RNA catalysis, thermodynamics and the origin of life.

The RNA World Hypothesis posits that the first self-replicating molecules were RNAs. RNA self-replicases are, in general, assumed to have employed nucleotide 5'-polyphosphates (or their analogues) as substrates for RNA polymerization. The mechanism by which these substrates might be synthesized with sufficient abundance to supply a growing and evolving population of RNAs is problematic for evolutionary hypotheses because non-enzymatic synthesis and assembly of nucleotide 5'-triphosphates (or other analogously activated phosphodiester species) is inherently difficult.

Highly efficient self-replicating RNA enzymes.

An RNA enzyme has been developed that catalyzes the joining of oligonucleotide substrates to form additional copies of itself, undergoing self-replication with exponential growth. The enzyme also can cross-replicate with a partner enzyme, resulting in their mutual exponential growth and enabling self-sustained Darwinian evolution. The opportunity for inventive evolution within this synthetic genetic system depends on the diversity of the evolving population, which is limited by the catalytic efficiency of the enzyme.

The origins of the RNA world.

The general notion of an "RNA World" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous.

Solving novel RNA structures using only secondary structural fragments.

The crystallographic phase problem is the primary bottleneck encountered when attempting to solve macromolecular structures for which no close crystallographic structural homologues are known. Typically, isomorphous "heavy-atom" replacement and/or anomalous dispersion methods must be used in such cases to obtain experimentally-determined phases. Even three-dimensional NMR structures of the same macromolecule are often not sufficient to solve the crystallographic phase problem.

Identification of dynamical hinge points of the L1 ligase molecular switch.

The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 5' to 3' phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert.

A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives.

The crystallographic phase problem [Muirhead & Perutz (1963), Nature (London), 199, 633-638] remains the single major impediment to obtaining a three-dimensional structure of a macromolecule once suitable crystals have been obtained. Recently, it was found that it was possible to solve the structure of a 142-nucleotide L1 ligase ribozyme heterodimer that possesses no noncrystallographic symmetry without heavy-atom derivatives, anomalous scattering atoms or other modifications and without a model of the tertiary structure of the ribozyme [Robertson & Scott (2007), Science, 315, 1549-1553]. Using idealized known RNA secondary-structural fragments such as A-form helices and GNRA tetraloops in an iterative molecular-replacement procedure, it was possible to obtain an estimated phase set that, when subjected to solvent flattening, yielded an interpretable electron-density map with minimized model bias, allowing the tertiary structure of the ribozyme to be solved.

In vitro selection using modified or unnatural nucleotides.

The use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool.

The structural basis of ribozyme-catalyzed RNA assembly.

Life originated, according to the RNA World hypothesis, from self-replicating ribozymes that catalyzed ligation of RNA fragments. We have solved the 2.6 angstrom crystal structure of a ligase ribozyme that catalyzes regiospecific formation of a 5' to 3' phosphodiester bond between the 5'-triphosphate and the 3'-hydroxyl termini of two RNA fragments.

The structure of a rigorously conserved RNA element within the SARS virus genome.

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-A resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3' end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome.

The importance of prebiotic chemistry in the RNA world.

In vitro selection experiments have clearly demonstrated that RNA can perform many of the functions necessary to support an RNA world. Moreover, it appears that novel functions could have readily evolved from existing functional RNA molecules. Therefore, diverse molecular ecosystems could potentially have arisen from an initial, small population of functional replicators.

In vitro selection of ribozymes dependent on peptides for activity.

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein.

Simultaneous detection of diverse analytes with an aptazyme ligase array.

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays.