RESUMEN
We evaluated the nuclear uptake of fluorescently labeled peptide nucleic acids and measured the binding of unlabeled peptide nucleic acids (PNAs) to the endogenous HER-2/neu promotor in digitonin-permeabilized SK-BR-3 cells. Fluorescently labeled PNAs readily enter the nucleus of digitonin-permeabilized cells, and binding to the chromosomal target sequence was detected with a bis-PNA. Nuclear uptake and target sequence binding were inhibited by N-ethylmaleimide (NEM) and GTPgammaS. We conclude that PNAs are transported into the nucleus through an energy-dependent process involving the nuclear pore complex.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Ácidos Nucleicos de Péptidos/farmacocinética , Secuencia de Bases , Línea Celular , Cromosomas/metabolismo , Colorantes Fluorescentes , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Poro Nuclear , Regiones Promotoras Genéticas , Receptor ErbB-2/genéticaRESUMEN
Peptide nucleic acids (PNAs) are promising tools for gene regulation. One of the challenges of using PNAs as gene regulators is the need to optimize the efficiency of interaction with critical sequences of DNA. To improve the efficiency of binding between PNAs and the HER-2/neu promoter, mono- and bis-pyrimidine-rich PNAs were conjugated to a nitrogen mustard at either the amino or carboxy terminus. Gel shift analysis demonstrated that conjugation to an alkylating agent slowed PNA binding and favored PNA:DNA:DNA triplex helix formation while preserving a high binding affinity. Sites of DNA alkylation were visualized by piperidine cleavage and showed PNA binding first by Hoogsteen bond formation with the target duplex to form a stable PNA:DNA:DNA triplex structure which is later converted to a PNA:DNA:PNA triple helix by strand invasion and Watson-Crick base pairing by a second PNA molecule. In this way, PNA-directed DNA alkylation was used to deduce the mode of PNA binding. Transient transfection experiments demonstrated that the PNA-nitrogen mustard conjugates suppressed HER-2/neu expression by up to 80%. In comparison with an unmodified mono-PNA or a bis-PNA, these results indicate that the covalent adducts stabilized PNA binding in cells and suggest that the conjugation of PNAs to nitrogen mustards is a robust strategy for developing antigene PNA oligonucleotides to prevent transcription.
Asunto(s)
Clorambucilo/farmacología , Compuestos de Mostaza Nitrogenada/farmacología , Ácidos Nucleicos de Péptidos/farmacología , Receptor ErbB-2/metabolismo , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Clorambucilo/química , ADN/química , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HeLa , Humanos , Compuestos de Mostaza Nitrogenada/química , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , Receptor ErbB-2/genética , TransfecciónRESUMEN
Artificial control of gene expression has great potential in the treatment of many human diseases, and peptide nucleic acids (PNAs) offer several potential advantages for silencing gene expression in mammalian cells. The pseudopeptide backbone of the PNA makes it resistant to enzymatic degradation, and PNAs bind complementary DNA and RNA with high affinity and specificity. PNAs are potentially leading agents for antigene and antisense therapeutics, but the application of PNAs in the in vivo setting is hampered by their poor intracellular delivery. This problem has been addressed by PNA conjugation to lipophilic moieties, peptides, and cell-specific receptor ligands. The biological activity of PNAs can also benefit from conjugation to DNA interactive compounds like intercalators and alkylators. Here we review the most interesting literature concerning PNA conjugation with small molecules, emphasizing synthetic approaches, properties and applications of the PNA conjugates.
Asunto(s)
Ácidos Nucleicos de Péptidos/química , Alquilantes/química , Colorantes Fluorescentes/química , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/síntesis químicaRESUMEN
Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.
Asunto(s)
Regulación de la Expresión Génica , Técnicas Genéticas , Regiones Promotoras Genéticas , Receptor ErbB-2/genética , Receptor ErbB-2/fisiología , Alquilantes/farmacología , Secuencia de Bases , Southern Blotting , Cationes , Línea Celular Tumoral , ADN/química , Cartilla de ADN/química , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Digitonina/farmacología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Mecloretamina/farmacología , Datos de Secuencia Molecular , Oligonucleótidos/química , Ácidos Nucleicos de Péptidos/química , Plásmidos/metabolismo , Unión Proteica , Receptor ErbB-2/metabolismo , TransfecciónRESUMEN
In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.