ABSTRACT
Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5' end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization.
Subject(s)
Diphosphates/chemistry , Protein Biosynthesis , RNA Cap Analogs , RNA Caps , RNA, Messenger/chemistry , RNA, Messenger/genetics , DNA-Binding Proteins/metabolism , Dendritic Cells , Humans , Molecular Structure , Protein Binding , RNA Cap Analogs/chemical synthesis , Transcription Factors/metabolismABSTRACT
The synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m(7)GTP derivatives, whereas 11 are 'two headed' tetraphosphate dinucleotides based on a m(7)Gppppm(7)G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between ß and γ phosphorus atoms. The compounds were prepared by divalent metal chloride-mediated coupling of an appropriate m(7)GMP analogue with a given P(1),P(2)-di(1-imidazolyl) derivative. The analogues were evaluated as tools for studying cap-dependent processes in a number of biochemical assays, including determination of affinity to eukaryotic initiation factor eIF4E, susceptibility to enzymatic hydrolysis, and translational efficiency in vitro. The results indicate that modification in the phosphate chain can increase binding to cap-interacting proteins and provides higher resistance to degradation. Furthermore, modified derivatives of m(7)GTP were found to be potent inhibitors of cap-dependent translation in cell free systems.
Subject(s)
Boranes/chemistry , Phosphates/chemistry , Phosphorothioate Oligonucleotides/chemistry , RNA Cap Analogs/chemistry , Boranes/chemical synthesis , Boranes/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Humans , Hydrolysis , Phosphates/chemical synthesis , Phosphates/metabolism , Phosphorothioate Oligonucleotides/chemical synthesis , Phosphorothioate Oligonucleotides/metabolism , Protein Biosynthesis , RNA Cap Analogs/chemical synthesis , RNA Cap Analogs/metabolism , RNA, Messenger/chemical synthesis , RNA, Messenger/chemistry , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Patch testing is an essential procedure in the investigation of eczema in children. OBJECTIVES: To analyse the frequency of contact hypersensitivity and allergic contact dermatitis among Polish children with eczema. PATIENTS/METHODS: During an allergy screening programme involving 9320 children aged 7 and 16 years, 12.6% reported symptoms of chronic/recurrent eczema. From this group, a representative sample of 229 eczema children underwent patch testing: 96 children aged 7 years and 133 teenagers aged 16 years. Patch testing was with 10 allergens: methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), nickel sulfate, mercury ammonium chloride, thimerosal, cobalt chloride, potassium dichromate, lanolin, fragrance mix I, Myroxylon pereirae (balsam of Peru), and colophonium. RESULTS: 49.4% tested children were found patch test (PT) positive. 43.8% of 7 year olds with eczema were PT positive, with sensitization to nickel sulfate (30.2%), thimerosal (10.4%), cobalt chloride (8.3%), fragrance mix I (7.3%), MCI/MI (6.3%), potassium dichromate (6.3%), M. pereirae (3.1%), mercury ammonium chloride (2.3%), and colophonium (1.0%). 52.6% teenagers were PT positive, with sensitization to nickel sulfate (23.3%), thimerosal (27.8%), cobalt chloride (10.5%), potassium dichromate (6.0%), mercury ammonium chloride (2.3%), M. pereirae (1.5%), and MCI/MI (0.8%). The final diagnosis of allergic contact dermatitis was confirmed in 36% of 7 year olds and 26% of 16 year olds. CONCLUSIONS: Every second child with eczema is PT positive, whereas every third child is finally diagnosed with allergic contact dermatitis.
