NMR Screen Reveals the Diverse Structural Landscape of a G-Quadruplex Library.

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.

Apollon: a deoxyribozyme that generates a yellow product.

Colorimetric assays in which the color of a solution changes in the presence of an input provide a simple and inexpensive way to monitor experimental readouts. In this study we used in vitro selection to identify a self-phosphorylating kinase deoxyribozyme that produces a colorimetric signal by converting the colorless substrate pNPP into the yellow product pNP. The minimized catalytic core, sequence requirements, secondary structure, and buffer requirements of this deoxyribozyme, which we named Apollon, were characterized using a variety of techniques including reselection experiments, high-throughput sequencing, comparative analysis, biochemical activity assays, and NMR.

Aurora: a fluorescent deoxyribozyme for high-throughput screening.

Fluorescence facilitates the detection, visualization, and tracking of molecules with high sensitivity and specificity. A functional DNA molecule that generates a robust fluorescent signal would offer significant advantages for many applications compared to intrinsically fluorescent proteins, which are expensive and labor intensive to synthesize, and fluorescent RNA aptamers, which are unstable under most conditions. Here, we describe a novel deoxyriboyzme that rapidly and efficiently generates a stable fluorescent product using a readily available coumarin substrate.

Pushing the Limits of Nucleic Acid Function.

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.

Optimizing the Chemiluminescence of a Light-Producing Deoxyribozyme.

Supernova is a chemiluminescent deoxyribozyme recently discovered in our group. It transfers the phosphate group from the 1,2-dioxetane substrate CDP-Star to its 5' hydroxyl group, which triggers a decomposition reaction and the production of light. Here we investigated the effects of reaction conditions on the ability of Supernova to generate a chemiluminescent signal (using a plate reader assay) and to phosphorylate itself (using a ligation assay).

Single-round deoxyribozyme discovery.

Artificial evolution experiments typically use libraries of ∼1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members.

Secondary Structure Libraries for Artificial Evolution Experiments.

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized.

Overlapping but distinct: a new model for G-quadruplex biochemical specificity.

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.

Structure and Function of Multimeric G-Quadruplexes.

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.

GTP-Dependent Formation of Multimeric G-Quadruplexes.

G-Quadruplexes are noncanonical nucleic acid structures made up of stacked guanosine tetrads connected by short loops. They are frequently used building blocks in synthetic biology and thought to play widespread biological roles. Multimerization can change the functional properties of G-quadruplexes, and understanding the factors that modulate this process remains an important goal.

Sequence Requirements of Intrinsically Fluorescent G-Quadruplexes.

G-Quadruplexes are four-stranded nucleic acid structures typically stabilized by GGGG tetrads. These structures are intrinsically fluorescent, which expands the known scope of nucleic acid function and raises the possibility that they could eventually be used as signaling components in label-free sensors constructed from DNA or RNA. In this study, we systematically investigated the effects of mutations in tetrads, loops, and overhanging nucleotides on the fluorescence intensity and maximum emission wavelength of >500 sequence variants of a reference DNA G-quadruplex.

Multimerization rules for G-quadruplexes.

G-quadruplexes can multimerize under certain conditions, but the sequence requirements of such structures are not well understood. In this study, we investigated the ability of all possible variants of the central tetrad in a monomeric, parallel-strand G-quadruplex to form higher-order structures. Although most of these 256 variants existed primarily as monomers under the conditions of our screen, ∼10% formed dimers or tetramers.

Altered biochemical specificity of G-quadruplexes with mutated tetrads.

A fundamental motif in canonical nucleic acid structure is the base pair. Mutations that disrupt base pairs are typically destabilizing, but stability can often be restored by a second mutation that replaces the original base pair with an isosteric variant. Such concerted changes are a way to identify helical regions in secondary structures and to identify new functional motifs in sequenced genomes.

Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling.

Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine.

A naturally occurring, noncanonical GTP aptamer made of simple tandem repeats.

Recently, we used in vitro selection to identify a new class of naturally occurring GTP aptamer called the G motif. Here we report the discovery and characterization of a second class of naturally occurring GTP aptamer, the "CA motif." The primary sequence of this aptamer is unusual in that it consists entirely of tandem repeats of CA-rich motifs as short as three nucleotides.

Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme.

The relationship between genotype and phenotype is often described as an adaptive fitness landscape. In this study, we used a combination of recombination, in vitro selection, and comparative sequence analysis to characterize the fitness landscape of a previously isolated kinase ribozyme. Point mutations present in improved variants of this ribozyme were recombined in vitro in more than 10(14) different arrangements using synthetic shuffling, and active variants were isolated by in vitro selection.

Discovery of widespread GTP-binding motifs in genomic DNA and RNA.

Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure.

New catalytic structures from an existing ribozyme.

Although protein enzymes with new catalytic activities can arise from existing scaffolds, less is known about the origin of ribozymes with new activities. Furthermore, mechanisms by which new macromolecular folds arise are not well characterized for either protein or RNA. Here we investigate how readily ribozymes with new catalytic activities and folds can arise from an existing ribozyme scaffold.