Patent Application: US-18120808-A

Abstract:
the invention relates to ribonucleic acids and oligonucleotide probes useful for detection and analysis of micrornas and their target mrnas , as well as small interfering rnas . the invention furthermore relates to oligonucleotide probes for detection and analysis of other non - coding rnas , mrnas , mrna splice variants , allelic variants of single transcripts , mutations , deletions , or duplications of particular exons in transcripts , e . g . alterations associated with human disease , such as cancer .

Description:
the present invention provides novel oligonucleotide compositions and probe sequences for the use in detection , isolation , purification , amplification , identification , quantification , or capture of mirnas , their target mrnas , stem - loop precursor mirnas , sirnas , other non - coding rnas , rna - edited transcripts or alternative mrna splice variants characterized in that the probe sequences contain a number of nucleoside analogues . in a preferred embodiment the number of nucleoside analogue corresponds to from 20 to 40 % of the oligonucleotide of the invention . in a preferred embodiment the probe sequences are substituted with a nucleoside analogue with regular spacing between the substitutions in another preferred embodiment the probe sequences are substituted with a nucleoside analogue with irregular spacing between the substitutions in a further preferred embodiment the detection probe sequences comprise a photochemically active group , a thermochemically active group , a chelating group , a reporter group , or a ligand that facilitates the direct of indirect detection of the probe or the immobilisation of the oligonucleotide probe onto a solid support . ( a ) the photochemically active group , the thermochemically active group , the chelating group , the reporter group , or the ligand includes a spacer ( k ), said spacer comprising a chemically cleavable group ; or ( b ) the photochemically active group , the thermochemically active group , the chelating group , the reporter group , or the ligand is attached via the biradical of at least one of the lna ( s ) of the oligonucleotide . ( a ) capture and detection of naturally occurring or synthetic single stranded nucleic acids such as mirnas , their target mrnas , stem - loop precursor mirnas , sirnas , other non - coding rnas , rna - edited transcripts or alternative mrna splice variants ; or ( b ) purification of naturally occurring single stranded nucleic acids such as mirnas , their target mrnas , stem - loop precursor mirnas , sirnas , other non - coding rnas , rna - edited transcripts or alternative mrna splice variants ; or ( c ) detection and assessment of expression patterns naturally occurring single stranded nucleic acids such as mirnas , their target mrnas , stem - loop precursor mirnas , sirnas , other non - coding rnas , rna - edited transcripts or alternative mrna splice variants by rna in - situ hybridisation , dot blot hybridisation , reverse ddot blot hybridisation , or in northern blot analysis or expression profiling by microarrays ( d ) functional analysis of naturally occurring single stranded nucleic acids such as mirnas , their target mrnas , stem - loop precursor mirnas , sirnas , other non - coding rnas , rna - edited transcripts or alternative mrna splice variants in vitro and in vivo in plants or animals , such as human , mouse , rat , zebrafish , caenorhabditis elegans , drosophila melanogaster , arabidopsis thaliana , rice and maize , by inhibiting their mode of action , e . g . the binding of mature mirnas to their cognate target mrnas . further embodiments includes the use of an lna modified oligonucleotide probe as an aptamer in molecular diagnostics or ( b ) as an aptamer in rna mediated catalytic processes or ( c ) as an aptamer in specific binding of antibiotics , drugs , amino acids , peptides , structural proteins , protein receptors , protein enzymes , saccharides , polysaccharides , biological cofactors , nucleic acids , or triphosphates or ( d ) as an aptamer in the separation of enantiomers from racemic mixtures by stereospecific binding or ( e ) for labelling cells or ( f ) to hybridise to non - protein coding cellular rnas , such as trna , rrna , snrna and scrna , in vivo or in - vitro or ( g ) to hybridise to non - protein coding cellular rnas , such as trna , rrna , snrna and scrna , in vivo or in - vitro or ( h ) in the construction of taqman probes or molecular beacons . the present invention also provides a kit for the isolation , purification , amplification , detection , identification , quantification , or capture of natural or synthetic nucleic acids , where the kit comprises a reaction body and one or more lnas as defined herein . the lnas are preferably immobilised onto said reactions body ( e . g . by using the immobilising techniques described above ). for the kits according to the invention , the reaction body is preferably a solid support material , e . g . selected from borosilicate glass , soda - lime glass , polystyrene , polycarbonate , polypropylene , polyethylene , polyethyleneglycol terephthalate , polyvinylacetate , polyvinylpyrrolidinone , polymethylmethacrylate and polyvinylchloride , preferably polystyrene and polycarbonate . the reaction body may be in the form of a specimen tube , a vial , a slide , a sheet , a film , a bead , a pellet , a disc , a plate , a ring , a rod , a net , a filter , a tray , a microtitre plate , a stick , or a multi - bladed stick . a written instruction sheet stating the optimal conditions for use of the kit typically accompanies the kits . once the appropriate target rna sequences have been selected , lna substituted detection probes are preferably chemically synthesized using commercially available methods and equipment as described in the art ( tetrahedron 54 : 3607 - 30 , 1998 ). for example , the solid phase phosphoramidite method can be used to produce short lna probes ( caruthers , et al ., cold spring harbor symp . quant . biol . 47 : 411 - 418 , 1982 , adams , et al ., j . am . chem . soc . 105 : 661 ( 1983 ). lna - containing - probes can be labelled during synthesis . the flexibility of the phosphoramidite synthesis approach furthermore facilitates the easy production of lnas carrying all commercially available linkers , fluorophores and labelling - molecules available for this standard chemistry . lna - modified probes may also be labelled by enzymatic reactions e . g . by kinasing using t4 polynucleotide kinase and gamma - 32 p - atp or by using terminal deoxynucleotidyl transferase ( tdt ) and any given digoxygenin - conjugated nucleotide triphosphate ( dntp ) or dideoxynucleotide triphosphate ( ddntp ). detection probes according to the invention can comprise single labels or a plurality of labels . in one aspect , the plurality of labels comprise a pair of labels which interact with each other either to produce a signal or to produce a change in a signal when hybridization of the detection probe to a target sequence occurs . in another aspect , the detection probe comprises a fluorophore moiety and a quencher moiety , positioned in such a way that the hybridized state of the probe can be distinguished from the unhybridized state of the probe by an increase in the fluorescent signal from the nucleotide . in one aspect , the detection probe comprises , in addition to the recognition