Molecules that reversibly bind DNA and trigger the formation of non-Watson-Crick secondary structures would be useful in the design of dynamic DNA nanostructures and as potential leads for new therapeutic agents. We demonstrate that coralyne, a small crescent-shaped molecule, promotes the formation of a duplex secondary structure from homo-adenine oligonucleotides. AFM studies reveal that the staggered alignment of homo-adenine oligonucleotides upon coralyne binding produces polymers of micrometers in length, but only 2 nm in height. A DNA duplex was also studied that contained eight A.A mismatches between two flanking 7-bp Watson-Crick helices. CD spectra confirm that the multiple A.A mismatches of this duplex bind coralyne in manner similar to that of homo-adenine oligonucleotides. Furthermore, the melting temperature of this hybrid duplex increases by 13 degrees C upon coralyne binding. These observations illustrate that the helical structure of the homo-adenine-coralyne duplex is compatible with the B-form DNA helix.

Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA-protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)*poly(dT) into triplex poly(dA)*poly(dT)*poly(dT) and a poly(dA) self-structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)16*(dT)16 by coralyne reverts over the course of hours if the sample is maintained at 4 degrees C. Coralyne-disproportioned (dA)32. (dT)(32), on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)16*(dT)16 sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)16 self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)n self-structure] binds coralyne in a length-dependent manner.

While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5′-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5′-aldehyde. We conclude that hydroxyl radical abstracts a 5′-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5′ deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2′-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.

Since the discovery of catalytic RNA two decades ago, [1] much attention has focused on the hypothesis that an early form of life used nucleic acids for both information storage and catalysis before the advent of proteins.[2] However, it is still a mystery how the first nucleic acid polymers assembled and replicated, as these tasks are carried out by protein enzymes in contemporary life. Decades of research have led to the inescapable conclusion that Watson-Crick base pairing alone does not sufficiently stabilize the assembly of mononucleo-