Abstract
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 β-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.
Highlights
Nanoscale engineering has advanced the fabrication and application of materials, with transformative impacts in a number of scientific fields
We further performed a series of optimization experiments to maximize the performance of enzymatic transcription, where the yield of represent hybrid materials consisting of organic (RNA) and PPi in the hybrid RNA flowers (hRNFs) products was found to be affected by the concentration of reaction components, including the concentrations of ribonucleotide triphosphates (rNTPs) (Figure S2), template DNA (Figure S3), and
We systematically investigated the composition and structure of hRNFs with combined characterization techniques including (S)transmission electron microscopy (TEM), focused ion beam (FIB)-scanning electron microscopy (SEM), and structured illumination microscopy (SIM) and showed that the hierarchical porous structures were formed as a hybrid composite of organic and inorganic RNA/Mg2PPi species
Summary
Nanoscale engineering has advanced the fabrication and application of materials, with transformative impacts in a number of scientific fields. The DNA origami field, for example, utilizes self-assembled DNA constructs with nanometer precision built on the basis of nucleic acid hybridization These sophisticated nanostructures can serve as excellent scaffolds to immobilize biomolecules and have been shown to effectively enhance catalytic activity[2,3] and enzyme stability.[2] the precise localization of enzyme cascades on DNA origami scaffolds[4] and the resulting enhancement of the cascade throughput have generated much excitement. Dimensional structures by adopting such properties including canonical or noncanonical base pairing, base stacking, and secondary or tertiary structural motifs This is typically found in many biological RNAs and found less often in DNAs. Functional RNAs such as RNA aptamers, ribozymes, riboswitches, siRNA, miRNA, and other protein-mimicking or noncoding RNA molecules can be fused or hybridized into RNA constructs, adding multiple functionalities to the tailored RNA nanostructures. We coupled β-galactosidase (β-gal) enzymes to hRNFs and observed enhanced enzymatic activity and improved stability in comparison to free enzymes
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