The present invention relates to a microRNA antisense PNA, a composition containing the same, and a method for using and evaluating the same, and more specifically, to a microRNA antisense PNA capable of inhibiting the activity or function of microRNA, also known as siRNA (small interfering RNA), a composition for inhibiting the activity or function of microRNA comprising the same, a method for inhibiting the activity or function of microRNA using the same, and a method for evaluating the same.
In 1993, some genes were found in Caenorhabditis elegans to regulate its developmental stages, among which let-7 and lin-4 were identified as small RNA fragments not translated into protein (non-coding RNA). These RNAs were commonly known as stRNA (small temporal RNA) because they are expressed in a specific developmental stage to regulate development. MicroRNA is a single-stranded RNA molecule of 21-25 nucleotides, which regulates gene expression in eukaryotes. Specifically, it is known to bind to 3′ UTR (untranslated region) of mRNA for a specific gene to inhibit its translation. All the animal microRNAs studied heretofore decrease protein expression without affecting the level of mRNA for a specific gene.
MicroRNA is attached to RISC (RNA-induced silencing complex) to complementarily bind with a specific mRNA, but the center of microRNA remains mismatched, so it does not degrade mRNA, unlike conventional siRNAs. Unlike animal microRNAs, plant microRNAs perfectly match target mRNA to induce its degradation, which is referred to as “RNA interference.”
Several plant microRNAs are involved in the translational regulation like animal microRNAs. Another report presents evidences that microRNAs induce methylation of chromatin in yeasts, including animals and plants, and so are involved in the transcriptional inhibition. Some of microRNAs are highly conserved inter-specifically, suggesting that they might be involved in important biological phenomena.
MicroRNA is produced through a two-step process. First, primary miRNA (pri-miRNA) is converted to pre-miRNA having step-loop structure of 70-90 nucleotides by an enzyme of RNase III type, Drosha, in a nucleus. Then, pre-miRNA is transported into cytoplasm and cleaved by an enzyme, Dicer, finally to form mature microRNA of 21-25 nucleotides. Recently, many researches have shown that microRNA plays an important role in cancer cells and stem cells as well as in cell proliferation, cell differentiation, apoptosis and control of lipid metabolism. However, many of microRNA functions remain unknown, for which studies are actively ongoing.
Researches on microRNA have been performed by investigating expression patterns by reporter gene analysis, microarray, northern blotting, and real-time polymerase chain reaction, or using antisense DNA or RNA (Boutla A, Delidakis C, and Tabler M. (2003) Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes. Nucleic Acids Res. 31(17): 4973-4980). Recently, 2′-O-Me RNA having higher binding affinity to RNA owing to its methyl group and having higher stability against nucleases than RNA itself, or 2′-O-methoxy oligonucleotide having even higher binding affinity than 2′-O-Me oligonucleotide have been synthesized and used as antisense against microRNA (Weiler J, Hunziker J and Hall J. (2006) Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease? Gene Therapy 13:496-502). To solve the drawback of DNA that it is readily degradable by nucleases, an oligonucleotide prepared by mixing LNA (Locked Nucleic Acid) and DNA has been used.
This oligonucleotide is known to have higher sensitivity and selectivity than DNA (Cha J A, Krichevsky A M and Kosik K S. (2005) microRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 65:6029-6033). In addition, RNA antagomir having the attached cholesterol has also been synthesized to investigate functions of microRNA (Krutzfeldt J, Rajewsky N, Braich R, Rajeev K G, Tuschl T, Manoharan M and Stoffel M. (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438:685-689). They are antisense against microRNA that interrupt functions of microRNA, and so are extremely important for studies on functions of microRNA.
As described above, to overcome the drawbacks of DNA and RNA, such chemically modified oligonucleotides as LNA and 2-O-methyl oligonucleotide have been used but they are still degraded by endo- or exo-nucleases in cells, or have decreased specificity or cause cytotoxicity due to their modified structures (Crinelli R, Bianchi M, Gentilini L, and Magnani M. (2002) Design and characterization of decoy oligonucleotides containing locked nucleic acids. Nucleic Acids Res. 30(11):2435-2443; Hutvágner G, Simard M J, Mello C C, Zamore P D. Hutvágner G, Simard M J, Mello C C, and Zamore P D. (2004) Sequence-specific inhibition of small RNA function. PLoS Biol. 2(4):E98). Therefore, there has been an eager demand on more efficient antisense oligonucleotides to interrupt functions of microRNA.
