WO2008127193A1

WO2008127193A1 - Metalled sirnas for facilitated targeting of suboptimal mrna sequences

Info

Publication number: WO2008127193A1
Application number: PCT/SE2008/050436
Authority: WO
Inventors: Sofi Elmroth
Original assignee: Forskarpatent I Syd Ab
Priority date: 2007-04-17
Filing date: 2008-04-17
Publication date: 2008-10-23
Also published as: WO2008127193A8

Abstract

The present invention relates to siRNAs with sense (passenger) strands deviating from that of a target gene by having introduced metallated bases in the sense (passenger) strand, as well as a method for locally decreasing the melting temperature and concomitant structural change of siRNA, and a method for targeting mRNA regions.

Description

TITLE

METALLATED SiRNAs FOR FACILITATED TARGETING OF SUBOPTIMAL mRNA SEQUENCES

DESCRIPTION

TECHNICAL FIELD

The present invention relates to the use of siRNAs for targeting mRNA The use has a particular interest in treating metastasising cancer, to prevent the occurrence of metastases The invention further relates to metallated siRNAs

BACKGROUND OF THE INVENTION

Long double-stranded RNAs (dsRNAs, typically >200 nt) can be used to silence the expression of target genes in a variety of organisms and cell types (e g , worms, fruit flies, and plants) Upon introduction, the long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway First, the dsRNAs get processed into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase Ill-like enzyme called Dicer (initiation step) Then, the siRNAs assemble into endoπbonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step) Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand In mammalian cells, introduction of long dsRNA (>30 nt) initiates a potent antiviral response, exemplified by nonspecific inhibition of protein synthesis and RNA degradation The mammalian antiviral response can be bypassed, however by the introduction or expression of siRNAs

The use of short interfering RNAs (siRNAs) for transient suppression of protein production has become a commonly used molecular biology technique The technique relies on use of the endogenous RNA inducing silencing complex (RISC) for processing and transport of the antisense strand of the siRNA to its complementary mRNA target For optimal processing, several factors have to be considered during siRNA design, the more important ones being ι) mRNA target accessibility and n) use of siRNA duplexes with melting properties favouring loading of the antisense strand to RISC In the latter case, siRNAs with hybridization properties favouring local melting of the 5' end of the antisense strand over melting of the 3' end have been shown to be particularly efficient The observation fits well with a picture of the process where the 5' ends of the siRNAs compete for loading onto RISC For highly symmetrical siRNAs, loading of both sense- and antisense strands might thus occur, with significant off-target effects as the result of the unwanted sense-mRNA interaction

For use of siRNAs as a transient gene knock-down reagent, the requirement of a low- melting antisense 5' end can usually be fulfilled, since the target mRNA can be chosen to fit this criteria However, when the mRNA region of interest is limited, or the siRNA is used to interfere with for example AU-rich regions in the untranslated regions (UTR) of the mRNA, the requirement of a low-melting 5' end might be more difficult to fulfil In some cases, introduction of mismatches have been used to modulate the melting properties of otherwise unfavourable siRNAs to facilitate loading of the antisense strand to RISC However, since the use mismatches for targeting of AU-rich sequences is likely to increase also the probability of microRNA (miRNA) induced activity, i e translational block rather than mRNA degradation, an alternative approach is highly warranted

WO 2005/116226 discloses a si-RNA molecule which has been modified by joining a nan particle of gold to one base or more of the sense strand This creates a molecule quite different from the one of the present invention

Holen, T et al discloses in "Tolerated wobble mutations in siRNAs decreases specificity, but can enhance activity in vivo", Nucleic Research, 2005, vol 33, no 15 p 4707-4710, a U G wobbling at the 5' terminal end of a RNA In the disclosure it is stated that there is no evidence suggesting that there is no absolute specificity of siRNAs

This statement is quite contrary to the present findings where selectivity is obtained and shown

Hagerlof, M et al in "More pronounced salt dependence and higher reactivity for platination of the hairpin r(CGCGUUGUUCGCG) compared with d(CGCGTTGTTCGCG)", J Biol lnorg Chem , 2006, vol 11 , p 974-990 discloses platination of RNA and DNA hairpins It is disclosed that the melt temperature of a double stranded RNA becomes lower by modifying guanine nucleotides using platinum SUMMARY OF THE INVENTION

