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WO2011161239A1 - Procédé pour mettre en évidence des acides ribonucléiques - Google Patents

Procédé pour mettre en évidence des acides ribonucléiques Download PDF

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Publication number
WO2011161239A1
WO2011161239A1 PCT/EP2011/060622 EP2011060622W WO2011161239A1 WO 2011161239 A1 WO2011161239 A1 WO 2011161239A1 EP 2011060622 W EP2011060622 W EP 2011060622W WO 2011161239 A1 WO2011161239 A1 WO 2011161239A1
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Prior art keywords
rna
nucleic acid
duplex
dna
modified
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PCT/EP2011/060622
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German (de)
English (en)
Inventor
Christoph Ritt
Martin Weber
Denis FLÜGGE
Stefan Pitsch
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Qiagen GmbH
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Qiagen GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to a method for the detection of ribonucleic acids by displacing a nucleic acid from a duplex comprising a nucleic acid modified on a sugar residue with a further sugar residue-modified nucleic acid or deoxyribonucleic acid by the ribonucleic acid to be detected and detection of the displacement event.
  • nucleic acids with a known nucleotide sequence For the specific detection of nucleic acids with a known nucleotide sequence, different methods are known from the prior art. Thus, for example, the complementary base pairing of single-stranded nucleic acids is exploited. In this process called hydrogen bonding between the complementary nucleotides of the respective single-stranded nucleic acids are formed, resulting in a stable duplex, which is composed of two nucleic acid single strands.
  • the principle of hybridization has been known in the art in various variations for the detection of specific nucleic acids for decades.
  • RNA molecules immobilized on a membrane.
  • specific RNAs are detected by hybridization with radioactively or otherwise labeled nucleic acid probes.
  • probes made of DNA are used for this purpose. Theoretically, in this detection method both sequence-complementary DNA and RNA would be detected, as it is not possible in this method to distinguish hybrids by hybridization between DNA-DNA and DNA-RNA.
  • purification steps which specifically accumulate mRNAs are carried out in practice before the hybridization step.
  • DNase digestion is generally carried out, ensuring that all DNA present in the sample is degraded so that it can not interfere with the detection of the RNA.
  • RNA A similar method for the detection of RNA is the dot blot.
  • the nucleic acids do not become prior to hybridization separated by electrophoresis. Rather, a small amount of RNA is applied to a nucleic acid-binding membrane and immobilized by the action of heat.
  • a nucleic acid probe which is for example radioactively labeled, it can now be determined whether a particular RNA sequence has been present in the nucleic acid immobilized on the membrane.
  • the dot blot it is also not possible with the dot blot to specifically detect RNA only by the hybridization conditions. Purification steps before immobilization and DNase digestion are needed to specifically detect RNA only.
  • An extension of the dot blot to the high-throughput method is the microarray.
  • the mRNA is detected by rewriting in cDNA by hybridization to short synthetic oligonucleotides or even to longer pieces of DNA. Even in this method, genomic DNA of the same sequence still present can make the comparative quantification of the originally present mRNA amount more difficult.
  • the cDNA synthesis has a fluctuating efficiency, which represents an additional potential source of error for comparative quantification in the microarray.
  • RNA The most frequently used method for the detection and quantification of RNA in the prior art is the quantitative RT-PCR.
  • the mRNA Before the amplification of the mRNA in the PCR, the mRNA must first be transcribed into cDNA by means of reverse transcriptase. There is a risk of artifacts due to the different efficiency of the reverse transcriptase when rewriting the mRNA into cDNA. This efficiency is primarily dependent on the secondary structure of the mRNA to be rewritten. Due to the exponential reaction kinetics in the amplification of the cDNA, the error propagation is also exponential.
  • the object of the present invention is to overcome the disadvantages of the prior art and to provide a cost-effective and less time-consuming method for the specific detection of ribonucleic acids without any DNA present during the detection interfering with this detection.
