G-quadruplexes are noncanonical nucleic acid structures formed by stacked guanosine tetrads. Despite their functional and structural diversity, a single consensus model is typically used to describe sequences with the potential to form G-quadruplex structures. We are interested in developing more specific sequence models for G-quadruplexes. In previous work, we functionally characterized each sequence in a 496-member library of variants of a monomeric reference G-quadruplex for the ability to bind GTP, promote a model peroxidase reaction, generate intrinsic fluorescence, and to form multimers. Here we used NMR to obtain a broad overview of the structural features of this library. After determining the 1H NMR spectrum of each of these 496 sequences, spectra were sorted into multiple classes, most of which could be rationalized based on mutational patterns in the primary sequence. A more detailed screen using representative sequences provided additional information about spectral classes, and confirmed that the classes determined based on analysis of 1H NMR spectra are correlated with functional categories identified in previous studies. These results provide new insights into the surprising structural diversity of this library. They also show how NMR can be used to identify classes of sequences with distinct mutational signatures and functions.

• Keywords
• DNA, G-quadruplex, Multimeric structures, NMR,
• MeSH
• G-Quadruplexes * MeSH
• Guanosine Triphosphate chemistry   metabolism   MeSH
• Magnetic Resonance Spectroscopy methods   MeSH
• Mutation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Guanosine Triphosphate MeSH

For many decades it was thought that information storage and information transfer were the main functions of nucleic acids. However, artificial evolution experiments have shown that the functional potential of DNA and RNA is much greater. Here I provide an overview of this technique and highlight recent advances which have increased its potency. I also describe how artificial evolution has been used to identify nucleic acids with extreme functions. These include deoxyribozymes that generate unusual products such as light, tiny motifs made up of fewer than ten nucleotides, ribozymes that catalyze complex reactions such as RNA polymerization, information-rich sequences that encode overlapping ribozymes, motifs that catalyze reactions at rates too fast to be followed by manual pipetting, and functional nucleic acids which are active in extreme conditions. Such motifs highlight the limits of our knowledge and provide clues about as of yet undiscovered functions of DNA and RNA.

• Keywords
• aptamer, deoxyribozyme, in vitro selection, ribozyme, supernova deoxyribozyme,
• MeSH
• DNA, Catalytic * metabolism   MeSH
• DNA MeSH
• Nucleic Acid Conformation MeSH
• Nucleotides MeSH
• Nucleic Acids * MeSH
• RNA, Catalytic * metabolism   MeSH
• RNA genetics   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA, Catalytic * MeSH
• DNA MeSH
• Nucleotides MeSH
• Nucleic Acids * MeSH
• RNA, Catalytic * MeSH
• RNA MeSH

G-quadruplexes are noncanonical nucleic acid structures formed by stacked guanine tetrads. They are capable of a range of functions and thought to play widespread biological roles. This diversity raises an important question: what determines the biochemical specificity of G-quadruplex structures? The answer is particularly important from the perspective of biological regulation because genomes can contain hundreds of thousands of G-quadruplexes with a range of functions. Here we analyze the specificity of each sequence in a 496-member library of variants of a reference G-quadruplex with respect to five functions. Our analysis shows that the sequence requirements of G-quadruplexes with these functions are different from one another, with some mutations altering biochemical specificity by orders of magnitude. Mutations in tetrads have larger effects than mutations in loops, and changes in specificity are correlated with changes in multimeric state. To complement our biochemical data we determined the solution structure of a monomeric G-quadruplex from the library. The stacked and accessible tetrads rationalize why monomers tend to promote a model peroxidase reaction and generate fluorescence. Our experiments support a model in which the sequence requirements of G-quadruplexes with different functions are overlapping but distinct. This has implications for biological regulation, bioinformatics, and drug design.

• MeSH
• DNA chemistry   MeSH
• Fluorescence MeSH
• G-Quadruplexes * MeSH
• Guanosine Triphosphate chemistry   MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Peroxidases chemistry   MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Guanosine Triphosphate MeSH
• Peroxidases MeSH

G-quadruplexes are noncanonical nucleic acid structures formed from stacked guanine tetrads. They are frequently used as building blocks and functional elements in fields such as synthetic biology and also thought to play widespread biological roles. G-quadruplexes are often studied as monomers, but can also form a variety of higher-order structures. This increases the structural and functional diversity of G-quadruplexes, and recent evidence suggests that it could also be biologically important. In this review, we describe the types of multimeric topologies adopted by G-quadruplexes and highlight what is known about their sequence requirements. We also summarize the limited information available about potential biological roles of multimeric G-quadruplexes and suggest new approaches that could facilitate future studies of these structures.

• Keywords
• DNA:RNA hybrid, G-quadruplex, R-loop, dimer, multimer, oligomer, promoter, telomere, tetramer,
• MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Nucleic Acid Conformation * MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• RNA chemistry   MeSH
• Telomere MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• RNA MeSH