As described above, the invention relates to the use of siRNAs for highly selective down regulation of a single protein, provided that the sense (passenger) strand is not complementary to other parts of the genome and able to act as a candidate for RISC- induced mRNA degradation If the latter is the case, then the siRNA candidate is normally discarded The idea of the present invention is to circumvent this additional sequence requirement on the passenger strand by introduction of siRNAs carrying metallated bases in the sense strand

The present invention includes the use of siRNAs with metallated sense-strands for local decrease of melting temperature and concomitant structural change of the siRNA The introduction of metallated bases allows for targeting of mRNA regions with sequences deviating from those typically considered as optimal ones

Besides platinum, palladium, gold, ruthenium, and osmium can be used in siRNAs

In particular the invention relates to siRNAs with sense (passenger) strands complementary to sequences deviating from that of a target gene by having introduced metallated bases in the sense (passenger) strand

In a preferred embodiment of the invention, metallation is introduced after coordination of a platinum-, palladium-, gold-, ruthenium- or osmium complex to the sense strand

In a preferred embodiment of the invention the metal coordination bonds are formed with one or more of guanine nucleotides present in the said siRNA

A further aspect of the invention relates to a method for providing a local decrease of melting temperature and concomitant structural change of a siRNA by introducing metallated sense-strands

In a preferred embodiment of the invention the metal used is selected from the group of platinum, palladium, gold, ruthenium, and osmium

In a preferred embodiment of the invention the metallate is present on one or more of guanine nucleotides present in the said siRNA In a further aspect of the invention it relates to a method for targeting mRNA regions with sequences deviating from those typically considered as optimal ones by introducing metallated sense-strands in a siRNA used

In a preferred embodiment of the invention the metallate is present on one or more guanine nucleotides present in the said siRNA

A still further aspect of the invention relates to a method for transient suppression of protein production by the administration of a therapeutically effective amount of small interfering RNAs (siRNAs), the sense strand of which siRNAs has been metallated

An aspect hereof is a method for suppression of growth of benign or malign tumours by suppressing the mobility of the tumour cells including optional metastases thereof by administering one or more siRNAs having a metallated sense-strand for targeting mRNA

A still further aspect hereof is a method for preventing occurrence of cancer metastases by administering one or more siRNAs having a metallated sense-strand for targeting mRNA

In a further aspect of the invention it relates to a use of one or more siRNAs having a metallated sense-strand for targeting mRNA in a therapeutic composition for transient suppression of protein production in illnesses dependent on such a protein production

In a preferred embodiment of the invention it relates to the use of one or more siRNAs having a metallated sense-strand for targeting mRNA in a therapeutic composition for suppression of growth of benign or malign tumours by suppressing the mobility of the tumour cells including optional metastases thereof

In a preferred embodiment of the invention it relates to the use of one or more siRNAs having a metallated sense-strand for targeting mRNA in a therapeutic composition for preventing occurrence of cancer metastases Thus the present invention can be used not only for preventing tumour cell growth, but is also contemplated to have a use in treatment of illnesses dependent of the overexpression or expression of certain illness promoting proteins, the inhibition or reduction of which facilitates the healing or treatment of the illness

The siRNAs used most probably carry 19 to 27 nucleotides in their strands They may be generally administered intravenously, or via hydrodynamic delivery (Herweyer, H et al, Gene Therapy (2007) 14, 99-107) It is also possible to use a Ringer's solution for administration Another delivery possibility is to use SNALPs ( stable nuclei acid lipid particles) Further possible administration form is in combination with cholesterol, in combination with F(ab), with an aptamer, with nanoparticles, or in combination with viruses, such as lentivirus, adenovirus, or adeno associated virus

The successful use is here exemplified by the design of platinated siRNAs targeting Wnt-5a mRNA (W-sιRNA33Pt, W-sιRNA139Pt, W-sιRNA147Pt and W-sιRNA156Pt)

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The aim of the present study has been to evaluate whether pre-platination of the sense strand of siRNAs can be used as a method to modulate siRNA efficacy For this purpose, the initial AU-rich 3' UTR of Wnt-5a was chosen as a target for the siRNAs employed The region (GenBank Accession No NM_003392, bases 1-259) belongs to a part of the mRNA, which serves as a potential protein binding site for HuR, a process that might prevent the mRNA from decay The four siRNAs studied here typically reduce protein production in cell free in vitro test systems to ca 20 - 40 % when used as unmodified double-stranded siRNA The present data reveals that platination of the sense-strand is compatible with siRNA activity with either similar or improved activity compared to the unmodified siRNAs Further, predicted off target effects were efficiently eliminated by use of a metallated sense strand

