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Sequence-specific gripNA™* probes provide researchers with the very latest technology available for gene silencing. This powerful new tool utilizes the high-affinity binding and sequence specificity of engineered peptide nucleic acids (PNAs) to improve gene targeting and reduce non-specific interactions. Active Motif’s custom gripNA synthesis service will accelerate your research by providing you with a unique alternative to the more traditional techniques used for gene silencing. Note: because each gripNAs order is a custom synthesis of the sequence you choose, to order you must complete and fax a copy of the gripNA Fax Order Form (Documents tab).
gripNA advantages
- Unsurpassed sequence specificity
- Resistant to nuclease degradation
- Easy delivery with Chariot™ II
- Flexible synthesis modifications
- Highly soluble
* Covered under U.S. Patent No. 6,962,906. Purchase includes the right to use for basic research only. Other-use licenses available, please contact your local Technical Services dept.
gripNA oligonucleotides cause their gene silencing effects by binding to the targeted mRNA molecule. Thus, the gripNA sequence must be complementary, or antisense, to the gene to be silenced. It should also be specific for the targeted gene. In order to design a gripNA probe that effectively and specifically silences a single target, researchers must have an accurate sequence of the gene that they wish to silence. GenBank or similar sources should be used to ensure that any selected gripNA sequence is targeted specifically to the gene of interest.
Blocking Translation
The following guidelines generally lead to gripNA probes that are effective for blocking translation of their targeted mRNAs. However, as research into gripNA continues, these parameters will be further refined and optimized.
1. Target the AUG
As ribosomes assemble at the AUG translational start site, Active Motif has focused its efforts on designing and testing gripNAs that bind in this region. Effective gene silencing has been shown both in vitro and in mammalian cells when gripNAs were targeted in the region from the 5´-cap structure to about 15 bases 3´ of the AUG translational start site of the spliced mRNA. To date, our experiments have not shown a statistically significant difference between the gene silencing effects of gripNAs that target different positions around the AUG. For example, 3 different gripNAs were synthesized to silence luciferase mRNA using an in vitro rabbit reticulocyte system. These were targeted so that the gripNA would bind either across or on the 5´ or 3´ end of the AUG in the luciferase mRNA:
5´-CAT-TTG-GAT-CCG-GGC-CCT-3´
5´-GTC-TTC-CAT-TTG-GAT-CCG-3´
5´-GTT-TTT-GGC GTC-TTC-CAT-3´
All 3 of these gripNAs were effective at silencing the luciferase. However, the secondary structures found in mRNA may have some effect on the relative efficacies of individual gripNA sequences. As new data becomes available, it will be posted here.
2. Minimize self-complementarity
Preferably, the sequence of your gripNA probe should not be able to form more than 4 contiguous intrastrand base pairings.
3. Sequence limitations.
Unlike other synthetic oligonucleotides, gripNAs have no sequence synthesis limitations that we have identified. gripNAs with a guanine content of 11 out of 18 bases (> 60%) have been made successfully. The solubility of gripNAs with high guanine content is also excellent due to the alternating negative charge on the gripNA backbone.
Blocking Nuclear Processing
The guidelines for effective blocking of nuclear processing events, particularly splicing, are essentially the same as those required for blocking translation. However, it should be noted that because some nuclear processing events occur rapidly after RNA transcription, gripNA probes will have only a brief time in which to interact with their targeted nuclear processing sequence before these sequences carry out their function. Effective blocking of such sequences may therefore require higher gripNA probe concentrations than would normally be required for blocking translation at the AUG translational start site.
Selection of Multiple Targets
To date, successful silencing has been achieved using the above method to select the target sequence, although the method is essentially random with respect to accounting for mRNA secondary structure. While gripNA silencing appears to be extremely effective by selecting a single target in the mRNA, it may be desirable to design and employ two independent gripNA probes to control for specificity of the silencing effect. It is as yet unknown if the targeting of a gene by two different gripNA duplexes is more effective than using a single gripNA probe. However, selecting multiple targets within your gene of interest will enable selection of the most efficient gripNA probe.
gripNA Oligo Length
gripNA synthesis is performed on a derivatized CPG support using an automated DNA synthesizer. In contrast to conventional phosphoramidite synthesis of DNA or RNA, gripNA synthesis utilizes phosphotriester coupling of dimer synthons. The gripNA dimer building blocks are composed of alternating HypNA-pPNA molecules. Due to the nature of the gripNA synthesis chemistry the total number of residues in any given sequence must be an even number, i.e. an 18-mer, 16-mer, 14-mer, etc. gripNA oligos are offered at a maximum length of 18 bases, which was found to be the optimal length to observe maximum antisense effect. While shorter gripNAs can be ordered, gripNA pricing is independent of length. We recommend ordering 18-mers, which should be more efficient.
3´-End Modifications
With each gripNA ordered, you can have one of three optional modifications added to the 3´-end of your sequence, if desired. These include:
- Addition of a 3´-primary amine.
- Addition of a 3´-Biotin.
- Addition of a 3´-Fluorescein.
The default for our gripNA Fax Order Form is for no 3´-end modification. Please be sure to check the appropriate box if you would like your gripNA sequence to contain one of the optional modifications.
