds to one or more of the following ribonucleoside triphosphates or deoxyribonulcleoside triphosphates alone or in combination:
The reactions mixtures for Examples 1 to 14 and Comparative Example 15 (including only unmodified rNTPs) were set up according to the following Table 2:
For RNA synthesis the reaction mixtures according to Examples 1 to 14 and Comparative Example 15 (no modified nucleotides present) were incubated at 30° C. for 2 h.
The abbreviations used in Table 2 above stand for the following nucleotides as outlined in Table 3.
Synthesis of Chemically Modified and Unmodified dsRNA Using Calicivirus RdRp
The reaction mixtures of Examples 1 to 14 and Comparative Example 15 were analysed by PAGE (polyacrylamide gel electrophoresis). Bands were detected by ethidium bromide staining. As shown in
These data suggest that RdRp of the Caliciviridae are capable of synthesizing dsRNA from various chemically modified building blocks.
Purification and Characterisation of Chemically Modified and Unmodified dsRNA Synthesised by Calicivirus RdRp
The products of the reaction mixtures of Comparative Example 15 (unmodified dsRNA-Product), Example 1 (dsRNA-Product+2-O—CH3—CMP) and Example 13 (dsRNA-Product+2-F-dGMP) were purified by ion exchange (IEX) chromatography and fractionated. Elution profiles are shown in
Melting Point Analysis of Chemically Modified and Unmodified dsRNA Synthesised by Calicivirus RdRp
The products of the reaction mixtures of Comparative Example 15 (unmodified dsRNA-Product), Example 1 (dsRNA-Product+2-O—CH3—CMP) and Example 13 (dsRNA-Product+2-F-dGMP) were incubated with a double-stranded RNA-intercalating agent (SYBR Green I) and the melting curves of the double-stranded RNA products were measured using the LightCycler device (Roche Diagnostics). The backbone-modified (dsRNA-Product+2-O—CH3—CMP and dsRNA-Product+2-F-dGMP, as indicated) and unmodified RNA (dsRNA-Product, as indicated) display various melting points as indicated in
The products of the reaction mixture of enzymatic synthesis using 2′-O—CH3—CTP (dsRNA product+2-O—CH3—CMP; Example 1) were analyzed by HPLC, with subsequent denaturation of the double stranded RNA leading to elution of the sense (template ssRNA) and antisense (complementary ssRNA) strands. The masses of the sense (template ssRNA, molecular weight of 7564.41) and the antisense (synthesized single stranded RNA complementary to the template ssRNA, molecular weight of 8080.73) were measured by ESI/MS. They were found to correspond exactly to the predicted mass of the template ssRNA and the incorporation of 2′-β-CH3—CMP in the antisense strand (
Sequences of the sense (template) and antisense (product) strand:
C residues in bold mark the 2′-O—CH3—CMP residues.
Calculated molecular weights (Da): ssRX21 for: 7564,6; ssRX21 rev: 8080,9
MS-Analysis was carried out by LCMS using a standard HPLC system from Agilent Technologies, Inc. (Santa Clara, Calif., USA), equipped with degasser, binary pump, temperature controllable autosampler, column oven and a DAD detector. The effluent from the DAD detector was coupled on-line with an accurate mass Q-TOF mass analyzer which was equipped with a dual spray electrospray source. An absolute amount of 10 mOD of total oligo was injected onto a reversed phase chromatography column and the oligonucleotides were separated using a methanol gradient.
Number | Date | Country | Kind |
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08010829.3 | Jun 2008 | EP | regional |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP2009/057119 | 6/9/2009 | WO | 00 | 7/27/2010 |