Materials and methods Chemicals and buffers

Autoclaved and sterile filtered H₂O of Millipore quality was employed for all applications Cisplatin (c/s-Pt(NH₃)₂CI₂) was bought from Sigma A MOPS buffer (50 mM MOPS, 140 mM NaOAc, 2 mM Mg(OAc)₂, pH 6 3) or phosphate buffer (100 mM NaCI (Sigma), 20 mM Na₂HPO₄/NaH₂PO₄ (Sigma) pH 6 5) were used for pH control Oligonucleotides.

All oligonucleotides were purchased from IBM (Germany) and PAGE purified by the manufacturer. The siRNAs were all designed as 19 nucleotide RNA duplexes with a d(TT)

3' overhang and an unmodified 5'-hydroxyl end. The siRNAs were numbered with respect to the location of their upstream, starting 5' hybridization site within the 3' UTR of Wnt-5a mRNA (GenBank Accession No. NM_003392), the latter renumbered from one starting at the base directly following the stop codon (base 1462).

The sequences used were the following:

W-siRNA33 sense: 5'-GGACCCGCUU AUUUAUAGAT T-3', antisense: 5'-UCUAUAAAUA AGCGGGUCCT T-3',

W-SiRNAI 39 sense: 5'-CCAUCUAAGA ACUCUGUGGT T-3', antisense: 5'- CCACAGAGUU CUUAGAUGGT T-3', W-siRNA147 sense: 5'-GAACUCUGUG GUUUAUUAUT T-3', antisense: 5'-AUAAUAAACC ACAGAGUUCT T-3',

W-SiRNAI 56 sense: 5'-GGUUUAUUAU UAAUAUUAUT T-3', antisense: 5'-AUAAUAUUAA UAAUAAACCT T-3'.

Platination of Sense Strands and Annealing.

Platination of the sense strands was performed in aqueous solution with a final oligonucleotide concentration of 2.3 μM and the ratio [oligonucleotide]:[c/s-PtCI₂(NH₃)₂] as 1 :1. The platination reaction was carried out in the dark at 37 ⁰C for 8 h, and was then quenched by quick freezing in liquid nitrogen. The obtained samples were kept at -80 ⁰C for no more then 24 hours before purification. The platinated products were separated by gel electrophoresis on a 20% denaturing polyacrylamide gel (PAGE). The products were visualized by UV-shadowing, excised and eluted by soaking in 1 M NaOAc (Riedel-de Haen) over night, followed by ethanol precipitation. The siRNAs were annealed in 1 x MOPS buffer (50 mM MOPS (Sigma) at pH 6.3 adjusted with 140 mM NaOAc (Reidel-de Haen) in the presence of 2.0 mM Mg(OAc)₂ (Sigma)) by heating 50 μM sense and 50 μM antisense strands for 5 minutes at 90 ⁰C, followed by slow cooling to room temperature over at least 20 minutes. The concentrations of the individual strands were determined by thir respective absorbance at 260 nm using calculated extinction coefficients based on the nearest-neighbour approximation.

Thermal Melt. Thermal melting studies were performed in 1 x MOPS on a Cary 4000 Uv-Vis spectrophotometer (Varian) equipped with a temperature control unit. The data was analyzed by use of on-line Cary WIN-UV software. The concentration of unplatinated siRNA was varied between 1.5 μM and 0.025 μM siRNA, while the concentration of platinated siRNA was kept constant at 0.6 μM (in ds siRNA). Prior to the melting analysis, the siRNAs were annealed by heating to 90 °C for 5 minutes followed by cooling to 20 ⁰C at a rate of 3 °C/min. The thermal melt data were collected from 20 ⁰C to 95 ⁰C at a rate of 0.2 °C/minute, with intervals of 0.5 ⁰C and a data average time of 2 seconds. The results were analyzed by both 1^st derivative- and hyperchromicity calculations. The ΔG-values were obtained using the van't Hoff equation.

In vitro protein expression in RRL.