element , first and second complementary sequences , which specifically hybridize to each other , when the probe is not hybridized to a recognition sequence in a target molecule , bringing the quencher molecule in sufficient proximity to said reporter molecule to quench fluorescence of the reporter molecule . hybridization of the target molecule distances the quencher from the reporter molecule and results in a signal , which is proportional to the amount of hybridization . in the present context , the term “ label ” means a reporter group , which is detectable either by itself or as a part of a detection series . examples of functional parts of reporter groups are biotin , digoxigenin , fluorescent groups ( groups which are able to absorb electromagnetic radiation , e . g . light or x - rays , of a certain wavelength , and which subsequently reemits the energy absorbed as radiation of longer wavelength ; illustrative examples are dansyl ( 5 - dimethylamino )- 1 - naphthalenesulfonyl ), doxyl ( n - oxyl - 4 , 4 - dimethyloxazolidine ), proxyl ( n - oxyl - 2 , 2 , 5 , 5 - tetramethylpyrrolidine ), tempo ( n - oxyl - 2 , 2 , 6 , 6 - tetramethylpiperidine ), dinitrophenyl , acridines , coumarins , cy 3 and cy 5 ( trademarks for biological detection systems , inc . ), erythrosine , coumaric acid , umbelliferone , texas red , rhodamine , tetramethyl rhodamine , rox , 7 - nitrobenzo - 2 - oxa - 1 - diazole ( nbd ), pyrene , fluorescein , europium , ruthenium , samarium , and other rare earth metals ), radio isotopic labels , chemiluminescence labels ( labels that are detectable via the emission of light during a chemical reaction ), spin labels ( a free radical ( e . g . substituted organic nitroxides ) or other paramagnetic probes ( e . g . cu 2 + , mg 2 + ) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy ). especially interesting examples are biotin , fluorescein , texas red , rhodamine , dinitrophenyl , digoxigenin , ruthenium , europium , cy5 , cy3 , etc . suitable samples of target nucleic acid molecules may comprise a wide range of eukaryotic and prokaryotic cells , including protoplasts ; or other biological materials , which may harbour target nucleic acids . the methods are thus applicable to tissue culture animal cells , animal cells ( e . g ., blood , serum , plasma , reticulocytes , lymphocytes , urine , bone marrow tissue , cerebrospinal fluid or any product prepared from blood or lymph ) or any type of tissue biopsy ( e . g . a muscle biopsy , a liver biopsy , a kidney biopsy , a bladder biopsy , a bone biopsy , a cartilage biopsy , a skin biopsy , a pancreas biopsy , a biopsy of the intestinal tract , a thymus biopsy , a mammae biopsy , a uterus biopsy , a testicular biopsy , an eye biopsy or a brain biopsy , e . g ., homogenized in lysis buffer ), archival tissue nucleic acids , plant cells or other cells sensitive to osmotic shock and cells of bacteria , yeasts , viruses , mycoplasmas , protozoa , rickettsia , fungi and other small microbial cells and the like . preferably , the detection probes of the invention are modified in order to increase the binding affinity of the probes for the target sequence by at least two - fold compared to probes of the same sequence without the modification , under the same conditions for hybridization or stringent hybridization conditions . the preferred modifications include , but are not limited to , inclusion of nucleobases , nucleosidic bases or nucleotides that have been modified by a chemical moiety or replaced by an analogue to increase the binding affinity . the preferred modifications may also include attachment of duplex - stabilizing agents e . g ., such as minor - groove - binders ( mgb ) or intercalating nucleic acids ( ina ). additionally , the preferred modifications may also include addition of non - discriminatory bases e . g ., such as 5 - nitroindole , which are capable of stabilizing duplex formation regardless of the nucleobase at the opposing position on the target strand . finally , multi - probes composed of a non - sugar - phosphate backbone , e . g . such as pna , that are capable of binding sequence specifically to a target sequence are also considered as a modification . all the different binding affinity - increasing modifications mentioned above will in the following be referred to as “ the stabilizing modification ( s )”, and the tagging probes and the detection probes will in the following also be referred to as “ modified oligonucleotide ”. more preferably the binding affinity of the modified oligonucleotide is at least about 3 - fold , 4 - fold , 5 - fold , or 20 - fold higher than the binding of a probe of the same sequence but without the stabilizing modification ( s ). most preferably , the stabilizing modification ( s ) is inclusion of one or more lna nucleotide analogs . probes from 6 to 30 nucleotides according to the invention may comprise from 1 to 8 stabilizing nucleotides , such as lna nucleotides . when at least two lna nucleotides are included , these may be consecutive or separated by one or more non - lna nucleotides . in one aspect , lna nucleotides are alpha and / or xylo lna nucleotides . the problems with existing detection , quantification and knock - down of mirnas and sirnas as outlined above are addressed by the use of the novel oligonucleotide probes of the invention in combination with any of the methods of the invention selected so as to recognize or detect a majority of all discovered and detected mirnas , in a given cell type from a given organism . in one aspect , the probe sequences comprise probes that detect mammalian mature mirnas , e . g ., such as mouse , rat , rabbit , monkey , or human mirnas . by providing a sensitive and specific method for detection of mature mirnas , the present invention overcomes the limitations discussed above especially for conventional mirna assays and sirna assays . the detection element of the detection probes according to the invention may be single or double labelled ( e . g . by comprising a label at each end of the probe , or an internal position ). in one aspect , the detection probe comprises two labels capable of interacting with each other to produce a signal or to modify a signal , such that a signal or a change in a signal may be detected when the probe hybridizes to a target sequence . a particular aspect is when the two labels comprise a quencher and a reporter molecule . in another aspect , the probe comprises a target - specific recognition segment capable of specifically hybridizing to a target molecule comprising the complementary recognition sequence . a particular detection aspect of the invention referred to as a “ molecular beacon with a stem region ” is when the recognition segment is flanked by first and second complementary hairpin - forming sequences which may anneal to form a hairpin . a reporter label is attached to the end of one complementary sequence and a quenching moiety is attached to the end of the other complementary sequence . the stem formed when the first and second complementary sequences are hybridized ( i . e ., when the probe recognition segment is not hybridized to its target ) keeps these two labels in close proximity to each other , causing a signal produced by the reporter to be quenched by fluorescence resonance energy transfer ( fret ). the proximity of the two labels is reduced when the probe is hybridized to a target sequence and