Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed "toolkits" utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.
The quinoprotein methylamine dehydrogenase (MADH), type I copper protein amicyanin, and cytochrome c-551i form a complex within which interprotein electron transfer occurs. It was known that complex formation significantly lowered the oxidation-reduction midpoint potential (Em) value of amicyanin, which facilitated an otherwise thermodynamically unfavorable electron transfer to cytochrome c-551i. Structural, mutagenesis, and potentiometric studies have elucidated the basis for this complex-dependent change in redox properties. Positively charged amino acid residues on the surface of amicyanin are known to stabilize complex formation with MADH and influence the ionic strength dependence of complex formation via electrostatic interactions. Altering the charges of these residues by site-directed mutagenesis had no effect on the Em value of amicyanin, ruling out charge neutralization as the basis for the complex-dependent changes in redox properties. The Em value of free amicyanin varies with pH and exhibits a pKa value for the reduced form of 7.5. The crystal structure of reduced amicyanin at pH 4.4 reveals that His95, which serves as a ligand for Cu2+, has rotated by 180 degrees about the Cbeta-Cgamma bond relative to its position in oxidized amicyanin and is no longer in the copper coordination sphere. At pH 7.7, the crystal structure of reduced amicyanin contains an approximately equal distribution of two active-site conformers. One is very similar to the structure of reduced amicyanin at pH 4.4, and the other is very similar to the structure of oxidized amicyanin at pH 4.8. Potentiometric analysis of amicyanin in complex with MADH indicates that its Em value is not pH-dependent from pH 6.5 to 8.5, and exhibits an Em value similar to that of free amicyanin at high pH. The structure of reduced amicyanin at pH 4.4, with His95 protonated and "flipped", was modeled into the structure of the complex of oxidized amicyanin with MADH. This showed that in the complex, the redox-linked pH-dependent rotation of His95 is hindered because it would cause an overlap of van der Waals' radii with residues of MADH. These results demonstrate that protein-protein interactions profoundly affect the redox properties of this type I copper protein by restricting a pH-dependent, redox-linked conformational change of one of the copper ligands.
Signaling initiated by Class Ia phosphatidylinositol-3-kinases (PI3Ks) is essential for cell proliferation and survival. We discovered a novel protein we call PI3K Interacting Protein 1 (PIK3IP1) that shares homology with the p85 regulatory PI3K subunit. Using a variety of in vitro and cell based assays, we demonstrate that PIK3IP1 directly binds to the p110 catalytic subunit and modulates PI3K activity. Our studies suggest that PIK3IP1 is a new type of PI3K regulator.
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