In vitro protein expression was performed using the T7 Rabbit Reticulocyte Lysate system (RRL, Promega) and the pGL3-Luc expression vector as a template for protein expression. The plasmids were constructed as previously described. Typically, 10 ng/μl of the pGL3-Luc or pGL3-Luc1-159 plasmids were used. A renilla plasmid was added to all reactions as an internal reference at a concentration of 10 ng/μl. The final reaction volume was 10 μl, and siRNAs were added with a final concentration of 5 μM. In the control lacking siRNA, a similar setup was made adding 1 x phosphate buffer instead of siRNA to reach a total volume of 10 μl. After 1.5 h incubation at 30 ⁰C the lysates were tested for Firefly Luciferase and Renilla Luciferase activities with the Dual-Luciferase Reporter Assay System

(Promega) according to the manufacturer's protocol. The reactions were analysed in at least triplicates. To obtained Firefly luciferase luminescence was normalized against the Renilla luciferase luminesence, and the ratio reported as Luciferase activity. The effect on the expression was examined both as a function of siRNA, and as a function of platinated versus non-platinated siRNA.

In vitro protein expression in HB2 cells.

The HB2, non-cancerous mammary epithelial cell line was used for the cellular in vitro studies. The cell line is a subclone of the MTSV-7 cell line originating and kindly received from the laboratory of Dr. J. Taylor-Papadimitriou (ICRF, UK) [']. Cells were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma), insulin (10 μg/ml) (Sigma) and hydrocortisone (50 nM) (Sigma) at 37 ⁰C and in a humidified atmosphere with 5% carbon dioxide The HB2 cells were transfected in 24-well plates using Lipofectamine 2000 (Invitrogen) in Transfection DMEM Transfection was performed in 24 well plates using 50 000 cells per well, 0 4 μg/well pMIR-Luc or pMIR-Luc/W-UTR(1-260), and 0 4 μg/well pMIR-Renilla Renatured W-siRNA (40 nM) was co-transfected with the plasmids The medium was changed to normal DMEM after 4 h, and the cells were incubated for 40 h prior to lysis in 100 μl lysis buffer (Promega) The lysed samples were immediately frozen in liquid nitrogen, and stored at -80 ⁰C until analysis The Luciferase activity was determined as described above

Results and Discussion

Platination of sense siRNA and product distribution

Exposure of the sense strands of W-sιRNA33, W-sιRNA147, and W-sιRNA156 to cisplatin gave rise to one single product only The resulting double stranded platinated siRNAs are denoted W-sιRNA33-Pt, W-sιRNA147-Pt, and W-sιRNA156-Pt In the case of W-sιRNA139, two distinct products were obtained, W-sιRNA139-Pt1 and W-sιRNA139-Pt2 The different product distribution patterns can be accounted for by consideration of the number and location of preferred GG- or AGA- platination sites present in the respective oligomers More specifically, for W-sιRNA147 and W-sιRNA156 only GG-site is present In the case of W-sιRNA33, the tentative GG and AGA sequences are both located at the end of the oligomer, i e equally disfavoured from an electrostatic point of view, thus allowing formation of the kinetically most favoured product, i e platination of GG In W-sιRNA139, the AGA-site is located in the middle of the oligomer whereas the GG-site is located at the 3'-end Under these circumstances two products are formed, indicating that the higher tendency for preaccumulation of the platinum complex in the middle of the oligomer is enough to compensate for the higher inherent reactivity exhibited by the GG-site

Influence of platination on thermal stability of W-siRNA A summary of the melting temperatures and related thermodynamic parameters obtained for the non-platinated siRNAs, together with the corresponding platinated ones is made in Table 1 As can be seen in the table, the melting temperatures (t_{m dem}) determined for the non-platinated siRNAs vary in the range 50 - 75 ⁰C, and agree well with the corresponding theoretically predicted ones {t_m, _hyPo) After platination, the f_m-values are decreased by ca 3 - 10 ⁰C As judged by the thermodynamic parameters, the decrease in t_m is a combined result of an increase in both AH and AS, i.e. due to both destabilization of the duplex formation and an increase of entropy, all in agreement with the structural change and loss of hydration that can be expected after adduct formation with the positively charged platinum centre.

