gripNAs™

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gripNA™ probes are a novel, negatively charged form of Peptide Nucleic Acids (PNAs) that have a number of properties that make them ideal for gene silencing. gripNAs combine high-affinity binding with much greater sequence specificity than that of other gene silencing reagents, such as siRNA and morpholinos.

PNAs are DNA analogs in which the nucleosides are attached to an N-(2-aminoethyl)glycine backbone instead of to deoxyribose, as in DNA. However, while traditional PNA molecules held great promise for gene silencing, poor water solubility and a tendency to self-aggregate limited their utility. Active Motif has overcome these shortcomings by developing negatively charged gripNAs^1-3 (Figure 1), which have superior solubility yet retain the PNA characteristics that are ideal for gene silencing.

 

Figure 1: Negatively charged gripNA.

gripNA probes are comprised of a backbone of alternating HypNA and pPNA monomers, with the bases attached through methylene carbonyl linkages.

Effective gene silencing

gripNAs have been shown to be highly effective in gene silencing experiments in both mammalian cells and in zebrafish. To illustrate, PNA and gripNA probes targeted against human cyclin B1 were delivered into HS-68 cells using Chariot II™. (Chariot II is a special formulation of Chariot, Active Motif’s novel protein delivery reagent, and is intended for use with gripNAs only. For delivery of proteins, peptides and antibodies, use the original Chariot.) Western blot analysis performed clearly indicates that cyclin B1 was silenced better by gripNAs than by PNAs (Figure 2).

Unsurpassed sequence specificity

A recurring problem of many gene silencing reagents is a lack of specificity. This can cause unintended phenotypes because these reagents may bind and silence more than just the intended target⁵. Experiments performed in zebrafish embryos by the Steve Farber lab (Thomas Jefferson University) demonstrate how this specificity can improve your results. While wild-type gripNA and morpholino probes were comparable in potency at silencing the chordin, uroD and no tail genes, the intentional inclusion of 2 and 4 base-pair mismatches in the morpholino probes caused non-specific phenotypic effects that were not triggered by comparable, mismatched gripNA probes⁶.

To demonstrate the high sequence specificity of gripNAs, in vitro mismatch discrimination experiments were performed. gripNA and DNA probes were annealed to a complementary oligonucleotide, as well as to oligonucleotides containing one- or two-base mismatches. After annealing, each duplex’s melting temperature (T_m) was determined. Table 1 shows that gripNAs are much more intolerant than DNA to mismatches. The higher T_m values indicate that gripNA/DNA duplexes are destabilized by mismatches much more than the DNA/DNA duplexes (especially when the mismatch is central in the sequence). In addition, the gripNA probe was unable to hybridize to several of the oligos with one mismatch and didn’t hybridize to any oligos with two mismatches. This clearly demonstrates gripNA's superior sequence specificity and emphasizes how using gripNA probes can minimize any non-specific gene interaction within the cell. Using gripNAs ensures your gene silencing experiment can be performed with confidence.

Table 1: Stringent binding specificity of gripNAs.

Identical 16-mer gripNA and DNA probes were synthesized with the sequence 5´-TGG-TCT-CAA-GTC-AGT-G-3´. These were annealed to a complementary DNA oligo (5´-CAC-TGA-CTT-GAG-ACC-A-3´) in Hybridization Buffer (20 mM Tris (pH 7.5), 500 mM NaCl, 10 mM MgCl₂). Each sample was heated to 90°C for 3 minutes, then cooled gradually to room temperature. The samples were then heated at a rate of 1°C per minute from 20°C to 100°C using a thermal control unit linked to a spectrophotometer. Changes in A₂₆₀ were recorded and a melting temperature (T_m) was calculated for the DNA/DNA and gripNA/DNA duplexes. The experiment was then repeated by hybridizing the gripNA and DNA probes to a series of DNA oligos containing one- or two-base mismatches (shown above, with the mismatches in bold & underlined). T_m values were measured for each DNA/DNA and gripNA/DNA duplex and a ΔT_m was calculated by subtracting the difference in the melting temperatures of the complementary and mismatched probes. Samples that are unable to form a stable duplex generate a “No T_m” value in this assay and no ΔT_m.

Simple, efficient gripNA delivery with Chariot II™

Delivery of inhibitory molecules into the cell is a key limitation for any gene silencing experiment. Classical delivery mechanisms such as microinjection and DNA/RNA transfection can be time consuming and inefficient. Their biggest drawback, however, is that these methods can induce cytotoxic responses within the target cells. Delivery of gripNAs with Chariot II is simple and provides high transfection efficiencies with minimal cytotoxicity(4).

To prove the effectiveness of Chariot II for gripNA delivery, fluorescently labeled gripNA was mixed with Chariot II and incubated for 30 min at 37°C, then overlaid onto cultured fibroblast cells for 1 hr (Figure 3). The results clearly show the high efficiency at which Chariot II is able to deliver gripNAs. Don’t waste time on microinjection and transfection when you can deliver your gripNAs by Chariot II.

 

Figure 3: Chariot II delivery of Fluorescein-tagged gripNA.

An 18-mer gripNA probe (1 µM) labeled on its 3´ end with Fluorescein was complexed with Chariot II in PBS and incubated for 30 minutes at 37°C, then overlaid onto cultured HS-68 for 1 hour. Cells were washed extensively prior to observation. (Data generously provided by Dr. L. Chaloin, Dr. M. Morris and Dr. G. Divita, Biophysics, CNRS, Montpellier, France.)

References

1. Efimov, V.A. et al. (1998) NAR 26: 566-575.
2. van der Laan et al. (1996) Tetrahedron Lett. 37: 7857-7860.
3. Efimov, V.A. et al. (1999) NAR 27: 4416-4426.
4. Morris, K.A. et al. (2003) Developmental Dynamics 228: 405-413.
5. Stein, C.A. (1999) Nature Biotech. 17: 209.
6. Urtishak, K.A. et al. (2004) Gene Therapy 11(9): 757-764.