  • NA1 contains at least one non-naturally occurring nucleotide having at least one modification to a sugar residue and wherein NA2 is also at least one non-natural containing occurring nucleotide having at least one modification to a sugar residue or is an unmodified deoxyribonucleic acid;
  • NA3 ribonucleic acid
  • the method according to claim 1 is particularly suitable for the detection of specific nucleic acids. It is preferred for this that the modified nucleic acid NA1 in step a) in claim 1 is present as duplex with a further nucleic acid NA2, which is either DNA or also a modified nucleic acid. It is very particularly preferred if the modified nucleic acid NA1 is present in step a) in claim 1 with a DNA as duplex.
  • the duplex between NA1 and NA2 is usually formed by two at least partially mutually complementary nucleic acids.
  • the complex is composed not only of two nucleic acids, but of two or more nucleic acids, each having overlapping regions, so that a nucleic acid double strand is likewise formed in the entirety , wherein the respective single strand of NAl or NA2 is not covalently connected over the full length.
  • the nucleic acid NA3 to be detected is a ribonucleic acid in the method according to the invention.
  • the ribonucleic acid NA3 may be a naturally occurring RNA, but may also be a non-naturally occurring RNA.
  • RNAs which can be detected in the method according to the invention include, for example, mRNAs, noncoding RNAs and viral RNAs.
  • Non-coding RNAs are understood to mean RNA molecules which, unlike the mRNA, are not translated into proteins but have a function after transcription. These include rRNAs, tRNAs, miRNAs, siRNAs, piRNAs, tiRNAs, antisense RNAs, riboswitches and ribozymes. Other non-coding RNAs are familiar to the person skilled in the art.
  • the RNAs to be detected are naturally occurring RNAs.
  • Non-naturally occurring RNAs can be prepared in an in vitro method known to those skilled in the art. These methods include, for example, in vitro transcription and nucleic acid synthesis.
  • nucleic acid of the duplex from step a) in claim 1 is coupled to a solid phase.
  • the NA1 of the duplex from step a) in claim 1 is coupled to a solid phase.
  • both the NA1 and NA2 of the duplex of step a) in claim 1 are coupled to different solid phases.
  • a solid phase for example, magnetic or non-magnetic particles, planar or curved surfaces, vessel walls or pipette tips come into question.
  • Other suitable solid phases are familiar to the person skilled in the art.
  • the duplex from step a) in claim 1 is marked in a single or multiple detectable manner.
  • the label or labels may be at the 5 'end, at the 3' end, or at any nucleotide of one or both of the nucleic acids of the duplex.
  • nucleic acids include, for example, fluorescent dyes, quenchers, radioactivity, chemiluminescence, nanoparticles of metals such as gold or silver, quantum dots, antibodies, biotin, avidin, streptavidin, streptactin, digoxigenin, and enzymes. Further possibilities of labeling nucleic acids are familiar to the person skilled in the art.
  • Corresponding methods for the detection and detection of nucleic acids are likewise known to the person skilled in the art.
  • Common methods include, for example, fluorescence microscopy, analysis with FACS instruments, spectrophotometers and other fluorescence readers, autoradiography, scintillation, enzyme-substrate reaction detection methods, e.g. by alkaline phosphatase or peroxidase, indirect detection methods e.g. with the help of antibodies, optical detection systems such as Surface Plasmon Resonance (SPR) and other interference-based methods.
  • SPR Surface Plasmon Resonance
  • the modification of the nucleotides is chosen so that the specificity of the base pairing is retained and the bases of the modified nucleic acid are not modified. Rather, the sugar residue of the nucleotides is modified.
  • the modification on the sugar residue is chosen so that the affinity of DNA and RNA to the Nucleotides with modified sugar residue differs and thus also result in differences in the melting temperature.
  • the modification is chosen such that the melting temperature of a duplex of modified nucleic acid and RNA is significantly higher than the melting temperature of a duplex of modified nucleic acid and DNA.
  • this sugar residue is a ribose.
  • the at least one modification is on
  • the modification at the 2'C atom of the sugar residue of a ribose belongs to the group of the hydroxyalkyl-oxymethyl substituents, moreover very particular preference is given to a 2'-O - [((2R, 3S) - 2,3,4-trihydroxy-butyl) oxymethyl substituent, hereinafter referred to as (DL) -C 4 .
