IDT miRNA Inhibitors

miRNA(MicroRNA)?

 
* Small non-coding RNAs
* Post-transcriptional gene regulations
* Cellular differentiation, cell death, ׸ cell metabolism ſ ۿ
* Precursor miRNA(pre-miRNA) 60-120 nucleotides
* Mature miRNA: precursor miRNA ߿ Ǵ 21-23 nucleotide
* pre-miRNA sequence larger primary transcript κ ̰ single pre-miRNA Ǵ 2 ̻ pre-miRNAs ִ.
* Following transcription, pre-miRNAs RNase III enzyme DROSHA ؼ stem-loop structure
* Stem-look pre-miRNA κ ͼ DICER/RISC complex mature miRNA ǰ regulatory ˴ϴ.
 

 

 

miRNA inhibitor?

 
MicroRNA (miRNA) Inhibitors mature miRNA پ ϴ Ѵ.
 

 

 

IDT miRNA Inhibitors

 
IDT miRNA Inhibitors oligonucleotides Ǵ ó ZEN modifications ִ 2'-O-methyl residues Ǿ ֽϴ.
Incorporating 2'-O-methyl residues ߰Ǹ endonuclease degradation ׼ ְ RNA targets binding affinity ݴϴ.  ݸ ZEN modification exonuclease degradation ְ binding affinity ִ մϴ.
 
 
Example:
 
5 nmol IDT miRNA Inhibitor
 
5'- mU/ZEN/mC mAmAmC mAmUmC mAmGmU mCmUmG mAmUmA mAmGmC mU/3ZEN/ -3'
 

 

 

IDT miRNA Inhibitor Ư¡

 
* In vitro ſ nanomolar 󵵷ε ϰ miRNA ȿ
* Knock down ϴ target ؼ specificity
* miRNA ϴ
* Nontoxic to mammalian cells 
* ֹ
 
IDT miRNA Inhibitors ֹ ϱ:
 
miRNA mature sequences ˻ , sequence miRNA inhibitor ֹâ
-> ڵ ֹ
 
miRBase : the miRNA database mature sequence ˻ Ʈ

 

 

IDT miRNA Inhibitors

 
MicroRNA (miRNA) Inhibitors are steric blocking oligonucleotides that hybridize to mature miRNAs, inhibiting their function. IDT miRNA Inhibitors are oligonucleotides comprised of 2'-O-methyl residues with ZEN modifications at or near the ends. Incorporating 2'-O-methyl residues confers resistance to endonuclease degradation and increases binding affinity to RNA targets, while the ZEN modification blocks exonuclease degradation and further increases binding affinity.
 

 

 

Features

 
* High potency provides effective inhibition of miRNA function in vitro at low nanomolar concentrations
* High specificity to ensure that the desired target is knocked down
* Uniform design for all miRNAs provides simplicity
* Nontoxic to mammalian cells
* Easy ordering
 
To order IDT miRNA Inhibitors, simply copy the mature sequences for the miRNAs from miRBase [1–4], the miRNA database, into the miRNA Inhibitor ordering tool.
 
Sequences for suggested controls for experiments in human, mouse, and rat cells are provided under the Support tab. To order, copy the sequences and paste them into the ordering tool.
 
Product Name Price ҿ
5 nm IDT miRNA Inhibitors          283,000 3-4
20 nm IDT miRNA Inhibitors          853,000 3-4
250 nm IDT miRNA Inhibitors       2,373,000 5-7
250 nm IDT miRNA Inhibitors (IE HPLC)       2,753,000 8-12
 
 
The standard method for inhibiting microRNA (miRNA) function is by steric blocking, using an oligonucleotide that is perfectly complementary to the mature miRNA target. These inhibitors form a duplex with the miRNA guide strand and prevent the miRNA from binding to its intended target.
 
For effective miRNA inhibition, the binding affinity between the oligo inhibitor and the miRNA must be significantly higher than that between miRNA guide strand and passenger strand. The miRNA inhibitor must be capable of binding to the miRNA guide strand either in single-stranded form or when bound to an Argonaute protein in the miRNA-induced silencing complex (miRISC). The inhibitor should also be capable of displacing the natural passenger strand in double-stranded miRNA.
 
Adding a 2 modification to ribose sugars in the RNA backbone can increase Tm and confer resistance to endonucleases [1]. 2-O-Methyl RNA (2OMe) is a naturally-occurring, nontoxic nucleic acid with a high binding affinity for RNA that provides the required resistance to mammalian endonucleases. Adding ZEN modifications to the termini of 2OMe-modified oligonucleotides further increases the binding affinity of the miRNA inhibitors and confers resistance to exonucleases [1].
 

