Delivery of antisense peptide nucleic acids to cells by conjugation with small arginine-rich cell-penetrating peptide (R/W)9.

Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine.

Flavin conjugates for delivery of peptide nucleic acids.

Oligonucleotides and their analogues, such as peptide nucleic acids (PNAs), can be used in chemical strategies to artificially control gene expression. Inefficient cellular uptake and inappropriate cellular localization still remain obstacles in biological applications, however, especially for PNAs. Here we demonstrate that conjugation of PNAs to flavin resulted in efficient internalization into cells through an endocytic pathway.

A steric blocker of translation elongation inhibits IGF-1R expression and cell transformation.

The insulin-like growth factor 1 receptor (IGF-1R) is involved in transformation, survival, mitogenesis and differentiation. It is overexpressed in many tumors and a validated target for anticancer therapy. In cell-free systems, polypyrimidic peptide nucleic acids (PNAs) can form triplex-like structures with messenger RNAs and halt the ribosomal machinery during the translation elongation.

Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA.

DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells.

WEBSAGE: a web tool for visual analysis of differentially expressed human SAGE tags.

The serial analysis of gene expression (SAGE) is a powerful method to compare gene expression of mRNA populations. To provide quantitative expression levels on a genome-wide scale, the Cancer Genome Anatomy Project (CGAP) uses SAGE. Over 7 million SAGE tags, from 171 human cell types have been assembled.

Oligonucleotides and oligonucleotide conjugates: a new approach for cancer treatment.

In this account we summarise recent studies on oligonucleotides and oligonucleotide derivatives and their utilisation in antigene, antisense and decoy approaches, with particular attention to peptide nucleic acids, locked nucleic acids and oligonucleotide conjugates, the most promising compounds in this field.

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis.

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets.

Screening of human bladder carcinomas for the presence of Ha-ras codon 12 mutation.

Contradictory results were obtained from previous studies aiming at defining the frequency of Ha-ras codon 12 mutations in bladder tumors. Differences in the sensitivities of the methods used could account for this discrepancy. In this study, we reevaluated the frequency of Ha-ras codon 12 mutations in a series of 87 human bladder tumors using a combination of two different methods.

Antisense PNA tridecamers targeted to the coding region of Ha-ras mRNA arrest polypeptide chain elongation.

We have previously described the rational design of mutation-selective antisense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. In order to further improve the biological efficacy of these unmodified oligonucleotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphoramidate linkage (PN). We show that PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro.

Analysis of antisense oligonucleotides by on-capillary isotachophoresis and capillary polymer sieving electrophoresis.

An attempt was made to evaluate the stability of an antisense oligonucleotide against nucleases present in HBL 100ras cells. To detect nanomolar concentrations of the oligonucleotide, a sensitive detection system was required. A combination of capillary electrophoresis/laser-induced fluorescence (CE-LIF) with fluorescence derivatization did not improve the sensitivity significantly and also resulted in loss of separation of the derivatized sample.

Antisense properties of end-modified oligonucleotides targeted to Ha-ras oncogene.

Phosphodiester oligodeoxyribonucleotides linked to an intercalating agent or a dodecanol tail or both complementary to the 12th codon region of Ha-ras mRNA were compared with the unmodified oligonucleotides of the same size and sequence with respect to their ability to induce RNaseH cleavage and antisense activity in cell culture. The hydrophobic tail not only protected the oligonucleotide from nucleases but also enhanced RNase H cleavage of the target. Oligonucleotides carrying both an acridine and a dodecanol substituent inhibited the proliferation of HBL100ras1 cells (human mammary cells stably transformed with the T24 Ha-ras gene carrying a G-->T point mutation in codon 12) at a 20-fold to 30-fold lower concentration than unmodified ones.

Transplatin-modified oligo(2'-O-methyl ribonucleotide)s: a new tool for selective modulation of gene expression.

