DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets, ease of synthesis and chemical modification, amenability to be interfaced with signal amplification schemes, and inherent biocompatibility. Here, we provide a historical perspective of how a transition from linear DNA structures toward more structurally complex nanostructures has revolutionized live-cell analysis. Modulating the structure gives rise to probes that can enter cells without the aid of transfection reagents and can detect, track, and quantify analytes in live cells at the single-organelle, single-cell, tissue section, and whole organism levels. We delineate the advantages and disadvantages associated with different probe architectures and describe the advances enabled by these structures for elucidating fundamental biology as well as developing improved diagnostic and theranostic systems. We also discuss the outstanding challenges in the field and outline potential solutions.