  • DL 2'-O - [((2R, 3S) - 2,3,4-trihydroxy-butyl) oxymethyl substituent
  • more than one nucleotide is modified in the modified nucleic acid, more preferably at least 30% of all nucleotides are modified in the modified nucleic acid, very particularly preferably at least 50% of all nucleotides in the modified nucleic acid are modified, moreover are very particularly preferred.
  • at least 80% of all nucleotides in the modified nucleic acid are modified, moreover at least 80% of all nucleotides in the modified nucleic acid are modified, moreover at least 90% of all nucleotides in the modified nucleic acid are modified, moreover very particularly
  • at least 95% of all nucleotides in the modified nucleic acid are modified, moreover, very particular preference is given to modifying all nucleotides in the modified nucleic acid.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • duplexes of modified nucleic acid with DNA or RNA differ markedly in their melting temperature, wherein the duplexes of modified nucleic acid and RNA have a significantly higher melting temperature than the duplexes of modified nucleic acid with DNA.
  • the exact difference in the melting temperature is dependent on the base sequence and the length of the duplex and the proportion of modified nucleotides in the nucleic acid.
  • nucleic acid consisting only of unmodified deoxyribo- or ribonucleotides binds both DNA and RNA with approximately the same affinity
  • affinity of a nucleic acid having at least one modification according to the invention is very different for DNA and RNA.
  • Duplexes of modified nucleic acid and RNA have a significantly higher melting temperature and thus higher affinity for each other than duplexes with the same Nukleotidab sequence of modified nucleic acid with DNA or duplexes of two modified nucleic acids.
  • steps a) and c) of claim 1 an unmodified DNA or RNA that hybridizes to nucleic acids with complementary base sequence would be used as NA1 in the method according to the invention, this would bind RNA and DNA with the complementary sequence in approximately equal amounts.
  • a modified nucleic acid is used as NA1 in steps a) and c) in the method according to the invention, the binding of a complementary RNA to this in the nucleotide sequence is much more likely than the binding of a complementary to this DNA, if these two nucleic acid species as a mixture Hybridization are offered.
  • the affinity of a modified nucleic acid for a complementary further modified nucleic acid is about the same order of magnitude as the affinity of a modified nucleic acid for a DNA.
  • either DNA or another modified nucleic acid can be used as NA2 to form a duplex with NA1.
  • a duplex of modified nucleic acid and DNA or of modified nucleic acid with a further modified nucleic acid in step a) of claim 1 is accordingly formed.
  • this duplex of NA1 and NA2 in step b) of claim 1 is brought into contact with a ribonucleic acid NA3 having an at least partial sequence homology to NA2, which may be a modified nucleic acid or DNA
  • this ribonucleic acid is due to its higher affinity for the modified nucleic acid NA1 able to displace the DNA or modified nucleic acid (NA2) from the duplex with the NA1 and form a duplex even with the modified nucleic acid NA1.
  • NA2 DNA or modified nucleic acid
  • the melting temperature of a duplex of NAl and NA3 is therefore higher than the melting temperature of a duplex of NAl and NA2.
  • RNA Due to the higher affinity of RNA for the modified nucleic acid compared to DNA, it is not necessary to previously remove DNA of the same or similar sequence from this mixture for the detection of a specific RNA from a nucleic acid mixture by the method according to the invention, ie no purification of the RNA is made before it is detected.
  • RNA and DNA with comparable affinity would bind to the NA1 in step c) and both RNA and DNA would be detected.
  • the method according to the invention allows for direct detection of specific RNA, is thus much less expensive, faster and has no artifacts, as they can occur due to uneven efficiency in the purification or amplification of different ribonucleic acids.
  • the temperature in forming the duplex in step a) of claim 1 is selected so that the temperature is below the melting temperature of the duplex between the NA1 and the NA2.
  • the nucleic acids NA1 and NA2 are heated to a temperature which is at least about 10 ° C above the melting temperature of their duplex, and allowed to cool slowly to room temperature. Once the reaction temperature is below the melting temperature of the duplex, the duplex begins to form between NA1 and NA2.