References

 
Lennox, KA, Owczarzy R, et al. (2013) Improved performance of anti-miRNA oligonucleotides using a novel non-nucleotide modifier. Mol Ther Nucleic Acids, 2:e117.
 

Further Reading

 
Lennox KA, Behlke MA. (2011) Chemical modification and design of anti-miRNA oligonucleotides. Gene Ther, 18:1111–1120.
Lennox KA, Behlke MA. (2010) A direct comparison of anti-microRNA oligonucleotide potency. Pharm. Res, 27(9):1788–1799.

Resuspension and Storage of IDT miRNA Inhibitors

 
1. Centrifuge tubes before opening. Some of the product may have been dislodged during shipping.
2. Resuspend miRNA Inhibitors in the appropriate volume of IDTE, pH 8 or TE buffer indicated below to obtain the desired concentration:
 
Product
Volume for 100 µM*
5 nmol IDT miRNA Inhbitor 50 µL
20 nmol IDT miRNA Inhibitor 200 µL
 
3. Further dilutions of IDT miRNA Inhibitors can be made using IDTE, pH 8 or TE buffer.
4. Store resuspended IDT miRNA Inhibitors at –20C for up to 24 months.
 

Transfection of IDT miRNA Inhibitors Into Cultured Cells

 
Successful miRNA modulation experiments require very high transfection efficiency of the miRNA inhibitors into the cells. Typically, miRNA inhibitors are transfected in by similar methods to siRNAs, using cationic lipids or by electroporation. We routinely use Lipofectamine 2000 for use in established cell lines because it works well in a variety of cell lines. Transfection into primary or more difficult to transfect cells needs to be established before using.
 
In order to ensure specificity, we recommend testing the cells with various miRNA inhibitors and use the lowest possible amount that results in the desired phenotype. As miRNA function is based on recognition of a seed region rather than complete homology between miRNA and target, a single miRNA can regulate tens to hundreds of genes whose sequences do not share exact complementarity with the miRNA. Therefore, inhibition of a single miRNA will affect the expression of many genes.
 

Where to Find miRNA Sequences

 
Mature miRNA sequences can be found in the miRNA database, miRBase [1–4]. Just copy the mature sequence for each miRNA and paste it into the IDT miRNA Inhibitor ordering tool.
 

Controls

 
We recommend using the following positive and negative controls for your miRNA modulation experiments.
 

Positive Controls

 
miRNAs are expressed at various levels in different cell types; therefore, it can be difficult to find a good positive control. Ensure that the positive control that you select is expressed at sufficiently high levels to enable measurement of response.
A good positive control is miR-21-5p, which is conserved in many species and expressed at high levels in HeLa cells. Protein modulation can be confirmed by measuring expression of endogenous miR-21 targets, such as PTEN [5] and PDCD4 [5,6,7,8], or using a reporter assay.
 
Species
Mature miR-21-5p Sequence*
(copy and paste into IDT miRNA Inhibitor ordering tool)
Human, Mouse, Rat uagcuuaucagacugauguuga
 
 

Negative Controls

 
A good negative control should be inert and not modulate any genes in the system under study. This is difficult to achieve; however, we propose 2 negative control sequences that we have used throughout our product development and validation process, and which we have found to work very well in vitro and in vivo.
 
Control
Sequence
(copy and paste into IDT miRNA Inhibitor ordering tool)
NC1 Negative Control (human) ucguuaaucggcuauaauacgc
NC5 Negative Control (mouse) accauauugcgcguauagucgc

 

References

 
Kozomara A, Griffiths-Jones S (2011) miRBase: Integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res, 39(Database Issue):D152–157.
Griffiths-Jones S, Saini HK, et al. (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res, 36(Database Issue):D154–158.
Griffiths-Jones S, Grocock RJ, et al. (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res, 34(Database Issue):D140–144.
Griffiths-Jones S (2004) The microRNA Registry. Nucleic Acids Res, 32(Database Issue):D109–D111.
Yang CH, Yue J, et al. (2011) MicroRNA miR-21 regulates the metastatic behavior of B16 melanoma cells. J Biol Chem, 286(45):39172–39178.
Frankel LB, Christoffersen NR, et al. (2008) Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem, 283(2):1026–1033.
Lu Z, Liu M, et al. (2008) MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene. Oncogene, 27(31):4373–4379.
Yao Q, Xu H, et al. (2009) MicroRNA-21 promotes cell proliferation and down-regulates the expression of programmed cell death 4 (PDCD4) in HeLa cervical carcinoma cells. Biochem Biophys Res Commun, 388(3):539–542.
The success of miRNA inhibition experiments is dependent on several factors, the most important of which are potency, specificity, stability, and toxicity. High potency ensures that only small doses are required to produce the desired phenotype, also reducing toxicity of the administered compound. miRNA inhibitors that are specific to their target reduce the incidence of off-target effects, ensuring that any observed phenotypes are the result of the effect on the target under investigation. Stability and low toxicity are essential for in vivo experiments.
The figures below demonstrate the high potency, specificity, stability, and extremely low toxicity of IDT miRNA Inhibitors compared to other modified oligonucleotides.

