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. 2007;35(13):4495-502.
doi: 10.1093/nar/gkm418. Epub 2007 Jun 21.

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Saïd Abes  1 , John J Turner, Gabriela D Ivanova, David Owen, Donna Williams, Andrey Arzumanov, Philippe Clair, Michael J Gait, Bernard Lebleu
Affiliations

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Saïd Abes et al. Nucleic Acids Res. 2007.

Erratum in

  • Nucleic Acids Res. 2007;35(21):7396

Abstract

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5-10 microM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 muM concentration of the R6Pen-PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen-PNA705 structure-function relationship have also been evaluated.

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Figures

Figure 1.
Figure 1.
Comparison of splice correction efficiencies by various CPP–PNA705 conjugates. HeLa pLuc705 cells were incubated for 4 h in OptiMEM in the absence (control), in the presence of 1 µM PNA705 alone, or in the presence of 1 µM CPP–PNA705 conjugates. Luciferase expression was quantified 20 h later and was expressed as RLU per microgram protein. Each experiment was made in triplicate and error bars (standard deviations) are indicated.
Figure 2.
Figure 2.
Splice correction specificity. HeLa pLuc705 cells were incubated for 4 h in OptiMEM in the absence (control) of correcting PNA, in the presence of the stably linked R6Pen–PNA705 splice correcting conjugate, or in the presence of its scrambled version at the indicated concentrations. Luciferase expression was quantified 20 h later and was expressed as RLU per microgram protein. Each experiment was made in triplicate and error bars (standard deviations) are indicated.
Figure 3.
Figure 3.
The effect of chloroquine on splice correction. HeLa pLuc705 cells were incubated for 4 h in OptiMEM with R6Pen–PNA705 correcting conjugates at the indicated concentrations in the absence (white bars) or in the presence (grey bars) of 100 µM chloroquine. Luciferase expression was quantified 20 h later and was expressed as RLU per microgram protein. Each experiment was made in triplicate and error bars (standard deviations) are indicated.
Figure 4.
Figure 4.
The effect of CPP–PNA705 linker stability on splice correction. HeLa pLuc705 cells were incubated for 4 h in OptiMEM in the absence (control) or in the presence of R6Pen–PNA705 conjugates at the indicated concentrations. The R6Pen and PNA705 moieties were conjugated by a stable thioacetyl or by a reducible disulfide linker. Luciferase expression was quantified 20 h later and was expressed as RLU per µg protein. Each experiment was made in triplicate and error bars (standard deviations) are indicated.
Figure 5.
Figure 5.
The effect of the number of arginine residues on splice correction. HeLa pLuc705 cells were incubated for 4 h in OptiMEM in the absence (control) or in the presence of RzPen–PNA705 conjugates (with z = 0, 3, 6 or 9) at the indicated concentrations. Luciferase expression was quantified 20 h later and was expressed as RLU per microgram protein. Each experiment was made in triplicate and error bars (standard deviations) are indicated.
Figure 6.
Figure 6.
The effect of a W→L Penetratin mutation on splice correction. HeLa pLuc705 cells were incubated for 4 h in OptiMEM in the absence (control) or in the presence of CPP–PNA705 conjugates at the indicated concentrations. Luciferase expression was quantified 20 h later and was expressed as RLU per microgram protein. Each experiment was made in triplicate and error bars (standard deviations) are indicated.
Figure 7.
Figure 7.
RT-PCR analysis of splice correction. (A) HeLa pLuc705 cells were incubated for 4 h in OptiMEM in the absence (control), in the presence of 1 µM PNA705 alone or in the presence of 1 µM CPP–PNA705 conjugates. Total RNA was extracted 20 h later and amplified by RT-PCR. PCR products from incorrectly (268 bp) and correctly (142 bp) spliced luciferase pre-mRNA were analysed on a 2% agarose gel. Lane 1: control, Lane 2: PNA705–alone, Lane 3: Pen-ss–PNA705, Lane 4: R6Pen-s-s–PNA705, Lane 5: R6Pen-s-s–scrambled PNA705, Lane 6: R6Pen(W–L)-s-s–PNA705. (B) Dose dependencies of splice correction using 1 µg of total RNA extracted, amplified by RT-PCR and analyses as in (a). Lane 1: control of untreated cells, Lanes 2–6: cells treated with 0.25, 0.5, 1, 2 or 4 µM R6Pen-s-s–PNA705 respectively and Lanes 7–11: cells treated with 0.25, 0.5, 1, 2 or 4 µM stably linked R6Pen–PNA705, respectively.
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