  • the temperature in the hybridization in step c) of claim 1 can in principle be carried out at any temperature which is below the melting temperature of the duplex of NA1 and NA2. Under these temperature conditions, it is possible that the RNA (NA3) displaces the NA2 from the duplex with the modified nucleic acid (NA1). If the reaction temperature were above the melting temperature of the duplex from NA1 and NA2, the NA2 would also dehybridize to NA1 without the presence of a specific RNA from the duplex, since its affinity to NA1 would be too low.
  • step c) of claim 1 can choose the temperature in step c) of claim 1 in such a way that optimal conditions for the reaction time and the specificity of the detection of the RNA are present for him. While the reaction kinetics are higher at a hybridization temperature only a few degrees below the melting temperature of the NA1-NA2 duplex, and thus the detection of specific RNA is faster, the specificity of detecting the specific RNA is higher when the hybridization temperature is around is more than about 10 ° C below the melting temperature of the duplex between NA1 and NA2.
  • nucleic acids for example, the length and the GC content of the nucleic acid, the salt concentration and the type of salts in the hybridization buffer and the presence of further agents such as denaturing substances. Further influencing factors are known to the person skilled in the art.
  • Figure 1 shows the structural formula of a ribonucleotide with 2 '-0 - [((2R, 3S) -2,3,4-trihydroxy-butyl) oxymethyl] substituent.
  • Figure 2 shows the melting curve analysis of a duplex between modified nucleic acid and RNA or modified nucleic acid and DNA. The temperature is plotted on the x-axis, and the optical density at 260 nm on the y-axis. Details of the experiment are given in Example 3.
  • Figure 3 shows the displacement of DNA (light bars) by RNA (dark bars) from the duplex with modified nucleic acid.
  • the x-axis shows the relative amount of RNA (in%) relative to the duplex, and the y-axis the relative fluorescence.
  • Figure 4 shows the displacement of fluorescently labeled DNA from the duplex with modified nucleic acid by unlabelled RNA in the presence of non-sequence homologous RNA and DNA.
  • the relative amount of RNA (in%) in relation to the duplex amount is indicated on the x-axis, and the relative fluorescence on the y-axis.
  • Figure 5 shows the displacement of fluorescently labeled DNA from the duplex with modified nucleic acid by unlabeled RNA in the presence of a cell lysate from human cell culture cells.
  • the relative amount of RNA (in%) in relation to the duplex amount is indicated on the x-axis, and the relative fluorescence on the y-axis.
  • Figure 6 shows the displacement of fluorescently labeled DNA from the duplex with modified nucleic acid labeled with a quencher by unlabeled RNA.
  • the figure shows the fluorescence of the released DNA.
  • the fluorescence amount of the free DNA was defined as 100%, and the fluorescent amount at which the entire DNA was present in the duplex with modified nucleic acid was 0%.
  • the x-axis shows the relative amount of RNA (in%) relative to the duplex, and the y-axis the relative fluorescence.
  • Figure 7 shows the detection of sequence-homologous RNA.
  • non-sequence-homologous RNA was used for the light bars, but not for the dark bars.
  • On the x Axis is the relative amount of RNA (in%) expressed in relation to the duplex, and the relative fluorescence on the y-axis.
  • Figure 8 shows the detection of sequence-homologous RNA.
  • the light bars additionally used cell lysate from human cell culture cells, but not at the dark bars.
  • the x-axis shows the relative amount of RNA (in%) relative to the duplex, and the y-axis the relative fluorescence.
  • Figure 9 shows the detection of long sequence homologous RNA.
  • the relative amount of RNA (in%) in relation to the duplex amount is indicated on the x-axis, and the relative fluorescence on the y-axis.
  • SeqA 5 -GUUGCAUCAGAUACdT (in the case of modified nucleic acid and RNA)
  • SeqB 5-GUAUCUGAUGCAACdT (in the case of modified nucleic acid and RNA)
  • SeqB 5 -GTATCTGATGCAACT (in the case of DNA)
  • SeqX 5 -TGCCAACCCCGAGAAGAAATG (in the case of DNA)
  • Paramagnetic microparticles with surface streptavidin functionalization were buffered in 50 ⁇ M buffer 1 (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.01% BSA).
  • buffer 1 10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.01% BSA.
