Synthesis of RNase H-sensitive porous supramolecular materials based on DNA-functionalized building blocks

Abstract

In the last decades, porous reticular materials with cavities capable of binding guest molecules, such as Metal-Organic Frameworks (MOFs), have drawn significant interest for applications including storage, separation, sensing and catalysis. However, these crystalline materials often face limitations such as poor processability, hardness, and marked insolubility or instability in solution, which hamper their use for applications like drug delivery or homogeneous catalysis. To address these limitations, a promising strategy has recently emerged that leverages preassembled Metal-Organic Cages (MOCs) as building blocks for the formation of soft porous materials through hierarchical self-assembly. This strategy exploits additional non-covalent interactions between the MOCs, orthogonal to the metal-ligand coordination that forms the cages, to construct extended supramolecular structures with intrinsic porosity. An alternative approach for achieving soft materials with periodic intermolecular porosity involves the supramolecular polymerization of carefully designed molecular building blocks into 2D or 3D network structures, known as Supramolecular Organic Frameworks (SOFs). In both cases, the building units are held together by reversible interactions, imparting dynamic properties in solution and enabling the development of stimuli-responsive materials. In DNA nanotechnology, the predictability and programmability of DNA interactions (i.e., DNA recognition and hybridization) have been widely used to design artificial DNA-based devices and self-assembled materials. The latter are formed through hierarchical self-assembly of building blocks functionalized with specific oligonucleotide sequences. Moreover, numerous systems capable of responding to input single-stranded DNA (ssDNA) or RNA (ssRNA) sequences have been developed. These systems are classified as dynamic DNA nanotechnology when operating under thermodynamic control, or as dissipative DNA nanotechnology when maintained out-of-equilibrium through an energy dissipation mechanism, such as continuous RNA enzymatic hydrolysis. The goal of this TFG project is to synthesize novel DNA-tethered building blocks for the preparation of enzyme-responsive porous supramolecular materials. These include a novel MOC functionalized with four ssDNA moieties on its periphery and a new tetra-DNA-functionalized Zn-porphyrin. The MOC will be employed to construct MOC-based hierarchical materials, while the DNA-functionalized porphyrin will be used to assemble 2D SOFs. The formation of these materials will be driven by DNA·RNA hybridization in the presence of a suitable ditopic RNA linker. Notably, these materials can be disassembled in the presence of RNase H, an enzyme that hydrolyzes RNA in DNA·RNA heteroduplexes, enabling potential biomedical applications and the implementation of dissipative control in these systems. To achieve this goal, we investigated the bioconjugation of DNA fragments (two different sequences) to a precursor azide-functionalized MOC using copper-free ´click´ chemistry, specifically the strain-promoted azide-alkyne cycloaddition (SPAAC). We compared post- and pre-functionalization approaches for preparing DNA-tethered MOCs, analyzing the reaction outcome using polyacrylamide gel electrophoresis (PAGE) and MALDI-TOF mass spectrometry. Notably, we identified a side reaction occurring, along with the ‘click’ reaction, when using the post-functionalization approach. In parallel, we synthesized a tetra-azide-functionalized Zn-porphyrin through a convergent multistep synthesis, exploring two alternative routes for the final steps. Lastly, we conducted preliminary bioconjugation studies of this porphyrin to ssDNA moieties using SPAAC.