PNA (peptide nucleic acid) is a polymeric compound having the similar structure to DNA, which is a nucleic acid in the form of protein, capable of binding with DNA and RNA (Nielsen P E, Buchardt O, Egholm M, Berg R H, U.S. Pat. No. 5,539,082, Peptide nucleic acids). The backbone of PNA has the structure of polypeptide (
To overcome the above described problems of the prior arts, the present inventors have conducted extensive studies to construct an antisense capable of specifically binding with microRNA, thereby inhibiting activity or function thereof, by using PNA having the above mentioned advantages. As a result, the present inventors developed an antisense PNA having superior and sustainable effect in cells, as compared with the conventional antisense DNA and RNA.
It is therefore an object of the present invention to provide a microRNA antisense PNA complementarily binding with microRNA, thereby inhibiting the activity or function thereof.
It is another object of the present invention to provide a composition for inhibiting activity or function of microRNA, containing the microRNA antisense PNA as an active ingredient.
It is still another object of the present invention to provide a method for inhibiting activity or function of microRNA by using the microRNA antisense PNA.
It is further still another object of the present invention to provide a method for evaluating the effectiveness of the microRNA antisense PNA.
It is a first aspect of the present invention to provide a microRNA antisense PNA, which consists of 10 to 25 nucleotides, and is capable of complementarily binding with microRNA, thereby inhibiting activity or function thereof.
It is a second aspect of the present invention to provide a composition for inhibiting activity or function of microRNA, containing the microRNA antisense PNA as an active ingredient.
It is a third aspect of the present invention to provide a method for inhibiting activity or function of microRNA, comprising the step of introducing into cells the microRNA antisense PNA.
It is a fourth aspect of the present invention to provide a method for evaluating the effectiveness of microRNA antisense PNA, comprising the step of measuring and comparing the expressions of microRNA, in presence and absence of the microRNA antisense PNA.
The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:
Hereinafter, the present invention will be described in detail.
The present invention relates to a microRNA antisense PNA complementarily binding with microRNA, thereby inhibiting the activity or function of microRNA. The antisense PNA of the present invention consists of 10 to 25 nucleotides, particularly, 15 nucleotides. It will be appreciated that short PNA of 10 to 14mer, long PNA of 16 to 25mer, and PNA containing a part of 5′ and 3′ regions, corresponding to seed region, of microRNA, can also sufficiently function as microRNA antisense, and thus, all of these PNAs fall within the scope of the present invention. In this invention, the microRNA includes any kind of microRNA, without limitation; for example, miR16, miR221, miR222, miR31, miR24, miR21, miR181a, miR23a, miR19b, miR20a, let7g, miR34a, miR30a, miR146a, miR130a, miR155, miR373, miR122a, miR145, miR191, and miR193b, but not limited thereto. The nucleotide sequence of antisense PNA of the present invention is not specifically limited, as long as it can complementarily bind to microRNA to inhibit the activity or function thereof. For example, the antisense PNA consists of one of the nucleotide sequences represented by SEQ. ID Nos. 1 to 82, preferably by SEQ. ID Nos. 1 to 4, 7, 11, 19, 21, 23, 26, 29 to 32, 34 to 36, 44, 47, 48, 51, 52, 54, 55, 59, 63, 65, 66, 68 to 80, and 82, as set forth in the following Table 1, but not limited thereto.
The PNA of the present invention can be introduced into cells, as it is, to inhibit the activity or function of microRNA. However, since PNA is electrically neutral, cellular lipids might interrupt its intracellular introduction. To overcome such problem, many studies have been conducted on its intracellular delivery system. As a result, various approaches have been known, for example, induction of cellular uptake by attaching cell penetrating protein (CPP) (Pooga M, Hallbrink M, Zorko M, and Langel U. (1998) Cell penetration by transportan.
Faseb J. 12: 67-77), Insulin-like growth factor I-receptor (Basu S, and Wickstrom E. (1997) Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insulin-like growth factor 1 for increased cellular uptake. Bioconjug. Chem. 8: 481-488), or asialoglycoprotein receptor (Zhang X, Simmons C G, and Corey D R. (2001) Liver cell specific targeting of peptide nucleic acid oligomers. Bioorg Med. Chem. Lett. 11: 1269-1271); or by using electroporation (Wang G, Xu X, Pace B, Dean D A, Glazer P M, Chan P, Goodman S R, and Shokolenko I. (1999) Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression. Nucleic Acids Res. 27(13):2806-2813) or liposome (Faruqi A F, Egholm M, and Glazer P M. (1998) Peptide nucleic acid-targeted mutagenesis of a chromosomal gene in mouse cells. Proc. Natl. Acad. Sci. USA. 95(4):1398-1403). CPP is generally classified into the following three groups. First group is Tat peptide consisting of amino acids in the position of 49 to 57 of Tat protein, which is involved in the transcription of HIV-I causing acquired immunodeficiency syndrome. Second group is penetratin, a peptide derived from homeodomain, which has been first discovered in homeodomain of antennapedia, homeoprotein of Drosophila. Third group is membrane translocating sequence (MTS) or signal sequence based peptide. Examples of peptide, which can be efficiently used for intracellular introduction of PNA, are shown in the following Table 2. Any one of them or one derived therefrom can be linked to PNA and used in the present invention.