the change in proximity produces a change in the interaction between the labels . hybridization of the probe thus results in a signal ( e . g . fluorescence ) being produced by the reporter molecule , which can be detected and / or quantified . the invention also provides a method , system and computer program embedded in a computer readable medium (“ a computer program product ”) for designing detection probes comprising at least one stabilizing nucleobase . the method comprises querying a database of target sequences ( e . g ., such as the mirna registry at http :// www . sanger . ac . uk / software / rfam / mirna / index . shtml ) and designing probes which : i ) have sufficient binding stability to bind their respective target sequence under stringent hybridization conditions , ii ) have limited propensity to form duplex structures with itself , and iii ) are capable of binding to and detecting / quantifying at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 % or at least about 95 % of all the target sequences in the given database of mirnas or other rna sequences . in one preferred aspect , the target sequence database comprises nucleic acid sequences corresponding to human , mouse , rat , drosophila melanogaster , c . elegans , arabidopsis thaliana , maize or rice mirnas . in another aspect , the method further comprises calculating stability based on the assumption that the recognition sequence comprises at least one stabilizing nucleotide , such as an lna molecule . in one preferred aspect the calculated stability is used to eliminate probes with inadequate stability from the database of virtual candidate probes prior to the initial query against the database of target sequence to initiate the identification of optimal probe recognition sequences . in another aspect , the method further comprises calculating the capability for a given probe sequence to form a duplex structure with itself based on the assumption that the sequence comprises at least one stabilizing nucleotide , such as an lna molecule . in one preferred aspect the calculated propensity is used to eliminate probe sequences that are likely to form probe duplexes from the database of virtual candidate probes . a preferred embodiment of the invention are kits for the detection or quantification of target mirnas , sirnas , rna - edited transcripts , non - coding antisense transcripts or alternative splice variants comprising libraries of detection probes . in one aspect , the kit comprises in silico protocols for their use . the detection probes contained within these kits may have any or all of the characteristics described above . in one preferred aspect , a plurality of probes comprises at least one stabilizing nucleotide , such as an lna nucleotide . in another aspect , the plurality of probes comprises a nucleotide coupled to or stably associated with at least one chemical moiety for increasing the stability of binding of the probe . the kits according to the invention allow a user to quickly and efficiently develop an assay for different mirna targets , sirna targets , rna - edited transcripts , non - coding antisense transcripts or alternative splice variants . in general , the invention features the design of high affinity oligonucleotide probes that have duplex stabilizing properties and methods highly useful for a variety of target nucleic acid detection methods ( e . g ., monitoring spatiotemporal expression of micrornas or sirnas or knock - down of mirnas ). some of these oligonucleotide probes contain novel nucleotides created by combining specialized synthetic nucleobases with an lna backbone , thus creating high affinity oligonucleotides with specialized properties such as reduced sequence discrimination for the complementary strand or reduced ability to form intramolecular double stranded structures . the invention also provides improved methods for detecting and quantifying ribonucleic acids in complex nucleic acid sample . other desirable modified bases have decreased ability to self - anneal or to form duplexes with oligonucleotide probes containing one or more modified bases . the invention will now be further illustrated with reference to the following examples . it will be appreciated that what follows is by way of example only and that modifications to detail may be made while still falling within the scope of the invention . the lna - substituted probes of example 2 to 11 were prepared on an automated dna synthesizer ( expedite 8909 dna synthesizer , perseptive biosystems , 0 . 2 μmol scale ) using the phosphoramidite approach ( beaucage and caruthers , tetrahedron lett . 22 : 1859 - 1862 , 1981 ) with 2 - cyanoethyl protected lna and dna phosphoramidites , ( sinha , et al ., tetrahedron lett . 24 : 5843 - 5846 , 1983 ). cpg solid supports derivatised with a suitable quencher and 5 ′- fluorescein phosphoramidite ( glen research , sterling , va ., usa ). the synthesis cycle was modified for lna phosphoramidites ( 250s coupling time ) compared to dna phosphoramidites . 1h - tetrazole or 4 , 5 - dicyanoimidazole ( proligo , hamburg , germany ) was used as activator in the coupling step . the probes were deprotected using 32 % aqueous ammonia ( 1 h at room temperature , then 2 hours at 60 ° c .) and purified by hplc ( shimadzu - spectrachrom series ; xterra ™ rp18 column , 10 ? m 7 . 8 × 150 mm ( waters ). buffers : a : 0 . 05m triethylammonium acetate ph 7 . 4 . b . 50 % acetonitrile in water . eluent : 0 - 25 min : 10 - 80 % b ; 25 - 30 min : 80 % b ). the composition and purity of the probes were verified by maldi - ms ( perseptive biosystem , voyager de - pro ) analysis . list of lna - substituted detection probes for detection of fully conserved vertebrate micrornas in all vertebrates . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methylcytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for detection of fully conserved vertebrate micrornas in all vertebrates . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methylcytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for detection of zebrafish micrornas . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for detection of drosophila melanogaster micrornas . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for detection of drosophila melanogaster and caenorhabditis elegans micrornas . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for detection of arabidopsis thaliana micrornas . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32 p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for detection of arabidopsis thaliana micrornas . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32 p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes useful as negative controls in detection of vertebrate micrornas . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for detection of human micrornas . lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine , pm perfect match to the mirna , mm one mismatch at the central position of the probe sequence . the detection probes can be used to detect and analyze conserved vertebrate mirnas by rna in situ hybridization , northern blot analysis and by silencing using the probes as mirna inhibitors . the lna - modified probes can be conjugated with a variety of haptens or fluorochromes for mirna in situ hybridization using standard methods . 5 ′- end labeling using t4 polynucleotide kinase and gamma - 32p - atp can be carried out by standard methods for northern blot analysis . in addition , the lna - modified probe sequences can be used as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh2 - c6 - or a nh2 - c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . list of lna - substituted detection probes for expression profiling of human and mouse micrornas by oligonucleotide microarrays lna nucleotides are depicted by capital letters , dna nucleotides by lowercase letters , mc denotes lna methyl - cytosine , pm perfect match to the mirna , mm one mismatch at the central position of the probe sequence , dir denotes the probe sequence corresponding to the mature mirna sequence , rev denotes the probe sequence complementary to the mature mirna sequence in question . the detection probes can be used t as capture sequences for expression profiling by lna oligonucleotide microarrays . covalent attachment to the solid surfaces of the capture probes can be accomplished by incorporating a nh 2 — c6 - or a nh 2 — c6 - hexaethylene glycol monomer or dimer group at the 5 ′- end or at the 3 ′- end of the probes during synthesis . in order to address mirna function in myoblastic differentiation , we designed a loss - of - function assay based on antisense oligonucleotides complementary to the mirna sequence . antisense oligonucleotides have been used to demonstrate a function for mirnas in drosophila ( boutla et al ., 2003 ). antisense oligonucleotides with a 2 ′- o - methyl modification have been shown to block risc activity ( hutvagner et al ., 2004 ; meister et al ., 2004 ). we chose to use oligonucleotides containing locked nucleotides ( lnas ) ( kumar et al ., 1998 ). these form highly stable duplexes with complementary rnas ( koshkin , 1998 ), even when using short sequences ( kurreck et al ., 2002 ). they trigger the degradation of target mrnas by rnase h , provided that the locked nucleotides are not located at the center of the sequence . ( wahlestedt et al ., 2000 ). a stable interaction between the antisense oligonucleotide and the mirna would be expected to facilitate the sequestration of the mirna in a duplex unable to interact with cellular protein complexes such as risc or the mirnps , or to anneal with complementary target mrnas . alternatively , it could induce the degradation of the mirna by rnase h , similar to what was described for lna / dna oligonucleotides targetting mrnas . mixed lna / dna oligonucleotides with lnas located either at the center or at the extremities of the sequence were transfected into myoblatsts , and mrnas were analyzed by northern blotting . results ( fig3 b ) showed that the target mirna became undetectable ( fig3 b ) in cells transfected with lna / dna antisense molecules , but not in cells transfected with a control sequence . this effect was independent of the position of the locked nucleotides , whether in the center ( wt125 : 8 ) or at the ends ( wt125 : 4 / 4 ) of the antisense oligonucleotides . thus it is unlikely that the lna induces mirna cleavage by rnase h . we favour a hypothesis in which the lna / dna oligonucleotides form a highly stable duplex with the mirna , that subsequently resists the denaturing conditions used for northern blot , thereby preventing microrna detection . supporting this hypothesis , in some experiments we observed a faint smear migrating above the mirna position that might correspond to melting mirna / lna duplexes ( see for example fig3 d or fig4 d ). furthermore , a duplex formed in vitro between a radiolabelled synthetic mirna and the 125 wt : 8 lna indeed resisted the denaturing conditions of the migration ( 7 m urea ), with about 50 % of the mirna annealed to the lna at a 1 to 1 ration of lna to mirna , and 100 % at a 3 to 1 ratio ( fig3 c ). in extracts from transfected cells , the lna also affected the apparent level of precursor ( fig2 b ). this effect , however , was only partial , as compared to the reduction observed with the mature mirna . this might be due to the fact that the target sequence is more accessible in the mature mirna than in the precursor , the latter having a secondary structure a priori more difficult to invade by the lna . alternatively , the lna might influence the actual level of precursor by affecting a regulatory loop in mirna metabolism . the effect was sequence specific and was not observed with a scrambled oligonucleotide ( fig3 a and b ). furthermore , inhibiting one mirna did not affect another mirna : lna / dna 10 complementary to mir - 125b affected mir - 125b but did not have any effect on mir - 181 and , conversely , lna / dna complementary to mir - 181 affected mir - 181 but had no effect on mir - 125b ( fig3 d ). a dose response analysis of the lna / dna oligonucleotide showed that the effect was dose dependent , and that 50 nm of lna / dna were sufficient to affect the target mirna ( fig3 e ). finally , lna - containing oligonucleotides are highly stable in physiological media ( kurreck et al ., 2002 ). consistent with this observation , inhibition of target mirnas lasted for at least several days in myoblastic cells ( fig3 f ), and the lna persisted for the same duration ( fig7 ), a timing compatible with the functional analysis of mirnas during in vitro differentiation of myoblastic cells . taken together , these data show that this approach is usable to address mirna functions during the differentiation process . mir - 181 has been described as having a role in b lymphocytes , and its upregulation during skeletal muscle differentiation ex vivo and during muscle regeneration in vivo ( fig1 and 2 ) was unexpected . in order to analyze its function , c2c12 cells were treated with the lna / dna oligonucleotide complementary to mir - 181 , or with a mutated sequence as a control ( fig4 a ), and myotube formation and muscle marker expression were monitored . cells treated with mir - 181 lna oligonucleotide formed few myotubes ( fig4 b ) and expressed low levels of muscle specific markers such as mhc ( fig4 b ) or mck ( fig4 c ), as compared to cells treated with mutant lna . in fact , most of the cells scoring positive for mhc in the mir - 181 lna - treated population were neither elongated nor fused to form multinucleated myotubes ( fig4 b ). an sirna directed against the precursor loop of mir - 181 ( fig4 a ), and designed as previously described ( zeng et al ., 2002 ), did not affect differentiation ( fig4 b and c ). monitoring mir - 181 expression , however , revealed that , in contrast to the lna , the sirna did not significantly decrease the levels of mature or precursor mir - 181 ( fig4 d ). this lack of activity might be due to the remarkably at - rich nature of the target loop . alternatively , it might be due to the apparent long half - life of mirna in these cells : indeed , down - regulating dicer , the enzyme involved in precursor processing , resulted in increased levels of precursor but not in reduced levels of mature mirna ( fig7 ). the level of expression achieved with a plasmid designed as previously described ( zeng et al ., 2002 ) was very low . rescue experiments were therefore performed by co - transfecting the mirna as a synthetic sequence . when cotransfected with the lna , synthetic mir - 181 was able to restore a significant level of differentiation in transfected cells , as assessed by the number of myotubes ( fig5 b ) and the expression of muscle markers . the mirna alone did not