RESUMO
Phosphorothioate (PS) group is a key component of a majority of FDA approved oligonucleotide drugs that increase stability to nucleases whilst maintaining interactions with many proteins, including RNase H in the case of antisense oligonucleotides (ASOs). At the same time, uniform PS modification increases nonspecific protein binding that can trigger toxicity and pro-inflammatory effects, so discovery and characterization of alternative phosphate mimics for RNA therapeutics is an actual task. Here we evaluated the effects of the introduction of several N-alkane sulfonyl phosphoramidate groups such as mesyl (methanesulfonyl) or busyl (1-butanesulfonyl) phosphoramidates into gapmer ASOs on the efficiency and pattern of RNase H cleavage, cellular uptake in vitro, and intracellular localization. Using Malat1 lncRNA as a target, we have identified patterns of mesyl or busyl modifications in the ASOs for optimal knockdown in vitro. Combination of the PSMA ligand-mediated delivery with optimized mesyl and busyl ASOs resulted in the efficient target depletion in the prostate cancer cells. Our study demonstrated that other N-alkanesulfonyl phosphoramidate groups apart from a known mesyl phosphoramidate can serve as an essential component of mixed backbone gapmer ASOs to reduce drawbacks of uniformly PS-modified gapmers, and deserve further investigation in RNA therapeutics.
RESUMO
Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21. A 22-nt anti-miR-21 mesyl phosphoramidate oligodeoxynucleotide specifically decreased the miR-21 level in melanoma B16 cells, induced apoptosis, reduced proliferation, and impeded migration of tumor cells, showing superiority over isosequential phosphorothioate oligodeoxynucleotide in the specificity of its biological effect. Lower overall toxicity compared with phosphorothioate and more efficient activation of RNase H are the key advantages of mesyl phosphoramidate oligonucleotides, which may represent a promising group of antisense therapeutic agents.
Assuntos
Amidas/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos/metabolismo , Fosfatos/metabolismo , Ácidos Fosfóricos/metabolismo , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , DNA/metabolismo , Melanoma Experimental , Camundongos , MicroRNAs/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismoRESUMO
Oligonucleotides carrying 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2'-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2'-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.
Assuntos
Etilenodiaminas , Oligonucleotídeos/síntese química , Aldeídos , Cisteína , Indicadores e Reagentes , Ácidos Nucleicos Peptídicos/síntese químicaRESUMO
Conjugates of amines or short peptides with oligonucleotides containing 5'-terminal carboxyl group were prepared by solid phase chemical synthesis. A correlation between the physicochemical parameters and retention times of the synthesized conjugates was established by ion-pair reversed-phase HPLC.
Assuntos
Química Orgânica/métodos , Cromatografia Líquida de Alta Pressão , Oligonucleotídeos/química , Aminas/química , Aminoácidos/química , Dióxido de Carbono/química , Ácidos Carboxílicos/química , Cromatografia Líquida de Alta Pressão/métodos , Oligonucleotídeos/síntese química , Peptídeos/químicaRESUMO
Combinations of the polyamine spermine and magnesium ions synergize to dramatically enhance cleavage of the hairpin ribozyme. Certain synthetic basic tripeptides stimulate hairpin cleavage significantly at limiting magnesium ion concentration, notably the tripeptide of L-diaminobutyric acid (Dab). Of a range of novel synthetic spermine-amino acid conjugates, L-Dab-spermine (but not D-Dab nor other amino acid conjugates) was more effective than spermine itself.
Assuntos
Aminoácidos/química , Magnésio/química , Peptídeos/química , RNA Catalítico/química , Espermina/química , Aminoácidos/metabolismo , Aminobutiratos/química , Bioquímica/métodos , Conformação de Ácido Nucleico , Peptídeos/metabolismo , RNA Catalítico/metabolismo , Espermina/metabolismoRESUMO
"Native ligation", a powerful method of joining peptide fragments, has been applied successfully to peptide-oligonucleotide conjugation. Novel reagents are described for the solid-phase synthesis of peptide N-terminal thioesters and 5'-cysteinyl oligonucleotides suitable for ligation reactions.
Assuntos
Cisteína/análogos & derivados , Oligonucleotídeos/síntese química , Peptídeos/síntese química , Oligonucleotídeos/química , Peptídeos/químicaRESUMO
Efficient methods of synthesis of peptide-oligonucleotide conjugates are badly needed for studies of uptake into cells in culture. We describe improvements to the procedures involved in "native ligation" conjugation in solution to extend the method to basic peptides. Further, we describe progress in development of a total solid-phase synthesis approach that makes use of a L-homoserine linker as the key reagent for growing of oligonucleotide and peptide chains on a single solid support.
Assuntos
Oligonucleotídeos/síntese química , Peptídeos/química , Sequência de Aminoácidos , Sequência de Bases , Homosserina/química , Oligonucleotídeos/químicaRESUMO
A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.
Assuntos
Oligonucleotídeos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Modelos Químicos , Dados de Sequência Molecular , Peptídeos/síntese química , Ácido TrifluoracéticoRESUMO
The preparation is described of four 2-cyanoethyl-N,N-diisopropyl phosphoramidites of N-alpha-Fmoc-S-protected cysteine hydroxyalkyl amides. The phosphoramidites were used in solid-phase synthesis of 5'-cysteinyl oligonucleotides, useful intermediates in the preparation of peptide-oligonucleotide conjugates through reaction with a maleimide peptide or with a peptide thioester via "native ligation".