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1.
Nucleic Acids Res ; 39(20): 9047-59, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21795380

RESUMO

A general procedure, based on a new activated alkyne linker, for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition ('click' reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into 'clickable' azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.


Assuntos
Química Click/métodos , Oligonucleotídeos/química , Peptídeos/química , Cobre/química , Organofosfonatos/química
2.
Nucleic Acids Res ; 37(6): 1925-35, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19208638

RESUMO

Accessing the nucleus through the surrounding membrane poses one of the major obstacles for therapeutic molecules large enough to be excluded due to nuclear pore size limits. In some therapeutic applications the large size of some nucleic acids, like plasmid DNA, hampers their access to the nuclear compartment. However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit. We report on the synthesis and possible applications of a natural RNA 5'-end nuclear localization signal composed of a 2,2,7-trimethylguanosine cap (m(3)G-CAP). The cap is found in the small nuclear RNAs that are constitutive part of the small nuclear ribonucleoprotein complexes involved in nuclear splicing. We demonstrate the use of the m(3)G signal as an adaptor that can be attached to different oligonucleotides, thereby conferring nuclear targeting capabilities with capacity to transport large-size cargo molecules. The synthetic capping of oligos interfering with splicing may have immediate clinical applications.


Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Guanosina/análogos & derivados , Oligorribonucleotídeos/química , Análogos de Capuz de RNA/química , RNA Nuclear Pequeno/química , Animais , Linhagem Celular , Guanosina/química , Humanos , Sinais de Localização Nuclear , Oligonucleotídeos Antissenso/química , Oligorribonucleotídeos/metabolismo , Oócitos/metabolismo , Análogos de Capuz de RNA/síntese química , Splicing de RNA , Estreptavidina/metabolismo , Transfecção , Xenopus
3.
Nucleic Acids Res ; 37(9): 2867-81, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19282453

RESUMO

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Estabilidade de RNA , RNA Interferente Pequeno/sangue , RNA Interferente Pequeno/toxicidade , Complexo de Inativação Induzido por RNA/metabolismo
4.
J Am Chem Soc ; 132(26): 8984-90, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20545354

RESUMO

DNA-cleaving restriction enzymes are well-known tools in biomedical and biotechnological research. There are, however, no corresponding enzymes known for RNA cleavage. There has been an ongoing development of artificial ribonucleases, including some attempts at sequence selectivity. However, so far these systems have displayed modest rates of cleavage, and in most cases, the cleaver has been used in excess or in stoichiometric amounts. In the current work, we present PNA-based systems (PNAzymes) that carry a Cu(II)-2,9-dimethylphenanthroline group and that act as site and sequence specific RNases. The general basis for the systems is that the target is cleaved at a nonbase paired region (RNA bulge) which is formed in the substrate upon binding of the PNAzyme. With this copper based system, cleavage takes place at virtually only one site and with a half-life of down to 30 min under stoichiometric conditions. Efficient turnover of RNA-substrate is shown with a 100-fold excess of substrate, thus, demonstrating true enzyme behavior. In addition, alteration of the sequence in the RNA bulge or a mismatch in the base-pairing region leads to substantial decreases in rate showing both kinetic resolution and binding discrimination in the substrate selectivity. The selectivity is further demonstrated by the substrates, with two potential cleavage sites differing in only one base, are cleaved only at the site that either does not have a mismatch or is kinetically preferred. We suggest that these systems can serve as a basis for construction of RNA restriction enzymes for in vitro manipulations.


Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Ribonucleases/metabolismo , Sequência de Bases , Oligorribonucleotídeos/química , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Compostos Organometálicos/química , Ácidos Nucleicos Peptídicos/genética
6.
Nucleic Acids Symp Ser (Oxf) ; (52): 27-8, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776236

RESUMO

The Bioplex technology allows the hybridization of functional entities to various forms of nucleic acids by the use of synthetic nucleic acid analogs. Such supramolecular assemblies can be made in a predetermined fashion and can confer new properties. The Zorro technology is based on a novel construct generated to simultaneously bind to both DNA strands. Such compounds may have gene silencing activity.


Assuntos
Nanoestruturas/química , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , DNA/química , Inativação Gênica , Hibridização de Ácido Nucleico , Ácidos Nucleicos/metabolismo , Plasmídeos/química
7.
Nucleic Acids Symp Ser (Oxf) ; (51): 149-50, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18029630

RESUMO

An efficient and selective synthesis of 2'-O-alkyl modified adenosine nucleosides carrying either a triple bond function or an azido group is reported. These nucleosides are thus armed for performing 1,3 dipolar cycloadditions (click chemistry)(1). Derivatives suitable for further conversion into different building blocks are reported.


Assuntos
Adenosina/análogos & derivados , Nucleotídeos de Adenina/química , Adenosina/síntese química , Adenosina/química , Oligonucleotídeos/síntese química , Oligonucleotídeos/química
8.
J Am Chem Soc ; 129(26): 8362-79, 2007 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-17552524

RESUMO

Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range 1H-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 degrees C and 1.5 degrees C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA ( approximately 85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.


Assuntos
Timidina/análogos & derivados , Timidina/química , Sequência de Bases , Carboidratos/química , Humanos , Estrutura Molecular , Desnaturação de Ácido Nucleico , Oligonucleotídeos , Oligonucleotídeos Antissenso/química , Compostos Organofosforados/química , Ribonucleases/química , Ribonucleases/metabolismo , Soro/química
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