  • Biotinylated oligonucleotides of the sequence SEQB added in two-fold excess relative to the binding sites on the particles and incubated for 25 min with shaking - for coupling to these microparticles 5 'were.
  • two washing steps were carried out with 500 ⁇ each of the buffer 1. Subsequently, the final uptake of the particles in 50 ⁇ buffer 1 was carried out.
  • ribonucleic acids modified with (DL) -C 4 were used on the 2 ' sugar residue.
  • complementary DNA oligonucleotides were added and hybridized.
  • These DNA oligonucleotides were labeled at the 5 ' end with the fluorescent dye FAM (6-FAM).
  • FAM fluorescent dye FAM
  • the duplex of modified oligonucleotide and labeled DNA oligonucleotide was designed with exclusion of light for 60 min at room temperature and shaking. It was then washed twice with 500 ⁇ M buffer 1 and the particles until their Use under the exclusion of light. In this state, the particles can be stored for several days before being used for the detection of specific ribonucleic acids.
  • the modified nucleic acid and liquid phase DNA duplex was hybridized in Buffer 1 (see Example 1).
  • DNA oligonucleotides with the sequence SeqA were used, which carried the fluorescence dye Alexa 532 at the 3 ' end and (DL) -C 4 ribonucleotides, which at the 5 ' end a black hole quencher BHQ1 (Biosearch Technologies Inc., Novato) carried so that no fluorescence was detectable after forming the duplex.
  • the modified nucleic acid and DNA duplex was stored under exclusion of light until used. In this state, the duplex can be stored for several days before being used for the detection of specific ribonucleic acids.
  • duplexes Two nucleic acid strands with complementary base sequence are below the melting temperature as duplex. Since single-stranded nucleic acids absorb more UV light of wavelength 260 nm than double-stranded nucleic acids of the same sequence, a UV melting peak at 260 nm can be recorded.
  • duplexes were formed consisting of modified nucleic acid of the sequence SeqA and DNA of the sequence SeqB, modified nucleic acid of the sequence SeqA and RNA of the sequence SeqB, modified nucleic acid of the sequence SeqB and DNA of the sequence SeqA and modified nucleic acid of the sequence SeqB and RNA of the Sequence SeqA. When modified ribonucleotides were used on the 2 ' sugar residue with (DL) -C 4 modified ribonucleotides.
  • the duplexes were transferred to a heatable cuvette (Beckmann) and absorbance at 260 nm was measured. Every 2 min, the temperature in this cuvette was up-regulated by 1 ° C by means of a heating pump (Julabo).
  • the course of the absorption curves in Figure 2 shows that the melting temperature of the duplexes was at different temperatures, depending on whether a complex of modified nucleic acid with DNA or with RNA was present. While the melting temperature of a complex of modified nucleic acid with DNA was 37 ° C, the melting temperature of a complex of modified nucleic acid with RNA was significantly higher at 54 ° C.
  • the binding of RNA with the 2 'sugar residue with (DL) -C 4 modified RNA has a significantly greater than the binding of DNA.
  • a duplex consisting of modified nucleic acid and DNA was prepared and coupled to a solid phase.
  • the sequence used was SeqB as modified nucleic acid, SeqA for the DNA.
  • the DNA was labeled at its 5 'end with the fluorescent dye FAM (6-FAM).
  • RNA of the sequence SeqA was added to this duplex at different concentrations, which at its 5 ' end was labeled with the fluorescent dye hex (6-carboxy-2 ' , 4, 4 ' , 5 ' , 7, 7 ' - hexachlorofluorescein-succinimidyl ester). was marked.
  • Example 5 After hybridization and washing, the microparticles were taken up in Buffer 1 (see Example 1) and the fluorescence on the particles measured in a Stratagene 3005p.
  • the detected FAM fluorescence is from DNA hybridized to the modified nucleic acid on the microparticles.
  • the detected hex fluorescence originates from RNA, which displaced the DNA from oligonucleotides modified with (DL) -C 4 at the 2 'sugar residue.
  • the test result is shown in FIG. 3 and shows a clear displacement of the DNA by the RNA from the duplex with modified nucleic acid.