Modification Key:

Name
Sequence1
IDT miRNA Inhibitors UzC A A C A U C A G U C U G A U A A G C U Az
DNA t c a a c a t c a g t c t g a t a a g c t a
2Ome U C A A C A U C A G U C U G A U A A G C U A
2OMe 3PS Ends U*C*A*A C A U C A G U C U G A U A A G*C*U*A
2OMe 3inZEN U C A A C A U C A G U C U G A U A A G C UzA
2OMe 5inZEN UzC A A C A U C A G U C U G A U A A G C U A
2OMe 3inC3 U C A A C A U C A G U C U G A U A A G C U+A
2OMe 5inC3 U+C A A C A U C A G U C U G A U A A G C U A
2OMe 5inC3, 3inC3 (2OMe 2XC3) U+C A A C A U C A G U C U G A U A A G C U+A
DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c*t*G*a*t*A*a*g*C*t*a
2OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*U*G*A*U*A*A*G*C*U*A
 
1 Lowercase = DNA; Uppercase = RNA; Red = LNA base; * = phosphorothioate linkage; z = ZEN insertion; + = C3 spacer.

 

High Potency

high potency.jpg
 
 
 
Figure 1. IDT miRNA Inhibitors Exhibit High Potency. Oligonucleotides designed to target miR-21 were transfected at 0.3–30 nM in the HeLa cells expressing the psiCHECK-miR-21 plasmid using Lipofectamine RNAiMAX transfection reagent. The cells were lysed after 24 hr and analyzed for luciferase activity. Results were normalized with the internal firefly luciferase (FLuc) control and are shown as fold change in Renilla luciferase (RLuc) compared with the lipid reagent control, which was set at 1. Tm values for the various oligos are shown above the respective profiles.

 

High Specificity

 
High Specificity.jpg
 
 
 
Mutant Type
miR-21 IDT miRNA Inhibitor Sequences (5' to 3')*
Wild Type (0 MUT) C A A C A U C A G U C U G A U A A G C U
 1 MUT C A A C A U C A G U C A G A U A A G C U
2 MUT C A A C C U C A G U C A G A U A A G C U
3 MUT C A A C C U C A G U C A G A U A A C C U
 
* Mutated bases are indicated by bold, red notation.
Figure 2. IDT miRNA Inhibitors are Highly Specific. Different miR-21 inhibitors (n=5) were synthesized, complementary to wild-type miR-21 or containing 1, 2, or 3 mismatched—0 MUT, 1 MUT, 2 MUT, and 3 MUT, respectively. The inhibitors were transfected into HeLa cells expressing the psiCHECK-miR-21 plasmid. After 24 hr, the cells were lysed and analyzed for luciferase activity. Values were normalized with internal firefly luciferase (FLuc) control and reported as a fold change in Renilla luciferase (RLuc) compared with lipid reagent control, which was set at 1.

 

High Stability

 
High Stability.jpg
 
Figure 3. IDT miRNA Inhibitors are Resistant to Nucleases. 1.1 nmol of each oligonucleotide was incubated in (A) 10% FBS, high exonuclease environment; or (B) 20% mouse liver protein extract, high endonuclease environment, for the indicated lengths of time. Each reaction was analyzed on a denaturing polyacrylamide gel stained with methylene blue.

 

Low Toxicity

 
Low Toxicity.jpg
 
Figure 4. IDT miRNA Inhibitors Exhibit Low Toxicity to Cells. Modified oligionucleotides corresponding to a nontargeting negative control sequence, NC1, unrelated to any known human miRNA were transfected into HeLa cells at concentrations of 10, 30, and 100 nM to investigate and compare toxicity. The cells were visualized by phase contrast microscopy (10X magnification).
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