Ambion - Harnessing the power of RNAI

RNAi has become a useful tool in target identification and validation. It provides critical functional data about alleged drug targets that is proving invaluable in drug discovery pipelines.

RNA interference (RNAi) is the biological process of inducing mRNA degradation and subsequent gene silencing in a sequence specific manner by the introduction of dsRNA. In mammalian cells and organisms, artificial induction of the natural RNAi pathway by delivery of short interfering RNAs (siRNAs; 21 bp dsRNA molecules complementary to a particular mRNA) is now routinely used to silence specific genes in cultured cells. The power and utility of RNAi for specifically silencing the expression of any gene for which sequence is available has driven its incredibly rapid adoption as a tool for reverse genetics in eukaryotic systems; in the last seven years, RNAi has become the cornerstone of many research programmes.


Accelerating discovery

Screening experiments with sets or libraries of siRNAs are increasingly popular for drug target identification and validation. RNAi-based target identification experiments require high quality libraries of siRNAs corresponding to either a subset of genes or the entire set of genes in the genome. Because of the large percentage of drugs targeting kinases, siRNA libraries targeting each gene within the human kinome are of particular interest, as are siRNA libraries targeting the druggable genome (these and other siRNA libraries are available from Ambion, now an Applied Biosystems Business).


Additional requirements for siRNA screening experiments include robust siRNA delivery methods and adequate experimental controls. When these components are combined with cell-based and other high-quality assays, a wealth of information can be gleaned about potential drug targets in a short amount of time.


Intelligent siRNA design

siRNA design is a critical factor for determining the efficacy of the siRNA as a silencing reagent. Therefore, siRNA library choice should be made cautiously to ensure use of effective, specific and potent siRNAs. Design algorithms have been developed that improve siRNA efficacy and help limit off-target effects. However, the most important attribute of any algorithm is ultimately its performance as tested experimentally, which is why early on Ambion embarked on a programme with Cenix BioScience to empirically test siRNAs to hundreds of endogenously expressed human targets. The resulting algorithm, which is used to design Ambion's Silencer® siRNAs, results in a high percentage of siRNAs that elicit strong silencing at low concentrations.


Although intelligent siRNA design algorithms improve siRNA efficacy, the majority of siRNAs in most libraries have not been experimentally validated. The use of three or four siRNAs per target is generally accepted to be the best approach for dealing with this situation. Employing three or more distinct individual siRNAs significantly decreases both false positive and false negative rates as compared with screening with pools of siRNAs. However, these pools can be used when assay costs are prohibitively high. To accommodate both approaches, Ambion provides siRNA libraries featuring three or four individual siRNAs per target, as well as pools of those same siRNAs.


Success stories

The value of siRNA libraries for functional genomics has already been demonstrated in several key published reports. In one, Marino Zerial and colleagues performed high throughput RNAi screening with an Ambion siRNA library to identify kinases involved in clathrin- and caveolae/raft-mediated endocytosis (Nature 2005; 436:78-86). Specific sets of kinases were found to regulate each endocytic route. The large number of kinases involved suggests that signalling functions such as those controlling cell adhesion, growth, and proliferation are built into the endocytosis pathway. Several other studies have been published, with hundreds more in progress.


The availability of genome-wide and custom siRNA libraries from Ambion and others has provided a powerful reverse genetics tool for use in human and rodent cells. RNAi experiments dramatically enhance the current toolbox of expression profiling and other functional genomics experiments to discern whether identified drug targets are of high value. Although RNAi alone will not solve every aspect of target identification and validation, RNAi does provide critical functional data about putative drug targets that is proving invaluable in drug discovery pipelines.


Company profile

Ambion is a market leader in the development and supply of innovative RNA-based life science research and molecular biology products. Ambion specialises in the development of products for stabilising, synthesising, handling, isolating, storing, detecting and measuring RNA.