affect differentiation . furthermore , differentiation was not restored by cotransfection of the lna with an irrelevant rna sequence . taken together , these results demonstrate a sequence - specific effect of the lna , and rule out non - specific effects potentially induced by the formation of an lna / rna duplex in the cells . in mammals , mirna targets have been predicted in silico ( lewis et al ., 2003 ), but have been validated in a very limited number of cases . among the potential targets identified in silico for mir181 , some may be relevant to muscle . in particular , hoxa11 , a homeodomain protein that is essential for limb ( small and potter , 1993 ) and kidney formation ( patterson et al ., 2001 ), is a repressor of myod function ( yamamoto and kuroiwa , 2003 ). immunofluorescence analysis demonstrated that it is expressed in nuclei of adult resting muscle , confirming previous observations ( takahashi et al ., 2004 ), but is undetectable in regenerating muscle ( fig6 a ), when mir181 expression is upregulated . in order to determine whether hox - a11 could be a target for mir181 in muscle , we used a synthetic sirna to knock down the protein ( fig6 b ). we reasoned that if hox - a11 is downstream of mir181 , then co - inhibiting mir181 and hox - a11 simultaneously should restore , at least in part , normal differentiation . indeed , whereas a control sirna had no effect on mck expression , the sirna against hox - a11 significantly rescued the differentiation phenotype ( fig6 c ). thus , knocking down hox - a11 resulted in what might be thought of as a phenotypic suppressive mutation . this results indicates that hox - a11 is in the same genetic pathway as mir181 , and supports a model in which hox - a11 is a direct target of this mirna . myoblastic c2c12 cells were maintained in dulbecco &# 39 ; s minimal essential medium ( dmem ), ( life technologies , inc .) supplemented with 15 % fetal bovine serum ( dominique dutscher ). for transfections , 5 × 10 4 cells were plated in 6 - well plates and transfected the next day with 1 μg of oligonucleotides using lipofectamine ( invitrogen ). to induce terminal differentiation , 24 h after transfection , growth medium was replaced by differentiation medium ( dmem , 2 % horse serum , sigma ). phenotypic differentiation was observed after 72 - 96 h . mouse es cells ( kind gift or dr . muriel vernet ) were cultured and maintained in an undifferentiated state as described ( zhuang et al ., 1992 ). for embryoid body ( eb ) formation they were diluted and kept in suspension for 4 days . to induce muscle differentiation eb were maintained an additional 2 days in presence of 1 % dmso in dmem with 10 % horse serum , then plated on tissue culture dishes in dmem with 2 % horse serum . myotubes were observed at 21 days of differentiation . 6 to 7 week - old balb / c mice were put to sleep with avertin as described in ( weiss and zimmermann , 1999 ), the leg was shaved , and injections were made into the tibialis anterior ( ta ) muscle , using a 29 g1 / 2 insulin syringe . cardiotoxin ( latoxan ) was diluted to 10 μm in pbs , and 25 μl were injected per ta muscle ( hosaka et al ., 2002 ). control animals received equal volumes of pbs . ta muscles were harvested at different time points , and total rna was extracted and used for northern blot . sirna , rna , dna and lna / dna mixed oligonucleotides were obtained from proligo , france . for northern blot analysis 30 μg of total rna were separated in 15 % denaturating polyacrylamide gels and electro - transferred to hybond - n + membranes . dna anti - sense probes were end - labeled with t4 polynucleotide kinase ( biolabs ) using γ - 32p - atp with high specific activity ( 7000 ci / mmol , icn ). hybridization and washing were carried out in rapid - hyb buffer ( amersham ) at 42 ° c . u6 expression was tested as loading control . the mirnain situ hybridization protocol by ambion was optimized for skeletal muscle . briefly , the tibialis anterior ( ta ) muscle was frozen in tissue - tek oct reagent , cryo - sections ( 12 μm ) were prepared , de - proteinized and acetylated as described in ( denardi et al ., 1993 ). digoxigenin ( dig )— labeled mirna probes ( mir181 : sequence complementary to the mature mirna ) were prepared according to the instructions for the mirvana probe construction kit ( ambion ). muscle sections were incubated with specific mirna probes ( 500 ng / ml ) overnight at 380 ° c ., washed with 2 × ssc at 38 ° c . for 20 min , and treated with 400 unit / ml ( 10 μg / ml ) rnase a at 37 ° c . for 30 min . after two washes with pbs , the slides were incubated with fitc - coupled anti - dig antibody ( roche ) for 4 hours at room temperature , washed , rinsed with 200 ng / ml hoecsht 33258 in pbs , mounted on glass slides with vectashield ( biovalley ), and analyzed by fluorescent microscopy . western blot and immunostaining were performed as described previously ( polesskaya et al ., 2001 ). rabbit anti - mck was kindly provided by dr . hidenory ito , anti hox - a11 was a kind gift of larry patterson , anti - mhc ( my - 32 ) and cy - 3 conjugated anti - mouse igg were purchased from sigma . synthetic mir - 181 a rna was end - labeled with 32p - atp using t4 polynucleotide kinase , purified on a g - 25 column and mixed with cold mir - 181 so that the final activity was 105 cpm / μmol rna . 10 μmol of labeled mir - 181 were mixed with increasing amounts of lna oligonucleotides in a total volume of 20 μl of depc - treated water containing 1 u / μl rnase inhibitor ( life technologies ). after 15 min incubation at room temperature , 10 μl of the olignucleotide mixture was mixed with 2 × urea loading buffer , incubated 15 min at 65 ° c . and separated on a 15 % denaturating polyacrylamide gel . rnas were electro - 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( 2003 ). the drosophila microrna mir - 14 suppresses cell death and is required for normal fat metabolism . curr biol 13 , 790 - 795 . yamamoto , m ., and kuroiwa , a . ( 2003 ). hoxa - 11 and hoxa - 13 are involved in repression of myod during limb muscle development . dev growth differ 45 , 485 - 498 . yekta , s ., shih , i . h ., and bartel , d . p . ( 2004 ). microrna - directed cleavage of hoxb8 mrna . science 304 , 594 - 596 . zeng , y ., wagner , e . j ., and cullen , b . r . ( 2002 ). both natural and designed micro rnas can inhibit the expression of cognate mrnas when expressed in human cells . mol cell 9 , 1327 - 1333 . zhuang , y ., kim , c . g ., bartelmez , s ., cheng , p ., groudine , m ., and weintraub , h . ( 1992 ). helixloop - helix transcription factors e12 and e47 are not essential for skeletal or cardiac myogenesis , erythropoiesis , chondrogenesis , or neurogenesis . proc natl acad sci usa 89 , 12132 - 12136 . the microrna mir - 181 targets the homeobox protein hox - a11 during mammalian myoblast differentiation deciphering the mechanisms underlying skeletal muscle - cell differentiation in mammals is an important challenge . cell differentiation involves complex pathways regulated at both transcriptional and post - transcriptional levels . recent observations have revealed the importance of small ( 20 - 25 base pair ) non - coding rnas ( micrornas or mirnas ) that are expressed in both lower organisms 1 and in mammals 2 , 3 . mirnas modulate gene expression by affecting mrna translation 4 or stability 5 . in lower organisms , mirnas are