Interference of W-siRNA and W-siRNA-Pt with protein translation in RRL and HB2. The four different siRNAs investigated in the present study were all designed to target the conserved AU-rich region containing a putative HuR binding site. With respect to internal thermodynamic base stability profile, W-siRNA33, W-siRNA147 and W-siRNA156 were designed with a high-melting region at the 5' end of the sense strand, compare also Table 2. However, only modest differences between the translational inhibitory efficacies of the unplatinated siRNAs are observed in RRL. The down regulation of protein expression lies between 20 and 40% with W-siRNA147 and W-siRNA156 as the most potent siRNAs, and W-siRNA33 as the least efficient one, see Figure 1. After platination of the sense strand, a different picture is obtained however. The most noticeable difference is observed for W- siRNA33-Pt, which reaches a suppression level around 50%, i.e. an improvement of the efficacy by more than 100% compared with the corresponding unplatinated W-siRNA33. As judged by primary fluorescence data (data not shown), the effect seems likely to be due to suppression of off-target effects caused by the sense strand. We therefore speculate that the presence of the platinated site on the siRNA sense strand serves as a block for loading onto RISC, thus can be utilized as a method for elimination of off-target effects. For the other three siRNAs, the introduction of the platinated sense strand has only marginal effect on the translational inhibition. In the case of W-siRNA139-Pt, the platination leads to a somewhat less efficient siRNA. The observation suggests that the thermal destabilization caused by the attached platinum compound, when located both in the middle of the siRNA and close to the 3' end of the sense strand, interferes with processing of the siRNA.

By changing the test environment from the RRL system to the HB2 cell line the sensitivity towards added siRNA is increased by 2 to 3 orders of magnitude, i.e. showing significant suppression levels in the nano-molar range, see Figure 2. With exception of W-siRNA156, all siRNAs exhibit suppression levels well above 80%, both as unplatinated and platinated W-siRNA. Interestingly, W-siRNA156 which partially overlaps with a tentative imiRNA target region ["], is the least efficient one. The lower efficacy of W-siRNA156 shows that this particular region is stabilized in the cellular environment and less prone to siRNA induced degradation. The observation is well in agreement with previous reports suggesting proteins such as HuR to be responsible for stabilization of the Wnt-5a mRNA. It might thus be possible that the high efficacy obtained for the other siRNAs might be a combined effect of destabilization of the HuR binding motif and siRNA induced degradation.

TABLES

Table 1 : Thermodynamic parameters for platinated and non platinated siRNAs W-siRNA33, W-siRNA33-Pt, W-siRNA139, W-siRNA139-Pt1 , W-siRNA139-Pt2, W-siRNA147, W- siRNAI 47-Pt, W-siRNA156and W-siRNA156-Pt obtained in 1 x MOPS buffer. siRNA ' mdenv ' mhypo ΔH ΔS ΔG₂₀ ΔG₂₅

/ °c / °c / kJmol^"1 / JmOl-¹K^" / kJmol^"1 / kJmol^"1

1

W-siRNA33 76.05 74.27 -463 -1184 -115.9 -110.0

W-siRNA33-Pt 72 71.44 -410 -1051 -102 -97

W-SiRNAI 39 75.06 73.16 -674.7 -1811 -144 -135

W-SiRNAI 39- 71.95 70.02 -437.9 -1151 -100 -94

Pt- 1

W-SiRNAI 39- 72.00 70.41 -398.4 -1038 -99 -94

Pt-2

W-SiRNAI 47 65.05 - -589.1 -1627 -112.16 -104.02

W-SiRNAI 47-Pt 55.00 - -463.5 -1292 -84.74 -78.28

W-SiRNAI 56 53.5 51.83 -673 -1947 -101 -92

W-siRNA156-Pt 50 50 -575 -1658 -85 -77

Table 2 Schematic overview of the siRNAs used and related properties.

5' sense strand position

" The column stating the sequence displays the sense strand, 5'- to 3¹ end, on top of the antiscnse strand, 3'- to 5' end. f^t The internal stability (AG (kcal/mol)) was determined from sfold [30] and displayed from the 5' position of the sense strand.

' Potential Pt(IT) binding sites: Number of GG and AGA sites in the sense strand.

''Number of plalinated products: The number of product bands that were visible on a PAA gel by UV shaddowing, following platination. e t_m: The first two columns give t_m for unplatinated siRNA, the third is W-siRNA platinated in the sense strand.

