A compact three-way junction oligonucleotide probe for widefield and super-resolution imaging of RNAs and proteins

Molecular imaging of RNAs and proteins requires methods that preserve nanoscale organization while enabling large-scale tissue analysis. However, weak or low-abundance targets often need signal amplification, which can increase labeling size, background, or signal spread and compromise super-resolution imaging. Here, we report a compact three-way junction (3WJ) oligonucleotide probe platform for multiscale RNA and protein imaging. The design couples paired target recognition with junction assembly and recruitment of preassembled amplifier complexes, while tunable amplification balances signal strength and labeling size. Using DNA origami and single-molecule localization microscopy, we show enhanced signal while retaining the ability to resolve nanoscale target patterns. The platform improves RNA detection and supports simultaneous RNA-protein super-resolution imaging in cells. It also enhances protein detection in formalin-fixed, paraffin-embedded tissues under low-numerical-aperture, large-field imaging conditions. Together, this programmable 3WJ platform enables sensitive RNA and protein detection across scales, supporting super-resolution analysis of molecular organization and large-field tissue mapping.