Adrian R. Ferré-D’Amaré

Rapid Construction of Empirical RNA Fitness Landscapes

Nonrandom Walks Fitness landscapes of RNA sequences can help us to see the connection between all possible phenotypes and all possible genotypes. In molecular evolution, the fitness landscape is formalized as the distribution of fitness in sequence space, a hyperdimensional object of staggering complexity. To better understand these complex processes, Pitt and Ferré-DAmaré (p. 376; see the Perspective by Kluwe and Ellington) used deep sequencing to analyze the composition of a population of variants of an RNA ligase ribozyme, both before and after one round of in vitro selection. Relating the sequences of individuals in the population to a measure of their corresponding fitness provides a detailed picture of an evolutionary fitness landscape from empirical data. Mapping between phenotype and genotype can be resolved by deep sequencing. Evolution is an adaptive walk through a hypothetical fitness landscape, which depicts the relationship between genotypes and the fitness of each corresponding phenotype. We constructed an empirical fitness landscape for a catalytic RNA by combining next-generation sequencing, computational analysis, and “serial depletion,” an in vitro selection protocol. By determining the reaction rate constant for every point mutant of a catalytic RNA, we demonstrated that abundance in serially depleted pools correlates with biochemical activity (correlation coefficient r = 0.67, standard score Z = 7.4). Therefore, enumeration of each genotype by deep sequencing yielded a fitness landscape containing ~107 unique sequences, without requiring measurement of the phenotypic fitness for each sequence. High-throughput mapping between genotype and phenotype may apply to artificial selections, host-pathogen interactions, and other biomedically relevant evolutionary phenomena.

RNA-modifying enzymes

A bewildering number of post-transcriptional modifications are introduced into cellular RNAs by enzymes that are often conserved among archaea, bacteria and eukaryotes. The modifications range from those with well-understood functions, such as tRNA aminoacylation, to widespread but more mysterious ones, such as pseudouridylation. Recent structure determinations have included two types of RNA nucleobase modifying enzyme: pseudouridine synthases and tRNA guanine transglycosylases.

Use of the spliceosomal protein U1A to facilitate crystallization and structure determination of complex RNAs.

The structure determination of complex RNA molecules such as ribozymes, riboswitches and aptamers by X-ray crystallography hinges on the preparation of well-ordered crystals. Success usually results from molecular engineering to facilitate crystallization. An approach that has resulted in 10 new RNA structures in the past decade is the use of the U1A crystallization module. In this approach, the cognate site for the U1A spliceosomal protein is introduced into a functionally dispensable location in the RNA of interest, and the RNA is cocrystallized with the basic RNA-binding protein. In addition to facilitating crystallization, the presence of U1A can be useful for de novo phase determination. In this paper, some general considerations for the use of this approach to RNA crystallization are presented, and specifics of the application of the U1A module to the crystallization of the hairpin ribozyme and the tetracycline aptamer are reviewed.

Dramatic Improvement of Crystals of Large RNAs by Cation Replacement and Dehydration

Compared to globular proteins, RNAs with complex 3D folds are characterized by poorly differentiated molecular surfaces dominated by backbone phosphates, sparse tertiary contacts stabilizing global architecture, and conformational flexibility. The resulting generally poor order of crystals of large RNAs and their complexes frequently hampers crystallographic structure determination. We describe and rationalize a postcrystallization treatment strategy that exploits the importance of solvation and counterions for RNA folding. Replacement of Li(+) and Mg(2+) needed for growth of crystals of a tRNA-riboswitch-protein complex with Sr(2+), coupled with dehydration, dramatically improved the resolution limit (8.5-3.2xa0Å) and data quality, enabling structure determination. The soft Sr(2+) ion forms numerous stabilizing intermolecular contacts. Comparison of pre- and posttreatment structures reveals how RNA assemblies redistribute as quasi-rigid bodies to yield improved crystal packing. Cation exchange complements previously reported postcrystallization dehydration of protein crystals and represents a potentially general strategy for improving crystals of large RNAs.

Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe Ç

Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7u2009Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies.

