High-Throughput Measurement of Metastable DNA Secondary Structures Using Multiplexed Low-Yield Bisulfite Sequencing (MLB-seq)

Predicting DNA secondary structures is critical to a broad range of applications involving single-stranded DNA (ssDNA), yet remains an open problem. Existing prediction models are limited by insufficient experimental data, due to a lack of high-throughput methods to study DNA structures, in contrast to RNA structures. Here, we present a method for profiling DNA secondary structures using multiplexed low-yield bisulfite sequencing (MLB-seq), which examines the chemical accessibility of cytosines in thousands of different oligonucleotides. By establishing a probability-based model to evaluate the consensus probability between MLB-seq data and structures proposed using NUPACK software, we identified the secondary structures of individual ssDNA molecules and estimated the distribution of multiple secondary structures in solution. We studied the structures of 1,057 human genome subsequences and experimentally confirmed that 84% adopted two or more structures. MLB-seq thus enables high-throughput ssDNA structure profiling and will benefit the design of probes, primers, aptamers, and genetic regulators.