A synthetic snRNA m3G-CAP enhances nuclear delivery of exogenous proteins and nucleic acids

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 (m3G-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 m3G 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

Mechanism of Histone H1-Stimulated Glucocorticoid Receptor DNA Binding In Vivo

Xenopus oocytes lack somatic linker histone H1 but contain an oocyte-specific variant, B4. The glucocorticoid receptor (GR) inducible mouse mammary tumor virus (MMTV) promoter was reconstituted in Xenopus oocytes to address the effects of histone H1. The expression of Xenopus H1A (H1) via cytoplasmic mRNA injection resulted in H1 incorporation into in vivo assembled chromatin based on (i) the appearance of a chromatosome stop, (ii) the increased nucleosome repeat length (NRL), and (iii) H1-DNA binding assayed by chromatin immunoprecipitation (ChIP). The H1 effect on the NRL was saturable and hence represents H1-binding to a specific site. A subsaturating level of H1 enhanced the hormone-dependent binding of GR to the glucocorticoid response elements (GREs) and the hormone-dependent MMTV transcription while it reduced the access to DNA as revealed by micrococcal nuclease (MNase) analysis. These H1 effects were lost at higher levels of H1. ChIP and MNase analysis revealed a hormone-dependent dissociation of H1 from the activated chromatin domain. The proposed mechanism of H1-induced GR binding is based on two effects: (i) a GR-induced asymmetric distribution of H1 in favor of inactive chromatin and (ii) an H1-induced reduction in DNA access. These effects results in increased concentration of free GR and, hence, in increased GR-GRE binding