Suppression in RRL:The plasmids pGL3-Luc/W-UTR( 1-259) or pGL3-Luc were incubated together with 5 μM W- siRNA, in RRL for 90 minutes at 30 ⁰C.

'Values refer to Luciferase activity obtained with pGL3-Luc/W-UTR( 1 -259) normalized to the Luciferase activity obtained with pGL3-L.

TABLE S1 Thermodynamic parameters for W-sιRNA33 in 1 x MOPS, as a function of siRNA concentration

Conc ^a -AS -AH -ΔG₂₀ -AG₂₅ ' m deπv ' m hypo

/ μM / Jmol V ¹ I kJmor¹ / kJmol ¹ I kJmol ¹ / °c / °c

2 33 1029 401 1 99 353 94 205 76 0 74 41

1 16 1 167 449 6 107 590 101 756 76 05 74 19

0 46 1337 507 9 109 394 116 077 75 09 72 55

0 23 969 6 382 8 93 681 98 528 74 12 71 50

0 15 702 9 289 1 79 582 83 096 73 16 70 34

0 038 - - - - 71 20 -

^! Cone = total concentration of strands Each strand is approximately half of this value TABLE S2 Thermodynamic parameters for W-sιRNA139 in 1 x MOPS as a function of siRNA concentration

Conc^a -AS -AH -AG₂₀ -AG₂₅ ' m denv ' hypo

/μM /JmOl¹K¹ / kJmol ¹ I kJmol ¹ I kJmol ¹ /°c /⁰C

256 1082 4195 10277 96867 7697 7558

129 1040 4063 101307 96105 7604 7504

070 1176 457 107993 102 7608 74

034 1183 4537 10694 101 74 7170

018 1305 4943 11189 105367 7304 7032

7208 a Conc = total concentration of strands Each strand is approximately half of this value

TABLE S3 Thermodynamic parameters for W-sιRNA147 in 1 x MOPS as a function of siRNA concentration

Conc^a -AS -AH -AG₂₀ -AG₂₅ ' m denv ' m hypo

/μM /Jmol¹K¹ I kJmol ¹ I kJmol ¹ I kJmol ¹ /°c /°c

323 6983 2811 76435 72943 7001 6864

129 7732 3051 78477 74611 6904 6904

026 5182 2221 70178 67587 6807 6565

0051 5650 1525 117694 110338 6610 6509

001 4567 2049 70964 6868 6617 6617

0002 6621 a Conc = total concentration of strands Each strand is approximately half of this value

TABLE S4 Thermodynamic parameters for W-sιRNA156 in 1 x MOPS as a function of siRNA concentration

Cone ^a -AH -AS -AG₂₀ -AG₂₅ ' mdeπv ' mhypo

/μM I kJmol ¹ /Jmol¹K¹ I kJmol ¹ I kJmol ¹ /°c /°c

206 6644 1915 1029 93349 5399 5306

103 6894 1998 1037 93697 5302 5302

041 6179 1764 1008 91998 5207 5111

024 5872 1676 9595 8757 5111 5029

018 5528 1573 9171 83846 5112 4948

011 5866 1674 95905 87536 5119 4912 ^aConc = total concentration of strands Each strand is approximately half of this value The invention has been tested above with the use of certain sequences appearing in metastasing breast cancer, but it will be equally valuable in the treatment of other cancer forms, such as skin cancer (using a composition penetrating the epidermis), lung cancer (using a local application strategy). Hereby, one should reach a concentration in the target muscle or other tissue of about 300 to 1500 ng/g tissue.

Abbreviations and key words

W-siRNA 33

Sense strand (passenger strand): δ'-GGACCCGCUUAUUUAUAGATT-S' (SEQ. ID. NO. 1) Antisense strand (guide strand): δ'-TTCCUGGGCGAAUAAAUAUCU-S' (SEQ. ID. NO. 2)

W-siRNA139Pt

Sense strand (passenger strand): δ'-CCAUCUAAGAACUCUGUGGTT-S' (SEQ. ID. NO. 3) Antisense strand (guide strand): δ'-TTGGUAGAUUCUUGAGACACC-S' (SEQ. ID. NO. 4)

W-siRNA 147Pt

Sense strand (passenger strand): δ'-GAACUCUGUGGUUUAUUAUTT-S' (SEQ. ID. NO.

5) Antisense strand (guide strand): δ'-TTCUUGAGACACCAAAUAAUA-S' (SEQ. ID.