  • Example 5 Example 5:
  • RNA 500 ng of total RNA and 50 ng of genomic DNA, which had each been isolated from human cell culture cells (Jurkat) with the aid of the RNeasy or DNeasy kit (QIAGEN), were added to the duplex of modified nucleic acid and DNA for hybridization added.
  • BLAST Basic Local Alignment Search Tool
  • BLAST Basic Local Alignment Search Tool
  • Example 4 The procedure was as described in Example 4. In addition, the lysate of approximately 500,000 human cell culture cells (Jurkat) obtained by mechanical lysis was added to the duplex of modified nucleic acid and DNA. The amount of RNA that was to displace the DNA from the duplex was added in different concentrations relative to the modified nucleic acid duplex with DNA. After washing, the fluorescence decrease of the DNA of the sequence SeqA from the duplex was measured as a function of the amount of RNA used of the sequence SeqA. The measurement was carried out as in Example 5 using FacsCalibur (Becton Dickinson). Figure 5 shows the result of this experiment.
  • FacsCalibur Becton Dickinson
  • Example 2 The procedure was as outlined in Example 2.
  • buffer 1 a duplex was formed consisting of SeqA DNA labeled with Alexa 532 at the 3 'end and SeqB modified nucleic acid separated at the 5' end by the Black Hole Quencher BHQ 1 (Biosearch Technologies ) was marked. Unlabeled RNA of the sequence SeqA in different concentrations was added to this duplex. Subsequently, the released Alexa 532 fluorescence was measured in a Stratagene 3005p. As long as the DNA is present in duplex with modified nucleic acid, the Alexa 532 fluorescence is deleted by the Black Hole Quencher BHQ 1.
  • RNA Displacement of DNA from the duplex with modified nucleic acid by RNA in the liquid phase in the simultaneous presence of further nucleic acids
  • the procedure was as described in Example 7, except that this time worked with the sequences SeqX for RNA and DNA and SeqY for modified nucleic acid.
  • 10 ⁇ l of a total RNA preparation from E. coli obtained with the aid of RNeasy (QIAGEN) were added to one part of the reaction mixtures.
  • Example 10 The procedure was as described in Example 8, except that this time instead of E. coli total RNA, the cell lysate of about 500,000 human cell culture cells (Jurkat), which were obtained by mechanical lysis, was used in the hybridization approach.
  • Figure 8 shows that the fluorescence profiles are almost identical, the components of a cell lysate such as proteins, other nucleic acids and sugars, therefore, have neither an effect on the specificity nor on the sensitivity of the test system.
  • Example 10 Example 10:
  • RNA oligonucleotides were detected using the method according to the invention. However, this method is not only suitable for short RNA oligonucleotides, also RNA that corresponds in length to the length of a typical mRNA can be detected using this method. The procedure was as described in Example 4, but the following nucleic acids were used for this experiment:
  • RNA In vitro transcribed 860 nucleotides long artificial RNA that contained the open reading frame of the human RANTES gene, a member of the interleukin-8 superfamily of cytokines.

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Abstract

La présente invention concerne un procédé pour mettre en évidence des acides ribonucléiques par expulsion d'un acide nucléique hors d'un duplexe se composant d'un acide nucléique modifié au niveau d'un radical sucre et d'un autre acide nucléique ou acide désoxyribonucléique modifié au niveau d'un radical sucre, et remplacement par l'acide ribonucléique à mettre en évidence, et détection de l'événement de remplacement.
PCT/EP2011/060622 2010-06-25 2011-06-24 Procédé pour mettre en évidence des acides ribonucléiques Ceased WO2011161239A1 (fr)

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DE102010025161A DE102010025161A1 (de) 2010-06-25 2010-06-25 Verfahren zum Nachweis von Ribonukleinsäuren
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2539354A4 (fr) * 2010-02-08 2013-08-07 Univ Pennsylvania Molécules d'acide nucléique codant pour rantes, compositions les comprenant et procédés les utilisant
US9034313B2 (en) 2010-02-08 2015-05-19 The Trustees Of The University Of Pennsylvania Nucleic acid molecules encoding rantes, and compositions comprising and methods of using the same

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