In situ target biomolecule analysis is of great significance for real‐time monitoring and regulation of endogenous biomarkers and elementary biomolecules in vivo. Gratifyingly, the rapid evolution of structural DNA nanotechnology during past decades has established an appealing toolbox for biological analysis and medical detection. The modulated self‐assembly and underlying canonical Watson‐Crick base‐pairing rules provide possibilities for accurate controlling of the topologies and functions of obtained nanomaterials. The probes composed of diverse DNA nanostructures and DNA‐nanoparticle complexes can create a confined space, which increases target accessibility and improves probe stability, sensitivity and specificity. In this minireview, we retrospect the research progress of in‐situ biomolecular analysis based on DNA nanostructures for intracellular and in vivo biosensors in confined space. The characteristics of distinct DNA nanomaterials are first introduced, and then the fundamentals of biosensing process of designed DNA nanostructures are emphasized. Moreover, we elucidate our perspective over the challenges of this field and discuss the potential directions of this kind of application‐oriented fabrication technique.

Proteins directly participate in tremendous physiological processes and mediate a variety of cellular functions. However, precise manipulation of proteins with predefined relative position and stoichiometry for understanding protein‐protein interactions and guiding cellular behaviors is still challenging. With superior programmability of DNA molecules, DNA origami technology is able to construct arbitrary nanostructures that can accurately control the arrangement of proteins with various functionalities to solve these problems. Herein, starting from the classification of DNA origami nanostructures and the category of assembled proteins, we summarize the existing DNA origami‐based protein manipulation systems (PMSs), review the advances on the regulation of their functions, and discuss their applications in cellular behavior modulation and disease therapy. Moreover, the limitations and potential directions of DNA origami‐based PMSs are also presented, which may offer guidance for rational construction and ingenious application.