SEWAL: an open-source platform for next-generation sequence analysis and visualization

Next-generation DNA sequencing platforms provide exciting new possibilities for in vitro genetic analysis of functional nucleic acids. However, the size of the resulting data sets presents computational and analytical challenges. We present an open-source software package that employs a locality-sensitive hashing algorithm to enumerate all unique sequences in an entire Illumina sequencing run (∼108 sequences). The algorithm results in quasilinear time processing of entire Illumina lanes (∼107 sequences) on a desktop computer in minutes. To facilitate visual analysis of sequencing data, the software produces three-dimensional scatter plots similar in concept to Sewall Wright and John Maynard Smith’s adaptive or fitness landscape. The software also contains functions that are particularly useful for doped selections such as mutation frequency analysis, information content calculation, multivariate statistical functions (including principal component analysis), sequence distance metrics, sequence searches and sequence comparisons across multiple Illumina data sets. Source code, executable files and links to sample data sets are available at http://www.sourceforge.net/projects/sewal.

A novel ARH splice site mutation in a Mexican kindred with autosomal recessive hypercholesterolemia

Autosomal recessive hypercholesterolemia (ARH) is characterized by elevated LDL serum levels, xanthomatosis, and premature coronary artery disease. Three loci have been described for this condition (1p35, 15q25-q26 and 13q). Recently, the responsible gene at the 1p35 locus, encoding an LDL receptor adaptor protein (ARH) has been identified. We studied a Mexican ARH family with two affected siblings. Sequence analysis of the ARH gene (1p35 locus) revealed that the affected siblings are homozygous for a novel mutation (IVS4+2T>G) affecting the donor splice site in intron 4, whereas both the parents and an unaffected sister are heterozygous for this mutation. The IVS4+2T>G mutation results in a major alternative transcript derived from a cryptic splice site, which carries an in-frame deletion of 78 nucleotides in the mature mRNA. The translation of this mRNA yields a mutant protein product (ARH-26) lacking 26 amino acids, resulting in the loss of β-strands β6 and β7 from the PTB domain. This is the first case where a naturally occurring mutant with an altered PTB domain has been identified.

Crystallization of the Hairpin Ribozyme

Conditions and techniques that result in successful crystallization differ from RNA to RNA. However, there are some general principles that facilitate crystallization of most RNAs. Three procedures that were instrumental in obtaining well-ordered crystals of the hairpin ribozyme are described in this chapter. These are: i) the design of a series of candidate crystallization constructs; ii) the evaluation of conditions to obtain monodisperse RNA; and iii) the use of seeding techniques to separate nucleation and growth events during crystallization. These procedures can be usefully adapted for the crystallization of other RNAs.

New molecular engineering approaches for crystallographic studies of large RNAs.

Crystallization of RNAs with complex three-dimensional architectures remains a formidable experimental challenge. We review a number of successful heuristics involving engineering of the target RNAs to facilitate crystal contact formation, such as those that enabled the crystallization and structure determination of the cognate tRNA complexes of RNase P holoenzyme and the Stem I domain of the T-box riboswitch. Recently, RNA-targeted antibody Fab fragments and Kink-turn binding proteins have joined the ranks of successful chaperones for RNA crystallization. Lastly, we review the use of structured RNAs to facilitate crystallization of RNA-binding proteins and other RNAs.

Crystallization of the glmS Ribozyme-Riboswitch

Procedures that were critical for crystallization of the glmS ribozyme-riboswitch RNA domain from the thermophilic Gram-positive bacterium Thermoanaerobacter tengcongensis are described. Experimental design based on screening multiple variant RNA sequences and techniques used to identify initial crystallization conditions were similar to those employed for most RNAs. However, serendipitous in-drop digestion of one RNA construct at a specific internucleotide linkage was crucial for the growth of high-quality glmS ribozyme crystals. Biochemical analysis of crystalline RNA identified the site of scission and guided design of an optimized bimolecular RNA construct. Finally, modifications of ionic strength and pH of solutions used for stabilization of the crystals were essential for optimal diffraction and binding of the activator glucosamine-6-phosphate, respectively. Although their details are specific to the glmS ribozyme, these general strategies may be useful for analyzing and improving crystals of other RNAs.