Repetitive DNA sequences are ubiquitous in life, and changes in the number of repeats often have various physiological and pathological implications. DNA repeats are capable of interchanging between different noncanonical and canonical conformations in a dynamic fashion, causing configurational slippage that often leads to repeat expansion associated with neurological diseases. In this report, we used single-molecule spectroscopy together with biophysical analyses to demonstrate the parity-dependent hairpin structural polymorphism of TGGAA repeat DNA. We found that the DNA adopted two configurations depending on the repeat number parity (even or odd). Transitions between these two configurations were also observed for longer repeats. In addition, the ability to modulate this transition was found to be enhanced by divalent ions. Based on the atomic structure, we propose a local seeding model where the kinked GGA motifs in the stem region of TGGAA repeat DNA act as hot spots to facilitate the transition between the two configurations, which may give rise to disease-associated repeat expansion.DNA tandem repeats | DNA slippage | single-molecule spectroscopy | X-ray crystallography D NA replication is a crucial process in all living organisms. Mishaps in the replication process generally lead to deleterious consequences but also drive biological evolution (1). Changes in the number of tandem copies of a specific DNA sequence within the genome are associated with devastating neuropathies and various types of cancer (2, 3). On the other hand, these changes also help shape normal genomic features such as microsatellite polymorphism, which are often used as markers for population biology studies (4).The unit sizes of repetitive DNA sequences involved in repeat number changes range from a single base (e.g., microsatellites) to dodecanucleotides (12 bases, e.g., in progressive myoclonic epilepsy type 1) (5, 6). DNA slippage is believed to be a primary mechanism driving the change in repeat number of various unit sizes. Repetitive DNA sequences often form alternative structures such as bulges and hairpin loops in addition to canonical DNA conformations (7,8). A repeat unit may slip between being part of a hairpin loop, a bulge, or a duplex in a dynamic fashion, which may alter the course of normal cellular DNA chemistry and ultimately lead to repeat expansion associated with neurological diseases (9). (TGGAA) n repeats, for example, may form noncanonical structures such as a hairpin arm (10, 11) or an antiparallel duplex (12). Expansion of this pentanucleotide sequence has been associated with spinocerebellar ataxia 31 (SCA31), an adult-onset autosomal-dominant neurodegenerative disorder (13).In this article, we probed the conformational heterogeneity and stability of hairpins composed of repetitive TGGAA sequences using single-molecule fluorescence resonance energy transfer [single-molecule FRET (smFRET)] spectroscopy and X-ray crystallography as primary tools. Remarkably, we were able to detect two distinct hairpin confi...

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.