early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations. Truncated p53 mutants modulated droplet formation, suggesting the importance of multivalent electrostatic interactions among the N-terminal and C-terminal domains. Second, FRET efficiency measurements for the dimer mutants of p53 revealed that distances between the core domains and between the C-terminal domains were modulated in an opposite manner within the droplets. Third, the molecular crowding agents were found to promote droplet formation, whereas ssDNA, dsDNA, and ATP, to suppress it. Finally, the p53 mutant mimicking posttranslational phosphorylation did not form the droplets. We conclude that p53 itself has a potential to form droplets that can be controlled by cellular molecules and by posttranslational modifications, suggesting that LLPS might be involved in p53 function. Tumor suppressor p53 is a multifunctional transcription factor that induces cell cycle arrest, DNA repair or apoptosis upon binding to its target DNA sequence. In 50% of human cancers, mutations on p53 are found to hamper its binding to the target sequence. Accordingly, extensive investigations have been conducted to characterize the functions as well as malfunctions of p53. However, an important aspect of p53, namely its involvement in liquid-like droplets, is still largely unresolved. In fact, p53 has long been known to be uptaken into cellular droplets such as Cajal and promyelocytic leukemia protein (PML) bodies. In this report, we describe that p53 itself can form liquid-like droplets upon the control of solution conditions, suggesting a possible involvement of the p53 droplets in the cellular environment. The primary function of p53 is the accommodation of various posttranslational modifications, termed activation, which in turn triggers the search for and the binding to its target DNA sequence, leading to the expression of downstream genes 1. p53 is composed of the N-terminal (NT) (residues 1-95), the core (95-293), the linker (293-326), the tetramerization (Tet) (326-357), and the C-terminal (CT) (357-393) domains. p53 slides along nonspecific DNA by attaching the CT domain to the DNA and by hopping the core domain 2-4. The sliding of p53 occurs in two modes 5,6 , in which the CT, core and linker domains are differently in contact with the DNA 7. The recognition efficiency of the target sequence by the sliding p53 is low, but is enhanced by the activation of p53 8. Furthermore, the sliding p53 can transfer from one DNA strand to another using the CT domain 9. In addition,

A novel pH-sensitive and targetable antisense ODN delivery system based on multimolecular assembly into polyion complex (PIC) micelles of poly(L-lysine) (PLL) and a lactosylated poly(ethylene glycol)-antisense ODN conjugate (Lac-PEG-ODN) containing an acid-labile linkage (beta-propionate) between the PEG and ODN segments has been developed. The PIC micelles thus prepared had clustered lactose moieties on their peripheries and achieved a significant antisense effect against luciferase gene expression in HuH-7 cells (hepatoma cells), far more efficiently than that produced by the nonmicelle systems (ODN and Lac-PEG-ODN) alone, as well as by the lactose-free PIC micelle. In line with this pronounced antisense effect, the lactosylated PIC micelles showed better uptake than the lactose-free PIC micelles into HuH-7 cells; this suggested the involvement of an asialoglycoprotein (ASGP) receptor-mediated endocytosis process. Furthermore, a significant decrease in the antisense effect (27 % inhibition) was observed for a lactosylated PIC micelle without an acid-labile linkage (thiomaleimide linkage); this suggested the release of the active (free) antisense ODN molecules into the cellular interior in response to the pH decrease in the endosomal compartment is a key process in the antisense effect. Use of branched poly(ethylenimine) (B-PEI) instead of the PLL for PIC micellization led to a substantial decrease in the antisense effect, probably due to the buffer effect of the B-PEI in the endosome compartment, preventing the cleavage of the acid-labile linkage in the conjugate. The approach reported here is expected to be useful for the construction of smart intracellular delivery systems for antisense ODNs with therapeutic value.

A novel cytoplasmic delivery system of antisense oligodeoxynucleotide (asODN) was developed by assembling a PEG-asODN conjugate with disulfide linkage (smart linkage) (PEG-SS-asODN) into polyion complex (PIC) micelles through the complexation with branched poly(ethylenimine) (B-PEI). The PIC micelle thus prepared showed a significant antisense effect against luciferase gene expression in HuH-7 cells, far more efficient than nonmicelle systems (asODN and PEG-SS-asODN in free form) and PIC micelle encapsulating the conjugate without the disulfide linkage. Use of poly(l-lysine) (PLL) instead of the B-PEI for PIC micellization led to a substantial decrease in the antisense effect. These results indicate that the PIC micelles formulated from PEG-SS-asODN conjugate and B-PEI is successfully transported from the endosomal compartment into the cytoplasm by the buffering effect of the B-PEI, releasing hundreds of active asODN molecules via cleavage of the disulfide linkage into the cellular interior, responding to a high glutathione concentration in the cytoplasmic compartment. Furthermore, the type of smart linkage (glutathione-sensitive SS linkage vs pH-sensitive linkage) in the conjugates substantially affected the antisense effect of the PIC micelles, depending on the nature of the counter polycation (B-PEI vs PLL).

No abstract

One mismatch is discriminated in a target mRNA sequence by an inducible alkylation system based on sulfide precursors to the nucleoside 2‐amino‐6‐vinylpurine (see scheme). The reactive oligonucleotides were delivered into the cell as poly(ethylene glycol) (PEG) conjugates in polyion‐complex (PIC) micelles and showed antisense activity of high selectivity and greater potency than that of the natural antisense oligonucleotide.