NO. 6)

W-siRNA 156Pt

Sense strand (passenger strand): δ'-GGUUUAUUAUUAAUAUUAUTT-S' (SEQ. ID.

NO. 7) Antisense strand (guide strand): δ'-TTCCAAAUAAUAAUUAUAAUA-S' (SEQ. ID. NO. 8)

Underlined bases refer to platination sites.

Platination reagent: cisplatin (c/s-Pt(NH₃)₂CI₂)

REFERENCE

[1] A. G. Mueller, H. G. Joost, Mouse ARF-related protein 1 : genomic organization and analysis of its promotor, Biochem. Biophys. Res. Commun. 292 (2002) 113-120.

FIGURE LEGENDS

FIG. 1 (A) Luciferase activity and (B) Normalized Luciferase activity obtained after incubation of pGL3-Luc (white columns) or pGL3-L/W-UTR (1-260) (gray colums) for 90 minutes at 30oC in RRL with W-siRNA. The plasmids were incubated in the absence (control) and presence of 5 μM W-siRNA33 (33), W-siRNA33-Pt (33-Pt), W-siRNA139 (139), W-siRNAI 39-Pt1 (139-Pt1 ), W-siRNA139-Pt2 (139-R2). W-siRNA147 (147), W- siRNAI 47-Pt (147-Pt), W-siRNAI 56 (156), and W-siRNA156-Pt (156-Pt). Buffer at pH 6.5 was used as control (contr.). FIG. 2 (A) Luciferase activity and (B) Normalized Luciferase activity obtained after transfection of HB2 cells with pMIR-Luc (white columns) or pMIR-Luc/W-UTR (1-260) (gray colums). The plasmids were incubated in the absence (0) and presence of 40 nM W- siRNA33 (33), W-siRNA33-Pt (33-Pt), W-siRNA139 (139), W-siRNA139-Pt1 (139-Pt1), W- siRNAI 39-Pt2 (139-Pt2). W-siRNAI 47 (147), W-siRNA147-Pt (147-Pt), W-siRNAI 56 (156), and W-siRNAI 56-Pt (156-Pt). Buffer at pH 6.5 was used as control (contr.).

Claims

1 siRNAs with sense (passenger) strands complementary to sequences deviating from that of a target gene by having introduced metallated bases in the sense (passenger) strand

2 siRNAs with passenger strands according to claim 1 , wherein metallation is introduced after coordination of a platinum-, palladium-, gold-, ruthenium-, or osmium complex to the sense strand

3 siRNAs according to claim 1 , wherein the metal coordination bonds are formed with one or more of guanine nucleotides present in the said siRNA

4 Method for providing a local decrease of melting temperature and concomitant structural change of a siRNA by introducing metallated sense-strands

5 Method according to claim 4, wherein the metal used is selected from the group of platinum, palladium, gold, ruthenium, and osmium

6 Method according to claim 5, wherein the metallate is present on one or more of guanine nucleotides present in the said siRNA

7 Method for targeting mRNA regions with sequences deviating from those typically considered as optimal ones by introducing metallated sense-strands in a siRNA used

8 Method according to claim 7, wherein the metal used is selected from the group of platinum, palladium, gold, ruthenium, and osmium

9 Method according to claim 8, wherein the metallate is present on one or more guanine nucleotides present in the said siRNA

10 Method for transient suppression of protein production by the administratation of a therapeutically effective amount of small interfering RNAs (siRNAs), the sense strand of which siRNAs has been metallated

11. Method for suppression of growth of benign or malign tumours by suppressing the mobility of the tumour cells including optional metastases thereof by administering one or more siRNAs having a metallated sense-strand for targeting mRNA.

12. Method for preventing occurrence of cancer metastases by administering one or more siRNAs having a metallated sense-strand for targeting mRNA.

13. Use of one or more siRNAs having a metallated sense-strand for targeting mRNA in a therapeutic composition for transient suppression of protein production in illnesses dependent on such a protein production.

14. Use of one or more siRNAs having a metallated sense-strand for targeting mRNA in a therapeutic composition for suppression of growth of benign or malign tumours by suppressing the mobility of the tumour cells including optional metastases thereof.

15. Use of one or more siRNAs having a metallated sense-strand for targeting mRNA in a therapeutic composition for preventing occurrence of cancer metastases.