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The four-way DNA junction that is a structural building block for DNA self-assemblies has been widely studied by diffraction methods and other physical techniques. However, terahertz (THz) spectroscopic characterization, which can reveal specific absorption fingerprints from different DNA structures and sequences, has not been reported yet. In this study, we present the spectral features of the four-way nucleic acid junction, its single- and double-stranded components, and their stoichiometric mixtures in the 10-25 cm-1 frequency range. We demonstrated that sub-THz vibrational spectroscopy can be used for sensing and characterizing the nanosize biomolecular architectures with well-defined composition and sequence. The results show that we can distinguish between the spectral features for all materials examined. Samples with amounts of material as low as 400 ng can be characterized. Thus, the resolved vibrational spectroscopy in the low-THz range can be used as a powerful tool to characterize the DNA junction structures and its components, as well as to monitor the J1 junction formation process.