-nitrobenzyl (ONB) groups based photoremovable protecting groups (PPGs) stand out as significant photocleavage components in oligonucleotides, owing to their straightforward synthesis, easy incorporation, and compatibility with a broad array of functional groups. These ONB-modified nucleic acids have found widespread applications in the area of biomedicine. In this perspective, we delve into the progress and study of PPGs derived from ONB for accurately controlling nucleic acid functions using light. Various chemical approaches are explored for incorporating ONB-based PPGs in different parts of single-stranded nucleic acids, including nucleobases, backbones, riboses, and phosphate groups, along with their distinct impacts on nucleic acid hybridization. Additionally, we evaluate the benefits and constraints of these alterations. Promising strategies, such as the molecular engineering of existing ONB derivatives, the integration of established PPGs with appropriate two-photon light-absorbing antennas in a modular setup, and the coupling of ONB protective groups with upconversion nanomaterials to extend their cleavage wavelengths into longer spectra are outlined and discussed. Subsequently, we systematically explore the biological applications of these light-responsive nucleic acids as versatile tools, specifically for light-triggered gene editing and as foundational elements for constructing 3D DNA-based nanomaterials for drug delivery. By leveraging the versatile chemistry of ONB alongside diverse nucleic acid modification techniques, we aim to provide an up-to-date overview while outlining existing challenges and proposing solutions within this evolving field. This Perspective seeks to foster innovation in the design of light-responsive nucleic acids within DNA nanotechnology for biomedical applications, underscoring the ongoing pursuit of novelty in this field.