Structural Enzymology, Phylogenetics, Differentiation, and Symbolic Reflexivity at the Dawn of Biology.

The reflexive translation of symbols in one chemical language to another defined genetics. Yet, the co-linearity of codons and amino acids is so commonplace an idea that few even ask how it arose. Readout is done by two distinct sets of proteins, called aminoacyl-tRNA synthetases (AARS).

In vitro selections with RNAs of variable length converge on a robust catalytic core.

In vitro selection is a powerful tool that can be used to understand basic principles of molecular evolution. We used in vitro selection to understand how changes in length and the accumulation of point mutations enable the evolution of functional RNAs. Using RNA populations of various lengths, we performed a series of in vitro experiments to select for ribozymes with RNA ligase activity.

Selection of 2'-Deoxy-2'-Fluoroarabino Nucleic Acid (FANA) Aptamers That Bind HIV-1 Integrase with Picomolar Affinity.

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2'-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants () of ∼50-100 pM in a buffer with 200 mM NaCl and 6 mM MgCl.

Big on Change, Small on Innovation: Evolutionary Consequences of RNA Sequence Duplication.

The potential for biopolymers to evolve new structures has important consequences for their ability to optimize function and our attempts to reconstruct their evolutionary histories. Prior work with in vitro systems suggests that structural remodeling of RNA is difficult to achieve through the accumulation of point mutations or through recombination events. Sequence duplication may represent an alternative mechanism that can more readily lead to the evolution of new structures.

Evolution of ribozymes in the presence of a mineral surface.

Mineral surfaces are often proposed as the sites of critical processes in the emergence of life. Clay minerals in particular are thought to play significant roles in the origin of life including polymerizing, concentrating, organizing, and protecting biopolymers. In these scenarios, the impact of minerals on biopolymer folding is expected to influence evolutionary processes.

In vitro evolution of distinct self-cleaving ribozymes in diverse environments.

In vitro evolution experiments have long been used to evaluate the roles of RNA in both modern and ancient biology, and as a tool for biotechnology applications. The conditions under which these experiments have been conducted, however, do not reflect the range of cellular environments in modern biology or our understanding of chemical environments on the early earth, when the atmosphere and oceans were largely anoxic and soluble Fe(2+) was abundant. To test the impact of environmental factors relevant to RNA's potential role in the earliest forms of life, we evolved populations of self-cleaving ribozymes in an anoxic atmosphere with varying pH in the presence of either Fe(2+) or Mg(2+).

Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, "UCAA-motif" RNA Aptamers.

RNA aptamers that bind the reverse transcriptase (RT) of human immunodeficiency virus (HIV) compete with nucleic acid primer/template for access to RT, inhibit RT enzymatic activity in vitro, and suppress viral replication when expressed in human cells. Numerous pseudoknot aptamers have been identified by sequence analysis, but relatively few have been confirmed experimentally. In this work, a screen of nearly 100 full-length and >60 truncated aptamer transcripts established the predictive value of the F1Pk and F2Pk pseudoknot signature motifs.

High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase.

Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT).

Broad-spectrum aptamer inhibitors of HIV reverse transcriptase closely mimic natural substrates.

A detailed understanding of how aptamers recognize biological binding partners is of considerable importance in the development of oligonucleotide therapeutics. For antiviral nucleic acid aptamers, current models predict a correlation between broad-spectrum inhibition of viral proteins and suppression of emerging viral resistance, but there is little understanding of how aptamer structures contribute to recognition specificity. We previously established that two independent single-stranded DNA aptamers, R1T and RT1t49(-5), are potent inhibitors of reverse transcriptases (RTs) from diverse branches of the primate lentiviral family, including HIV-1, HIV-2 and SIV(cpz).

Conformational dynamics of single pre-mRNA molecules during in vitro splicing.

The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites.

Molecular dynamics and quantum mechanics of RNA: conformational and chemical change we can believe in.

Structure and dynamics are both critical to RNA's vital functions in biology. Numerous techniques can elucidate the structural dynamics of RNA, but computational approaches based on experimental data arguably hold the promise of providing the most detail. In this Account, we highlight areas wherein molecular dynamics (MD) and quantum mechanical (QM) techniques are applied to RNA, particularly in relation to complementary experimental studies.

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme.

Despite numerous structural and biochemical investigations, the catalytic mechanism of hairpin ribozyme self-cleavage remains elusive. To gain insight into the coupling of active site dynamics with activity of this small catalytic RNA, we analyzed a total of approximately 300 ns of molecular dynamics (MD) simulations. Our simulations predict improved global stability for an in vitro selected "gain of function" mutation, which is validated by native gel electrophoretic mobility shift assay.

A rugged free energy landscape separates multiple functional RNA folds throughout denaturation.

The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis).

Focus on function: single molecule RNA enzymology.

The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches.