Avidin [HRP]

Programmable nucleic acids have emerged as powerful building blocks for the bottom-up fabrication of two- or three-dimensional nano- and microsized constructs. Here we describe the construction of organic-inorganic hybrid RNA flowers (hRNFs) via rolling circle transcription (RCT), an enzyme-catalyzed nucleic acid amplification reaction. These hRNFs are highly adaptive structures with controlled sizes, specific nucleic acid sequences, and a highly porous nature. We demonstrated that hRNFs are applicable as potential biological platforms, where the hRNF scaffold can be engineered for versatile surface functionalization and the inorganic component (magnesium ions) can serve as an enzyme cofactor. For surface functionalization, we proposed robust and straightforward approaches including in situ synthesis of functional hRNFs and postfunctionalization of hRNFs that enable facile conjugation with various biomolecules and nanomaterials (i.e., proteins, enzymes, organic dyes, inorganic nanoparticles) using selective chemistries (i.e., avidin-biotin interaction, copper-free click reaction). In particular, we showed that hRNFs can serve as soft scaffolds for beta-galactosidase immobilization and greatly enhance enzymatic activity and stability. Therefore, the proposed concepts and methodologies are not only fundamentally interesting when designing RNA scaffolds or RNA bionanomaterials assembled with enzymes but also have significant implications on their future utilization in biomedical applications ranging from enzyme cascades to biosensing and drug delivery.

The temperature sensitivity of vaccines and therapeutic proteins forces the distribution of life-saving treatments to rely heavily on the temperature-controlled (usually 2-8 degrees C) supply and distribution network known as the cold chain. Here, using avidin as a model, we demonstrate how surface engineering could significantly increase the thermal stability of therapeutic proteins. A combination of spectroscopic (Fourier transform infrared, circular dichroism, and ultraviolet-visible) and scattering techniques (dynamic light scattering, small-angle, and wide-angle X-ray scattering) were deployed to probe the activity, structure, and stability of the model protein. Temperature-dependent synchrotron radiation circular dichroism spectroscopy was used to demonstrate a significant increase in thermal stability, with a half denaturation temperature of 139.0 degrees C and reversible unfolding with modified avidin returning to a 90% folded state when heated to temperatures below 100 degrees C. Accelerated aging studies revealed that modified avidin retained its secondary structure after storage at 40 degrees C for 56 days, equivalent to 160 days at 25 degrees C. Furthermore, binding studies with multiple ligands revealed that the binding site remained functional after modification. As a result, this approach has potential as a storage technology for therapeutic proteins and the elimination of the cold chain, enabling the dissemination of life-saving vaccines worldwide.