essential for cell differentiation during development 6 - 9 ; some mirnas are involved in maintenance of the differentiated state . here , we show that mir - 181 , a microrna that is strongly upregulated during differentiation , participates in establishing the muscle phenotype . moreover , our results suggest that mir - 181 downregulates the homeobox protein hox - a11 ( a repressor of the differentiation process ), thus establishing a functional link between mir - 181 and the complex process of mammalian skeletal - muscle differentiation . therefore , mirnas can be involved in the establishment of a differentiated phenotype — even when they are not expressed in the corresponding fully differentiated tissue . muscle precursors , or myoblasts , are derived from multipotent precursor cells and acquire the myoblastic phenotype during the first step of skeletal muscle differentiation . they subsequently exit from the cell cycle and enter terminal differentiation , fusing into large multinucleated myotubes and expressing muscle - specific marker proteins . differentiation can be partially recapitulated in two in vitro models : using either totipotent embryonic stem cells oriented toward the muscle lineage 10 , or established myoblastic cell lines that enter the skeletal muscle differentiation pathway by default when they are deprived of growth factors . we screened for mirnas that were differentially expressed during the two main steps of muscle differentiation and found that mir - 181 is one of the most strongly upregulated mirnas when terminal differentiation is induced in either of the two models . in myoblastic cells , mir - 181 is upregulated before , or concomitant with , expression of differentiation - specific proteins such as muscle creatine kinase ( mck ; fig8 a ). chemically modified locked nucleic acid ( lna ) oligonucleotides 11 were used as probes to discriminate between the three previously characterized mir - 181 isoforms ( http :// microrna . sanger . ac . uk /), and mir - 181a and mir - 181b ( fig8 b ), but not mir - 181c ( fig1 ) were detected . as one of the mir - 181b precursors , mir - 181b2 , is located close to mir - 181a on mouse chromosome 2 , it is likely that the two isoforms are co - induced as a long primary transcript ( pri - mirna ). in vivo , mir - 181 was barely detectable in the tibialis anterior muscle of adult mouse , as previously described 9 , 12 , but was strongly upregulated on regeneration of muscle fibres ( fig8 c ). cells expressing mir - 181 were characterized by immunofluorescent in situ hybridization ( fish ) experiments ( fig8 d , e ): mir - 181 was detected by fish , and embryonic myosin heavy chain ( emhc , a well - characterized protein marker of regenerating fibres ) was detected by immunofluorescence microscopy . nuclei were counterstained with dapi to delineate the regeneration area ( indicated by a high number of nuclei with a disorganized topology ). mir - 181 - positive cells were mostly multinucleated in regenerating fibres , but some mononucleated cells were also labelled . whether multinucleated or mononucleated , all of the mir - 181 - positive cells were also positive for emhc , indicating that they were differentiating , non - proliferating muscle cells . only about 80 % of the cells positive for emhc were also positive for mir - 181 . this can be explained by the sensitivities of the two detection assays : fish uses such short probes that it is less sensitive than immunofluorescence microscopy . although mir - 181 returned to basal levels at the end of the regeneration process , it was still detectable for a long period after the disappearance of emhc ( fig8 f ). mir - 181 seems to have a long half - life in muscle cells , as suggested by its long - term ( over several days ) persistence in dicer - depleted myoblastic cells , which may explain its long - term presence during regeneration . together , these data indicate that mir - 181 , though poorly expressed in terminally differentiated muscle , is highly expressed in regenerating muscle , and raised the possibility that it may function during muscle differentiation . to address this issue , we designed an antisense - based loss - of - function assay 13 , 14 using lna - dna - mixed - antisense oligonucleotides 15 : these oligonucleotides form highly stable sequence - specific duplexes with their target mirna sequences , and are potent , specific and long - lasting inhibitors of these molecules . an lna - dna antisense oligonucleotide complementary to mir - 181a ( for sequence , see fig9 a ) abrogated mir - 181 detection ( both a and b isoforms ) in northern blots ( fig9 b , fig1 a ), most likely through sequestration of the target microrna . mir - 181 inhibition was also evident at a functional level , when a luciferase reporter construct harboring a sequence complementary to mir - 181 was assayed . in c2c12 cells , the reporter activity was inhibited on differentiation . inhibition was released by mir - 181 antisense lna , but not by a mutant form of the antisense oligonucleotide , whereas a reporter harbouring a mutated sequence was not affected ( fig9 c ). these results , along with similar data for a green fluorescent protein ( gfp ) target sequence , indicate that antisense lnas can be used to analyse mirna function in live cells . the mir - 181 antisense lna oligonucleotides dramatically affected c2c12 myoblast differentiation , as assessed by both myotube formation and by the expression of sarcomeric myosin heavy chain ( mhc ; fig9 d ) or mck ( fig9 e ), both markers for terminal differentiation . a mutated antisense lna did not affect differentiation - marker expression . moreover , the differentiation phenotype was rescued by transfecting the mirna as a synthetic double - stranded sequence ( fig9 f ), under conditions that result in high levels of cellular mirna ( monitored by northern blot , fig1 b ). this demonstrated that the effect of the lna - dna antisense oligonucleotide was specifically due to mir - 181 inhibition . the synthetic mirna alone did not dramatically affect differentiation ( fig9 f ). taken together , these data functionally link mir - 181 to myoblast differentiation , even though mir - 181 is barely detectable in resting muscle cells . this suggests that mir - 181 is involved in the establishment of the differentiated phenotype , but probably not in its maintenance . interestingly , during regeneration , a similar expression profile is observed for the transcription factors that orchestrate the differentiation process , the basic helix - loop - helix ( bhlh ) myogenic proteins myod and myogenin : these essential proteins are not detected in resting muscle and are expressed only on regeneration 16 . during differentiation of c2c12 cells in vitro , myod induces myogenin and triggers the entire differentiation programme . in mir - 181 - depleted c2c12 cells , myod expression was inhibited ( fig9 g ), as was the expression of its downstream targets , myogenin ( fig9 h ) and p21cip1 ( fig1 c ). therefore , mir - 181 acts upstream of myod in the differentiation pathway . one of the targets consistently predicted for mir - 18117 - 19 ( see http :// www . microrna . org / and http :// pictar . bio . nyu . edu ) is the homeobox protein hox - a11 . this protein is involved in urogenital tract development 20 but is also important for limb muscle patterning 21 , 22 and can inhibit myod expression 