Bruce E. Eaton

Selection of RNA amide synthases

BACKGROUND It is generally accepted that, during evolution, replicating RNA molecules emerged from pools of random polynucleotides. This prebiotic RNA world was followed by an era of RNA-mediated catalysis of amide-bond formation. RNA would thus have provided the machinery responsible for the assembly of peptides and the beginning of the protein world of today. Naturally occurring ribozymes, which catalyze the cleavage or ligation of oligonucleotide phosphodiester bonds, support the idea that RNA could self-replicate. But was RNA constrained to this path and were RNA-acylated carriers required before RNA could catalyze the formation of amide bonds? RESULTS We have isolated RNA catalysts that are capable of mediating amide-bond synthesis without the need for specifically designed templates to align the substrates, and we have kinetically characterized these catalysts. The rate enhancement observed for these RNA amide synthases exceeds the noncatalyzed amidation rate by a factor of approximately 10(4). In addition, Cu2+ ions caused a change in the affinity of RNA for the substrate rather than being directly involved in amide-bond formation. CONCLUSIONS The discovery of these new amide synthases shows how functionally modified nucleic acids can facilitate covalent-bond formation without templating. Previously unforeseen RNA-evolution pathways can, therefore, be considered; for example, to guide amide-bond formation, en route to the protein world, it appears that substrate-binding pockets were formed that are analogous to those of protein enzymes.

Let's get specific: the relationship between specificity and affinity

The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.

Post-SELEX combinatorial optimization of aptamers.

In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.

The joys of in vitro selection: chemically dressing oligonucleotides to satiate protein targets

Oligonucleotide in vitro selection provides surprisingly specific aptamers to protein targets. Synthetic chemistries for modifying nucleotides have been developed to enhance aptamer binding affinity. The diversity of nucleosides amenable to either triphosphate synthesis or phosphoramidite activation is now sufficiently broad to rival any oligomeric combinatorial library.

DRESSED FOR SUCCESS : REALIZING THE CATALYTIC POTENTIAL OF RNA

In this manuscript the catalytic ability of RNA is examined and compared to other biopolymers. Despite having considerably fewer catalytically enabling properties when compared to proteins, the power of in vitro selection has allowed for RNA and DNA catalysts to be isolated. RNA catalysis has been expanded by incorporating modified bases to enrich the structural and functional diversity of RNA. Successful examples of new RNA chemistry using base modifications include carbon–carbon bond forming reactions and creation of highly specific active sites that are capable of recognizing small organic molecules without the need for nucleic acid templating or intercalation. In fact, the scope of functional modifications available for use in the RNA platform may eventually surpass those that are found in proteins and there are already hints that well chosen modifications allow nucleic acid catalysts to take advantage of mechanisms not available to selected protein catalysts for similar reactions. The chemical versatility of RNA is just emerging and future research directions will likely entail more creative methods for functional modification that will lead to new catalysts.

Biomolecules in the synthesis and assembly of materials for energy applications

Biomolecules such as RNA, DNA, peptides, and proteins are emerging as powerful chemical tools for the synthesis of inorganic nanoparticles. Specific biomolecule sequences have been isolated that afford remarkable control over the size, shape, polymorph, and hierarchical assembly of nanoparticles. Such exquisite control over nanoparticle growth and integration has already produced materials with unexpected photophysical properties and battery devices with improved performance. Continued exploration of biomolecule-mediated materials synthesis portends further advances in materials for the energy sciences. This review surveys the use of biomolecules in the synthesis and assembly of materials with a primary focus on methods that allow vast landscapes of biomolecule sequence space to be sampled simultaneously to discover unique sequence codes for new materials.

In vitro selection of RNA sequences capable of mediating the formation of iron oxide nanoparticles

In vitro selection experiments involving RNA, phagemids, or whole cells can yield biomolecules that bind tightly to or mediate the formation of inorganic materials. Herein we show that RNA sequences that mediate the formation of metal oxide nanoparticles can be isolated from iterative cycles of RNA selection and amplification. In contrast to prior work, the in vitro selection described within was based upon a desired materials property. In order to be isolated from a starting random sequence pool, an RNA sequence was required to mediate the assembly of Co and/or Fe into a solid that responded to a magnetic field. Sequences isolated from this selection were able to mediate the formation of iron oxide nanoparticles containing small amounts of Co under atypical synthesis conditions of temperature and pH.

Scanning Probe‐based Fabrication of 3D Nanostructures via Affinity Templates, Functional RNA, and Meniscus‐mediated Surface Remodeling

Developing generic platforms to organize discrete molecular elements and nanostructures into deterministic patterns on surfaces is one of the central challenges in the field of nanotechnology. Here we review three applications of the atomic force microscope (AFM) that address this challenge. In the first, we use two-step nanografting to create patterns of self-assembled monolayers (SAMs) to drive the organization of virus particles that have been either genetically or chemically modified to bind to the SAMs. Virus-SAM chemistries are described that provide irreversible and reversible binding, respectively. In the second, we use similar SAM patterns as affinity templates that have been designed to covalently bind oligonucleotides engineered to bind to the SAMs and selected for their ability to mediate the subsequent growth of metallic nanocrystals. In the final application, the liquid meniscus that condenses at the AFM tip-substrate contact is used as a physical tool to both modulate the surface topography of a water soluble substrate and guide the hierarchical assembly of Au nanoparticles into nanowires. All three approaches can be generalized to meet the requirements of a wide variety of materials systems and thereby provide a potential route toward development of a generic platform for molecular and materials organization.

Cooperativity between two selected RNA Pdases in the synthesis of Pd nanoparticles

We have made the surprising discovery that the crystallinity of nanoparticles formed from solutions containing RNA depends upon the presence of sequence mixtures. That is, a single sequence selected from the original random RNA sequence library produced mostly amorphous hexagonal nanoparticles, while a combination of sequences that emerged from the selection yields crystalline material as determined by SAED. To our knowledge this is the first example in which two biomolecules (RNA, DNA, or peptides) selected in vitro work together to provide a unique chemical outcome. In addition, this article provides a rigorous examination of the chemistry of Pd nanoparticle formation using RNA and the organometallic precursor complex Pd2(DBA)3 (DBA is dibenzylideneacetone). These studies have identified the specific conditions required for the successful RNA-mediated synthesis of Pd nanoparticles from aqueous solutions (10% THF : 90% H2O) containing Pd2(DBA)3, as well as conditions that led to anomalous results. A variety of techniques were employed to characterize materials formed under different solution conditions including SEM, AFM, TEM, selected area electron diffraction (SAED), and a chemical reactivity test. These analysis methods support the formation of Pd particles by RNA mediation when accounting for and controlling the important variables in the execution of the experiments. It is now clear that nanoparticles formed from RNA sequences isolated viain vitro selection can be dependent on many factors and it is understood that the specific sequence or sequence mixtures must be taken into account to fully understand RNA mediation of nanoparticle formation.