DF-c [CFDWKTC] T
99mTc chelating peptide
D[GGGGCSKC] (D: D type)
In addition, other known or novel peptides, effectively used for PNA, can be linked to PNA and used. Those peptide can be directly linked with PNA, but is preferably linked with PNA via an appropriate linker, such as 8-amino-3,6-dioxaoctanoic acid linker (O-linker), E-linker represented by the following formula 1, and X-linker represented by the following formula 2.
In addition to the above enumerated peptides, polyarginine, penetratin, and α-aminoacridine are known to enhance intracellular introduction of PNA. So, any of them can be linked with PNA in this invention. In one embodiment, modified Tat peptide, particularly, R peptide consisting of the amino acid sequence represented by SEQ. ID No: 83 (RRRQRRKKR), or K peptide consisting of the amino acid sequence represented by SEQ. ID No: 84 (KFFKFFKFFK) may be used to enhance intracellular introduction of PNA.
In this invention, the microRNA antisense PNA can be introduced into cells, thereby inhibiting the activity or function of microRNA. The microRNA antisense PNA can be introduced into cells by using cationic lipid, such as Lipofectamine 2000 (Invitrogen). In addition, other methods, such as electroporation or use of liposome, can be applied for intracellular introduction of the antisense PNA, and in such case, PNA with or without linked peptide may be used to act as microRNA antisense.
Further, the present invention provides a composition for inhibiting the activity or function of microRNA, containing the microRNA antisense PNA as an active ingredient. For example, the composition of the present invention can be used as a preventive or therapeutic agent for microRNA mediated diseases. The effective dose of the microRNA antisense PNA can be suitably determined by considering age, sex, health condition, type and severity of disease, etc. For example, for an adult, it may be administered at 0.1˜200 mg per time, and once, twice or three times a day. For administration, any conventional gene therapy, for example, ex vivo or in vivo therapy, may be used without limitation.
In this invention, the effectiveness of the antisense PNA can be evaluated by measuring and comparing the expressions of microRNA, in presence and absence of the antisense PNA. For measuring expressions, any conventional methods known in the art can be used. For example, reporter gene, Northern blot, microarray, real time PCR, in vivo/in situ hybridization, or labeling can be used. In one embodiment, in case of measuring expressions by using report gene, the effectiveness of microRNA antisense PNA can be evaluated by the method comprising the following steps:
(a) mixing the antisense PNA with a control vector containing a reporter gene (ex: Renilla luciferase), not a target microRNA binding sequence, and an experimental vector containing another reporter gene (ex: firefly luciferase) and the target microRNA binding sequence, and then, introducing the mixture into cells; and,
(b) measuring and comparing the expressions from the reporter genes in the control vector and the experimental vector of step (a).
The experimental vector can be constructed by introducing the target microRNA binding sequence into a vector containing the reporter gene (ex: firefly luciferase).
Hereinafter, the present invention will be described in more detail with reference to the following examples, which are provided only for the better understanding of the invention, and should not be construed to limit the scope of invention in any manner.
To investigate the antisense effect of PNA against microRNA, the antisense PNAs having the complementary sequences with specific target microRNAs, i.e. miR16, miR221, miR222, miR31, miR24, miR21, miR181a, miR23a, miR19b, miR20a, let7g, miR34a, miR30a, miR146a, miR130a, miR155, miR373, miR122a, miR145, miR191, miR193b and miR802, were synthesized.
In general, microRNAs consist of 21 to 25 nucleotides, among which 2nd to 8th nucleotides are known as seed sequence.
PNAs having various sequences, for example, complementary with 1st to 15th, 2nd to 16th, or 3rd to 17th nucleotides of target microRNA, were synthesized so that they could complementarily bind with the target microRNA. Modified HIV-1 Tat peptide (R-peptide, RRRQRRKKR) was linked to those PNAs via O-linker. To evaluate the effect of the modified Tat peptide, antisense PNAs were also linked with K-peptide (KFFKFFKFFK), known to enhance intracellular introduction of PNA into E. coli, not into animal cells. The control PNAs (con-K, con-R and con-2R) having no antisense activity were also synthesized. The synthesized antisense PNAs and the control PNAs are shown in the following Table 3.
To evaluate function of the antisense PNA and effect of binding peptide thereon, HeLa cells were spread onto a 24 well plate at the density of 6×104 cells/well, and cultivated for 24 hours. The cells were transformed with pGL3-control vector (Promega) having firefly luciferase gene and the cloned miR16 binding sequence (see
Control PNAs (con-K and con-R) were also transformed in the above manner. Expressions of reporter genes were measured to evaluate the effectiveness of the antisense PNA.
The results are shown in
To compare the effects of antisense PNAs with and without the linked modified Tat peptide, HeLa cells were spread onto a 24 well plate at the density of 6×104 cells/well, and cultivated for 24 hours. The cells were transformed with pGL3-control vector (Promega) having firefly luciferase gene and the cloned miR16 binding sequence (see
The results are shown in
To investigate the effect of the antisense PNA against microRNA, an experimental vector containing miR16 binding sequence was used. For this, pGL3-control vector (Promega) containing firefly luciferase gene was used. To compare the level of transformation, the control vector (Promega) containing Renilla luciferase gene was used as well.
The experimental vector was constructed by inserting miR16 binding sequence into XbaI site in 3′ UTR of luciferase gene of pGL-3 control vector. The sequence of miR16 was determined with reference to miR Base Sequence Database (http://microRNA.sanger.ac.uk/sequences/) (Table 4).
The corresponding complementary DNA having the same length as the microRNA was synthesized to include XbaI site in 5′ and 3′ regions (Table 5), and then, cloned into pGL3-control vector.
To compare efficiencies of the conventional microRNA antisense and the PNA antisense, miRCURY™ LNA Knockdown probe (Exiqon) against miR16 and miRIDNA (Dharmacon) against miR16 were purchased, and their effects were compared at the concentration of 200 nM. For the antisense PNA, each 100 nM of 2 kinds (#1 and #7) of PNA, which had been shown to have high efficiency at the concentration of 200 nM, as shown in
HeLa cells were cultivated for 24 hours, and transformed with the experimental vector containing miR16 binding sequence and the control vector containing Renilla luciferase gene, together with the microRNA antisense PNA, miRCURY™ LNA Knockdown probe (Exiqon) against miR16, or miRIDNA (Dharmacon) against miR16, by using Lipofectamine 2000 (Invitrogen). To confirm the microRNA inhibitory effect, the control PNA (con-R), miRCURY™ LNA Knockdown probe (Exiqon) against miRNA181b and miRIDNA (Dharmacon) against miRNA181b having the sequences not complementary with that of miR16 were also transformed in the above manner. After the transformation, the cells were cultivated for 48 hours. Then, the expressions of firefly luciferase and Renilla luciferase were measured by using Dual luciferase assay system (Promega). The results are shown in
To investigate the effect of the antisense PNA against microRNA 16 at its various concentrations, HeLa cells were cultivated for 24 hours. The cells were transformed with the experimental vector containing the inserted miR16 binding sequence and the control vector containing Renilla luciferase gene, together with various concentrations (50, 100, 200 and 300 nM, respectively) of the antisense PNA (mixture of #1 and #7), by using Lipofectamine 2000 (Invitrogen). The control PNA (conR) was also transformed in the above manner. After the transformation, the cells were cultivated for 48 hours. Then, the expressions of firefly luciferase and Renilla luciferase were measured by using Dual luciferase assay system (Promega).
The results are shown in
To investigate the effect of the antisense PNA against microRNA 16 with the lapse of time, HeLa cells were cultivated for 24 hours. Then, the cells were transformed with the experimental vector containing the inserted miR16 binding sequence and the control vector containing Renilla luciferase gene, together with 200 nM of the antisense PNA against miR16 (mixture of 100 nM of miR16-1 and 100 nM of miR16-7) and 200 nM of miRCURY™ LNA Knockdown probe against miR16, by using Lipofectamine 2000 (Invitrogen). To confirm the effect of the microRNA inhibitory effect, the control PNA (con-R) and miRCURY™ LNA Knockdown probe (Exiqon) against miRNA181b having the nucleotide sequence not complementary with that of miR16 were also transformed in the above manner. After the transformation, the cells were cultivated for 24, 36 and 48 hours, respectively. Then, the expressions of firefly luciferase and Renilla luciferase were measured by using Dual luciferase assay system (Promega).