23 . in adult humans , hox - a11 is detected in various organs , including skeletal muscle 24 . the pattern of expression of hox - a11 protein is complementary to that of mir - 181 in muscle : hox - a11 is highly expressed in cells with low mir - 181 levels , resting muscle in vivo ( fig1 a ) and undifferentiated myoblasts in vitro ( fig1 b ), whereas hox - a11 downregulation coincides with mirna induction during terminal differentiation in both models ( fig1 a , b ). in differentiating c2c12 cells , despite reduced hox - a11 protein levels , a concomitant decrease in hox - a11 mrna ( fig1 c ) was not observed , suggesting a post - transcriptional regulation mechanism . whether or not mir - 181 could affect hox - a11 cellular protein levels was examined and it was found that hox - a11 protein was downregulated by ectopic mir - 181a in proliferating myoblasts ( fig1 a ). conversely , hox - a11 was upregulated by inhibition of mir - 181 ; absolute levels of hox - a11 protein were higher in cells treated with the lna antisense oligonucleotides than in control cells ( fig1 b ). twofold upregulation of the hox - a11 protein was estimated by semi - quantitative analysis of the western blots , similarly to previously published data on mir - 375 and its target , myotrophin 25 . the lna - dna antisense oligonucleotide did not , however , abolish hox - a11 downregulation on differentiation , suggesting that there are additional mechanisms of regulation . the level of hox - a11 mrna was not affected by inhibition of mir - 181 with the lna antisense oligonucleotide ( fig1 a ), even though the protein level increased , supporting a post - transcriptional mechanism . these data are consistent with the hypothesis that hox - a11 is a direct target of mir - 181 in muscle cells . to test this hypothesis , a standard reporter assay in mir - 181 - negative cells , using plasmids with tandem repeats of hox - a11 predicted target sequences ( fig1 c ) inserted downstream of the firefly luciferase gene , was used . insertion of wild - type sequences rendered the reporter sensitive to ectopic mir - 181a ( fig1 d ). mutation of the target sequences abolished this effect . moreover , a reporter harbouring an irrelevant target sequence ( mir - 196 target sequence from hox - b8 ) 5 was not affected by mir - 181a , and was inhibited only by its cognate microrna , mir - 196 . interestingly , the effect on the hox - a11 target sequence was far more pronounced with mir - 181a than with mir - 181b . moreover , co - transfection of the mir - 181b isoform did not increase inhibition by the mir - 181a isoform in the luciferase assay ( fig1 b ). in the literature , mir - 181 is consistently predicted to bind to hox - a11 mrna , although with differing scores ; the best - ranked isoform varies between different studies . our experimental data best fit with the predictions that identified the a isoform of mir - 181 as a mirna that potentially binds to hox - a11 ( refs 17 - 19 ). taken together , our data indicate that hox - a11 is a direct target of mir - 181 during mammalian muscle differentiation . this was confirmed at the functional level by simultaneously inhibiting mir - 181 and hox - a11 ; an sirna against hox - a11 partly rescued the differentiation phenotype created by the anti - mir - 181 antisense lna oligonucleotide ( fig1 e - f ). moreover , differentiation was inversely correlated with hox - a11 expression ( fig1 g ); hox - a11 was absent in control cells that differentiated normally , and upregulated in lna - treated cells that did not differentiate . the sirna reduced hox - a11 protein levels in lna - treated cells , resulting in an intermediate level of hox - a11 protein , as well as an intermediate level of differentiation . thus hox - a11 is a critical target of mir - 181 . as hox - a11 is a repressor of myoblast terminal differentiation in vitro 23 , our data support a model in which mir - 181 participates in differentiation by alleviating repression by hox - a11 , which in turn results in myod induction and triggers the expression of muscle markers ( fig1 ). we note , however , that as inhibition of mir - 181 did not completely abolish hox - a 11 downregulation ( fig1 b ), additional mechanisms must control cellular hox - a 11 levels during myoblastic differentiation . a plausible hypothesis is that hox - a11 mrna translation is controlled by more than one mirna . indeed , in the reporter assay described in fig1 d , insertion of only one target sequence did not influence reporter activity , and inhibition required multiple targets in the reporter . this is generally the case for mirnas acting at the translational level , as documented previously 26 , 27 , and is consistent with the observation that several mirnas generally bind to natural target mrnas . a number of other mirnas are predicted to bind to the natural hox - a113 ′ untranslated region ( utr ). among these , several are expressed and / or upregulated in terminally differentiating myoblasts ( mir23 a , mir188 , mir339 and mir30b ; fig1 c ). the coordinate action of these mirnas may lead to the complete disappearance of hox - a11 on differentiation . our results also suggest that hox - a 11 is not the only target of mir - 181 . first , restoration of the lna - induced differentiation phenotype by treatment with anti - hox - a11 sirna was only partial ( fig1 f ), perhaps due to the residual level of hox - a11 protein observed in these cells ( fig1 g ). second , the involvement of other protein targets in mir - 181 pathways would be consistent with the marked phenotype created by downregulating this mirna . indeed , mirnas do seem to have multiple targets , including in mammals 28 . from in silico analysis , mir - 181a is suspected to have a number of targets in addition to hox - a11 , some of which are relevant to skeletal - muscle terminal differentiation , such as id2 ( a bhlh - related inhibitor of differentiation and dna binding ) or eya1 ( a homologue of the drosophila eyes absent transcription factor ; see http :// www . microrna . org /). as mir - 181 is also involved in b lymphocyte differentiation 9 , it will be important to determine whether the pathways in which it participates are common to these two cell types , and are more widespread . taken together , our data demonstrate that mir - 181 is required for skeletal myoblast terminal differentiation , during which an important target is the homeobox protein hox - a11 . however , neither upregulation of mir - 181 , nor downregulation of hox - a11 , triggered terminal differentiation of proliferating myoblasts ( fig1 d ). thus , mir - 181 is necessary , but not sufficient , for differentiation . mir - 181 is expressed at very low levels in adult muscle , in contrast to other mirnas , such as mir - 133 or mir - 1 , which are readily detected in adult muscle 12 . a reasonable explanation is that mir - 133 and mir - 1 are involved in the maintenance of the muscle phenotype , whereas mir - 181 is involved in its establishment . our results underscore the importance of dynamic analysis of mirnas during cell differentiation . dna constructs . the reporter plasmid containing the mir - 181a complementary sequence was constructed by inserting a synthetic double - stranded oligonucleotide into the sali - xbai site of piso vector ( kind gift of d . bartel ) downstream of the firefly luciferase coding sequence . reporter plasmids