RESUMEN
Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.
Asunto(s)
Imitación Molecular , Ácidos Nucleicos/química , Ácidos Nucleicos de Péptidos/química , Sulfatos/química , Línea Celular , Proteínas de Unión al ADN/química , Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , SolubilidadRESUMEN
Peptides isolated from natural fonts are the object of several studies aimed at finding new molecules possessing antibacterial activity. We focused our studies on peptides originally isolated from the Royal Jelly, the jelleins and on some analogs having a UV reporter at the N- or C-terminus. We found that jelleins are mainly active against gram-positive bacteria; interestingly, they act in synergy with peptides belonging to the family of temporins such as temporin A and temporin B against Staphylococcus aureus A170 and Listeria monocytogenes.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Ácidos Grasos/química , Péptidos/química , Péptidos/farmacología , Animales , Péptidos Catiónicos Antimicrobianos , Dicroismo Circular , Hemólisis/efectos de los fármacos , Listeria monocytogenes/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Proteínas/química , Proteínas/farmacología , Staphylococcus aureus/efectos de los fármacosRESUMEN
The discovery of siRNAs as the mediators of RNA interference has led to an increasing interest in their therapeutic applications. Chemical modifications are introduced into siRNAs to optimize the potency, the stability and the pharmacokinetic properties in vivo. Here, we synthesize and test the effects of RNA-3'-PNA chimeras on siRNA functioning and stability. We demonstrate that the chemical modifications are compatible with the siRNA machinery, because all the PNA-modified siRNAs can efficiently mediate specific gene silencing in mammalian cells. Furthermore, we find that the modification on the sense strand of siRNA results in an increased persistence of the activity, whereas modification on both strands results in enhanced nuclease resistance in serum.
RESUMEN
Peptide nucleic acids (PNAs) are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudo-peptide backbone. Among PNA-based molecules, PNA-DNA conjugates characterized by tracts of DNA bound to N and/or C terminus of PNA are very soluble in aqueous media, are able to recognize exclusively single strands of DNA and RNA in antiparallel fashion, activate RNAse H, bind to transcription factors and are more stable than DNA to nucleases degradation. Very little information is available on chimeras constituted of alternating monomers of PNA and DNA. In this article, we describe a simple fully automated strategy for the synthesis of 6-mer and 10-mer alternate PNA-DNA chimeras consisting of polythymine oligomers, stability assays in fetal calf serum, UV and CD studies of the single strand alternate chimeras and of alternate chimera/DNA and alternate chimera/RNA duplexes. Evidences supporting the formation of duplex hybrids were found. Furthermore, the ability of forming Hoogsteen base pairing with duplex DNA was investigated. Finally, we tested the ability of the PNA-DNA alternates in (a) interfering with reverse transcription of eukaryotic mRNA and (b) inhibiting DNA-protein interactions.
Asunto(s)
ADN/química , Ácidos Nucleicos de Péptidos/química , Animales , Emparejamiento Base/efectos de los fármacos , Sitios de Unión , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Dicroismo Circular/métodos , ADN/antagonistas & inhibidores , Ensayo de Cambio de Movilidad Electroforética/métodos , Células Eucariotas/química , Sangre Fetal/química , Humanos , Estructura Molecular , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/farmacología , Proteínas/antagonistas & inhibidores , ARN/química , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Espectrofotometría Ultravioleta/métodos , Timina/química , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
[structure: see text] A new and versatile on-line automated solid-phase approach to obtain cyclic PNA (I and III) and cyclic PNA-DNA chimeras (II) in highly pure form has been developed. Starting from a Tentagel matrix functionalized with a 3-chloro-4-hydroxyphenylacetic linker, the synthesis of representative, new cyclic molecules by standard peptide and phosphoramidite-based chemistry has been achieved.