The results are shown in
To construct a vector having miR221 binding sequence, a modified pGL3-control vector was used. Specifically, a synthetic oligomer containing EcoRI restriction site in 5′ region and PstI restriction site in 3′ region was cloned into its EcoRI/PstI site (see Tables 6 and 7).
To compare the PNA antisense with the conventional antisense against microRNA, miRCURY™0 LNA Knockdown probe (Exiqon) was used as well. HeLa cells were cultivated for 24 hours, and transformed with the experimental vector containing miR221 binding sequence and the control vector containing Renilla luciferase gene, together with 200 nM of the antisense PNA against miR221 and 200 nM of miRCURY™ LNA Knockdown probe (Exiqon) against miR221, by using Lipofectamine 2000 (Invitrogen). To confirm the microRNA inhibitory effect, the control PNA (con-R) and miRCURY™ LNA Knockdown probe (Exiqon) against miRNA181b having the nucleotide sequence not complementary with that of miR221 were also transformed in the above manner. After the transformation, the cells were cultivated for 48 hours. Then, the expressions of firefly luciferase and Renilla luciferase were measured by using Dual luciferase assay system (Promega).
The results are shown in
To construct a vector having miR222 binding sequence, a modified pGL3-control vector was used. Specifically, a synthetic oligomer containing EcoRI restriction site in 5′ region and PstI restriction site in 3′ region was cloned into its EcoRI/PstI site (see Tables 8 and 9).
To compare the PNA antisense with the conventional antisense against microRNA, miRCURY™ LNA Knockdown probe (Exiqon) was used. HeLa cells were cultivated for 24 hours, and transformed with the experimental vector containing miR222 binding sequence and the control vector containing Renilla luciferase gene together with 200 nM of the antisense PNA against miR222 and 200 nM of miRCURY™ LNA Knockdown probe (Exiqon) against miR222, by using Lipofectamine 2000 (Invitrogen). To confirm the microRNA inhibitory effect, the control PNA (con-R) and miRCURY™ LNA Knockdown probe (Exiqon) against miRNA181b having the nucleotide sequence not complementary with that of miR222 were also transformed in the above manner. After the transformation, the cells were cultivated for 48 hours. Then, the expressions of firefly luciferase and Renilla luciferase were measured by using Dual luciferase assay system (Promega).
The results are shown in
Each DNA with the same length as and complementary with miR31, miR24, miR21, miR181a, miR23a, miR19b, miR20a, let7g, miR34a, miR30a, miR146a, miR130a, miR155, miR373, miR122a, miR145, miR191, miR193b and miR802 was cloned into pGL3-control vector, according the same procedures as described in Example 3.
HeLa cells were cultivated for 24 hours, and transformed with the experimental vector containing each microRNA binding sequence and the control vector containing Renilla luciferase gene, together with 200 nM of each microRNA antisense PNA, by using Lipofectamine 2000 (Invitrogen). To confirm the microRNA inhibitory effect, the control PNA (con-2R) having the nucleotide sequence complementary with none of the microRNAs was also transformed in the above manner. After the transformation, the cells were cultivated for 48 hours. Then, the expressions of firefly luciferase and Renilla luciferase were measured by using Dual luciferase assay system (Promega).
The results are shown in
The microRNA antisense PNA of the present invention, an artificially synthesized DNA analogue, which can complementarily bind with DNA or RNA with a higher strength, specificity and sensitivity than DNA or RNA itself, and has high stability against not only biological degradative enzymes, such as nucleases and proteases, but also physicochemical factors, such as pH and heat, shows higher and more sustained effect in cells, and can be stored for a longer period of time, than the conventional antisense DNA or RNA. The antisense PNA of the present invention could be applied in studies for functions of microRNA to understand the regulation of gene expression in eukaryotes, and for microRNA metabolic or functional defect mediated diseases, and used as novel therapeutic agents for such diseases.
SEQ. ID Nos. 1 to 82 show the nucleotide sequences of miRNA antisense PNAs;
SEQ. ID No. 83 shows the amino acid sequence of R peptide;
SEQ. ID No. 84 shows the amino acid sequence of K peptide;
SEQ. ID Nos. 85 and 86 show the nucleotide sequences of control PNAs;
SEQ. ID Nos. 87 to 89 show the nucleotide sequences of miRNAs; and
SEQ. ID Nos. 90 to 95 show the nucleotide sequences of miRNA target sequence cloning oligomers.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
Number | Date | Country | Kind |
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10-2007-0120459 | Nov 2007 | KR | national |
10-2008-0116856 | Nov 2008 | KR | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/KR08/06926 | 11/24/2008 | WO | 00 | 5/7/2010 |