with four copies of the pre - dicted hox - a11 target sequence for mir - 181 ( position of the binding nucleus , 1361 in mrna , nm — 005523 ), either wild type or mutated , were constructed by cloning four tandem repeats of a 64 base pair sequence containing the target into the sal i - xbai sites of piso . details of construction are available on request . the piso reporter plasmid containing the mir196 target site from hox - b8 was a kind gift of d . bartel . oligonucleotides . sirna , rna , and dna oligonucleotides were obtained from various sources ; lna probes discriminating mir - 181a , b and c isoforms were from exiquon ( vedbaek , denmark ). lna - dna mixed oligonucleotides were from proligo ( paris , france ). the anti - hox - aii sirna sequence was : 5 ′- gagcucggccaacgucuactt - 3 ′. cell culture and transfection . myoblast c2c12 cells were maintained in dulbecco &# 39 ; s minimal essential medium ( dmem ; gibco - invitrogen , paisley , uk ) supplemented with 15 % fetal bovine serum ( dominique dutscher , brumath , france ). for transfections , 5 × 10 4 cells were plated in 6 - well plates and transfected the next day with 1 μg of oligonucleotides using lipofectamine ( gibco - invitrogen ). to induce terminal differentiation , 24 h after transfection , growth medium was replaced by differentiation medium ( dmem , 2 % horse serum , invitrogen ). phenotypic differentiation was observed after 72 - 96 h . for luciferase assays , hela s3 cells were grown in complete dmem in 24 - well plates . cells were transfected , using lipofectamine 2000 , with firefly luciferase reporter vectors ( 0 . 05 μg ), together with a renilla luciferase control vector ( 0 . 1 μg ) ( promega , lyon , france ) and equal amounts ( as assessed by a260 and by gel analysis ) of synthetic mirna duplexes ( 0 . 1 μg ). luciferase activity was measured 24 h post transfection and was normalized using renilla luciferase activity . c2c12 cells were transfected with firefly luciferase reporter constructs containing a sequence complementary to mir - 181 in their 3 ′ utr , or a mutated version of this sequence , along with a renilla construct to monitor transfection efficiency , and placed under differentiation conditions ; reporter activities were measured after 2 days in differentiation medium . mouse embryonic stem cells ( kind gift of m . vernet ) were cultured and maintained in an undifferentiated state using standard procedures . for embryoid body formation they were diluted and kept in suspension for 4 days . to induce muscle differentiation , embryoid bodies were maintained an additional 2 days in 1 % dmso in dmem with 10 % horse serum10 , then plated on tissue culture dishes in dmem with 2 % horse serum . myotubes were observed at 21 days of differentiation . cardiotoxin - induced regeneration assay . six - to seven - week - old balb / c mice were anaesthetized with avertin ( sigma - aldrich , steinheim , germany ), the leg was shaved , and injections were made into the tibialis anterior muscle , using a 29 g ½ insulin syringe . cardiotoxin ( latoxan , valence , france ) was diluted to 10 μm in pbs , and 25 μl were injected per muscle 29 . control animals received equal volumes of pbs . tibialis anterior muscles were harvested at different times , and total rna was extracted and used for northern blots , or muscles were frozen as described below for in situ hybridization . northern blot . for northern blot analyses , 30 μg of total rna were separated on 15 % denaturing polyacrylamide gels and electro - transferred to hybond - n + ( amersham biosciences , little chalfont , uk ) membranes . dna anti - sense probes were end - labelled with t4 polynucleotide kinase ( biolabs , hitchin , uk ) using γ - 32p - atp with high specific activity ( 6 , 000 ci mmol - 1 , amersham biosciences ). hybridization was carried out in rapid - hyb buffer ( amersham biosciences ) at 42 ° c . washing was carried out at 42 ° c . ( dna probes ) or at 65 ° c . ( lna probes ). u6 expression was used as a loading control . in situ hybridization and immuno - fish . the mirna in situ hybridization protocol by ambion ( austin , tex .) was optimized for skeletal muscle . briefly , the tibialis anterior muscle was frozen in tissue - tek oct reagent , cryo - sections ( 12 μm ) were prepared , de - proteinized and acetylated as previously described30 . digoxigenin ( dig )- labelled mirna probes ( mir - 181 : sequence complementary to the mature mirna ) were prepared according to the instructions for the mirvana probe construction kit ( ambion ). muscle cross - sections were incubated with specific mirna probes ( 500 ng / ml ) overnight at 38 ° c ., washed with 2 . ssc at 38 ° c . for 20 min , and treated with 400 units / ml ( 10 μg / ml ) rnase a at 37 ° c . for 30 min . after two washes with pbs , the slides were incubated with fitc - coupled anti - dig antibody ( roche , mannheim , germany ) for 4 h at room temperature , washed , rinsed with 200 ng / ml dapi in pbs , mounted on glass slides with vectashield ( biovalley , burlingame , calif . ), and analysed by fluorescent microscopy . for immuno - fish experiments , the cryo - sections were fixed with 4 % phosphate - buffered paraformaldehyde ( pfa ) for 10 min , treated for 10 min with 1 μg / ml proteinase k , and post - fixed in 4 % pfa for 10 more min . in situ hybridization was performed as described above , and then the sections were incubated 4 h to overnight at room temperature with anti - embryonic mhc antibody ( f1 . 652 , developmental studies hybridoma bank , iowa city , iowa ), at 2 μg / ml in pbs - 1 % bsa - 5 % newborn calf serum . detection was performed by 1 h incubation with anti - mouse - tritc anti - body ( sigma - aldrich ), diluted 1 : 1 , 000 in blocking solution . for all fish and immuno - fish experiments , 25 - 30 cross - sections from four independent regenerating tibialis anterior muscles were analysed . negative controls with secondary antibodies alone were performed in all cases , and gave no detectable staining . real - time rt - pcr . poly - a + mrna was isolated with a magna pure lc mrna isolation kit ( roche ). one - step real time q - rt - pcr was carried out with a hot start lc rna master sybr green kit ( roche ), using the following primers : hox - a11 , forward 5 ′- tctctaaggctccagcctac - 3 ′, reverse gcttaaccacggagatctga ; mck , forward 5 ′- caccatgccgttcggcaaca - 3 ′, reverse 5 ′- ggttgtccaccccagtct - 3 ′; 36b4 ( housekeeping gene used for rna normalization ), forward 5 ′- atgtgcagctgataaagactgg - 3 ′, reverse 5 ′- aggccttgaccttttcagtaag - 3 ′. western blotting and immunofluorescence microscopy . western blotting and immunostaining were performed using standard procedures . rabbit anti - mck was kindly provided by h . ito , anti - hox - a11 was a kind gift of l . patterson . anti - tubulin , anti - actin , anti - mhc ( my - 32 ) and cy - 3 conjugated anti - mouse igg were purchased from sigma , and anti - myod antibodies ( c20 ) were purchased from santa cruz ( santa cruz , calif .). 1 . ambros , v . micrornas : tiny regulators with great potential . cell 107 , 823 - 826 ( 2001 ). 2 . lagos - quintana , m . et al . identification of tissue - specific micrornas from mouse . curr . biol . 12 , 735 - 739 ( 2002 ). 3 . lagos - quintana , m . et al . new micrornas from mouse and human . rna 9 , 175 - 179 ( 2003 ). 4 . olsen , p . h . & amp ; 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