MicroRNA: Small regulators with global impact

MicroRNAs (miRNAs) are an important class of small RNA molecules that are expressed in eukaryotes. Although the first miRNA was identified in a genetic screen in 1993 1, it was not until 2001 that the breadth of the miRNA gene class was recognised with the cloning and sequencing of more than 100 miRNAs from worms, humans and mice 1-4. These evolutionarily conserved, non-coding RNA molecules regulate translation of mRNAs through base pair interactions 5. With some exceptions, worm and human miRNAs inhibit the translation of specific mRNAs, while plant miRNAs induce mRNA degradation.

In cells, the ~22 nt mature, functional miRNAs are produced by a recently described process (see Figure 1). A primary transcript (pri-mRNA) that can be more than 1000nt in length is first produced in the nucleus. An RNA hairpin (precursor miRNA, pre-miR) comprising ~80nt, including the mature miRNA, results from digestion of pri-miRNA by the double-strand-specific ribonuclease, Drosha 6. The resulting pre-miRNA is transported to the cytoplasm via a process that involves Exportin-5 7,8. The pre-miRNA is further digested by Dicer 6,8 to generate a short, partially double-stranded RNA wherein one strand is the mature miRNA. The mature miRNA is taken up by a protein complex that is similar to, if not identical to, the RNA-induced silencing complex (RISC) that supports RNA interference (RNAi) 9,10 and the bound complex functions to regulate translation.

As a unique class of small RNA molecules, miRNAs require special tools for accurate and sensitive analysis. Ambion's scientists have developed a portfolio of products that provide a complete solution to rapidly identifying and characterising miRNA. The most recent addition is the Pre-miR miRNA precursor molecules and Anti-miR miRNA inhibitors for the regulation of miRNA expression in cells.

MicroRNA functional analysis can be performed with protocols that are similar to standard genes. Up-regulation of the miRNAs can be conducted to identify gain-of-function phenotypes; down-regulation or inhibition can be conducted to identify loss-of-function phenotypes. The combination of up- and down-regulation can be used to identify genes that are regulated by specific miRNAs, as well as to identify cellular processes that are affected by specific miRNAs. Key applications include:

  • miRNA target site validation
  • miRNA target site identification
  • Screening for miRNAs that regulate the expression of a gene
  • Screening for miRNAs that affect a cellular process
  • The pMIR-REPORT™ miRNA expression reporter vector provides accurate, quantitative, in-cell measurement of miRNA expression. This miRNA-validated reporter system contains Luciferase, under the control of a mammalian promoter/terminator system, with a miRNA-cloning region following the termination of translation sequence. This vector is designed for the cloning of specific, putative sequence binding sites for miRNA. The pMIR-REPORT miRNA expression reporter vector can also be used as a screening tool, in which random or non-random sequences are inserted into the reporter to identify miRNA targets, or coupled with a library of miRNA that can be used to screen a target sequence with all known miRNAs.
  • The use of these new tools can help lead advances in therapeutics as well as basic research into the functioning of development and protein expression. Look to Ambion as the complete miRNA solution provider.

Footnotes

  • 1.Lee R, Feinbaum R, and Ambros V (1993) The heterochronic gene lin-4 of C. elegans encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843-54
  • 2. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294: 853-8
  • 3. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294: 858-62
  • 4. Lee RC and Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science294: 862-4
  • 5. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281-97
  • 6. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S, Kim VN (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425: 415-9
  • 7. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear export of microRNA precursors.Science 303(5654): 95-8
  • 8. Yi R, Qin Y, Macara IG, Cullen BR (2004) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17(24): 3011-6
  • 9. Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science297(5589): 2056-60
  • 10. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, Dreyfuss G (2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs.Genes Dev 16(6): 720-8

 

Products and Services

Contact Details

Ambion
www.ambion.com

Make An Enquiry
First Name

Last Name

Email Address

Enquiry


Privacy Policy
We have updated our privacy policy. In the latest update it explains what cookies are and how we use them on our site. To learn more about cookies and their benefits, please view our privacy policy. Please be aware that parts of this site will not function correctly if you disable cookies. By continuing to use this site, you consent to our use of cookies in accordance with our privacy policy unless you have disabled them.