Orthogonal L-DNA and R-DNA Probes Enable Fast, Multiplexed Imaging of Cellular Proteins

Abstract The intracellular molecular crowding and organelle compartmentalization demand high-performance tools for subcellular protein imaging with minimal background. DNA-barcoded antibodies enable multiplexed and ultrasensitive detection but suffer from nonspecific binding to endogenous nucleic acids and limited coding sequences, especially in the nucleus. Here, we develop an immunostaining platform using left-handed DNA-conjugated antibodies (Immuno-L-DNA) that greatly reduces cross-hybridization with cellular nucleic acids, yielding ultralow background fluorescence for nuclear protein imaging. L-DNA probes retain specific antibody recognition and multiplexing capability while drastically reducing nonspecific signals. Moreover, orthogonal hybridization of L-DNA and R-DNA expands coding sequence space for parallel multitarget detection. This strategy improves imaging quality for low-abundance nuclear proteins and is compatible with conventional immunostaining workflows, offering a robust tool for high-performance spatial proteomics.