In the reaction between trans-diamminedichloroplatinum(II) and single-stranded oligo(2'-O-methyl ribonucleotide)s containing the sequence GNG (N being a nucleotide residue), the 1,3-trans-{Pt-(NH3)2[GNG]} cross-links are formed. The 1,3-intrastrand cross-links are inert within the single-stranded oligonucleotides. By contrast, they rearrange into interstrand cross-links when the platinated oligonucleotides are paired with their complementary RNA strands.

Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-ras mRNA in human cells.

Antisense oligodeoxynucleotides targeted to Ha-ras mRNA have been designed to discriminate between the codon 12-mutated oncogene and the normal proto-oncogene. An in vitro assay using two different sources of RNase H (rabbit reticulocyte lysates and nuclear extract from HeLa cells) was used to characterize oligonucleotide binding to normal and mutated Ha-ras mRNA. Short oligonucleotides (12- or 13mers) centered on the mutation had a very high discriminatory efficiency.

Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets.

Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G<-->U) in codon 12 of the Ha-ras mRNA. DNA and RNA fragments, 27 nucleotides in length, and containing the complementary sequence of the 12mers, were used to compare the reactivity of the activatable dodecamers (cleavage of the target by the phenanthroline-12mer conjugates; photo-induced cross-linking of psoralen-12mer conjugates to the target). The reactivity of the RNA with the dodecamers was weaker than that of the DNA target.

Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice.

Ras oncogenes owe their transforming properties to single point mutations in the sequence coding for the active site of the p21 protein. These mutations lead to changes in cellular proliferation and induce tumorigenic properties. Point mutations represent a well-defined target for antisense oligonucleotides that can specifically suppress the translation of the targeted mutant mRNA.

Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake.

Oligonucleotides can be adsorbed on polyisohexylcyanoacrylate nanoparticles in the presence of hydrophobic quarternary ammonium salts. Oligonucleotides bound to nanoparticles are protected from nuclease attack both in buffer and in cell culture media. Cellular uptake of oligonucleotides is increased when they are adsorbed onto nanoparticles as a result of the capture of nanoparticles by an endocytic phagocytic pathway.

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation.

A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence.

Oligonucleotide-directed triplex formation within upstream regulatory sequences is envisioned as a potential tool for gene inhibition. However, this approach requires that triple helix-forming oligonucleotides are chemically modified, so that the triplex is stable under physiological conditions. Here, we have compared several chemical modifications of an oligonucleotide, targeted to a natural 15-base pair homopyrimidine.

Triple-helix formation by alpha oligodeoxynucleotides and alpha oligodeoxynucleotide-intercalator conjugates.

Base-pair sequences in double-stranded DNA can be recognized by homopyrimidine oligonucleotides that bind to the major groove at homopurine.homopyrimidine sequences thereby forming a local triple helix. To make oligodeoxynucleotides resistant to nucleases, we replaced the natural (beta) anomers of the nucleotide units by the synthetic (alpha) anomers.

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells.

Inhibition of restriction endonuclease cleavage via triple helix formation by homopyrimidine oligonucleotides.

A 17-mer homopyrimidine oligonucleotide was designed to bind to the major groove of SV40 DNA at a 17 base pair homopurine-homopyrimidine sequence via Hoogsteen base pairing. This sequence contains the recognition site for the class II-S restriction enzyme Ksp 632-I. The oligonucleotide was shown to inhibit enzymatic cleavage under conditions that allow for triple helix formation.

Sequence-specific intercalating agents: intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide-intercalator conjugates.

An acridine derivative was covalently linked to the 5' end of a homopyrimidine oligonucleotide. Specific binding to a homopurine-homopyrimidine sequence of duplex DNA was demonstrated by spectroscopic studies (absorption and fluorescence) and by "footprinting" experiments with a copper phenanthroline chelate used as an artificial nuclease. A hypochromism and a red shift of the acridine absorption were observed.