The use of calorimetry in the biophysical characterization of small molecule alkaloids binding to RNA structures

BackgroundRNA has now emerged as a potential target for therapeutic intervention. RNA targeted drug design requires detailed thermodynamic characterization that provides new insights into the interactions and this together with structural data, may be used in rational drug design. The use of calorimetry to characterize small molecule–RNA interactions has emerged as a reliable and sensitive tool after the recent advancements in biocalorimetry.Scope of the reviewThis review summarizes the recent advancements in thermodynamic characterization of small molecules, particularly some natural alkaloids binding to various RNA structures. Thermodynamic characterization provides information that can supplement structural data leading to more effective drug development protocols.Major conclusionsThis review provides a concise report on the use of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) techniques in characterizing small molecules, mostly alkaloids–RNA interactions with particular reference to binding of tRNA, single stranded RNA, double stranded RNA, poly(A), triplex RNA.General significanceIt is now apparent that a combination of structural and thermodynamic data is essential for rational design of specific RNA targeted drugs. Recent advancements in biocalorimetry instrumentation have led to detailed understanding of the thermodynamics of small molecules binding to various RNA structures paving the path for the development of many new natural and synthetic molecules as specific binders to various RNA structures. RNA targeted drug design, that remained unexplored, will immensely benefit from the calorimetric studies leading to the development of effective drugs for many diseases. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Opportunities in the design and application of RNA for gene expression control

The past decade of synthetic biology research has witnessed numerous advances in the development of tools and frameworks for the design and characterization of biological systems. Researchers have focused on the use of RNA for gene expression control due to its versatility in sensing molecular ligands and the relative ease by which RNA can be modeled and designed compared to proteins. We review the recent progress in the field with respect to RNA-based genetic devices that are controlled through small molecule and protein interactions. We discuss new approaches for generating and characterizing these devices and their underlying components. We also highlight immediate challenges, future directions and recent applications of synthetic RNA devices in engineered biological systems.     

Chemical and functional diversity of small molecule ligands for RNA

Functional RNAs such as ribosomal RNA and structured domains of mRNA are targets for small molecule ligands that can act as modulators of the RNA biological activity. Natural ligands for RNA display a bewildering structural and chemical complexity that has yet to be matched by synthetic RNA binders. Comparison of natural and artificial ligands for RNA may help to direct future approaches to design and synthesize potent novel scaffolds for specific recognition of RNA targets.     

Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.     

A small RNA activates CFA synthase by isoform‐specific mRNA stabilization

Small RNAs use a diversity of well‐characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq‐associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation‐independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot‐exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E‐mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA‐controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.     

A protocol for designing siRNAs with high functionality and specificity

Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.     

Exploring the repertoire of RNA secondary motifs using graph theory; implications for RNA design

Understanding the structural repertoire of RNA is crucial for RNA genomics research. Yet current methods for finding novel RNAs are limited to small or known RNA families. To expand known RNA structural motifs, we develop a two-dimensional graphical representation approach for describing and estimating the size of RNA’s secondary structural repertoire, including naturally occurring and other possible RNA motifs. We employ tree graphs to describe RNA tree motifs and more general (dual) graphs to describe both RNA tree and pseudoknot motifs. Our estimates of RNA’s structural space are vastly smaller than the nucleotide sequence space, suggesting a new avenue for finding novel RNAs. Specifically our survey shows that known RNA trees and pseudoknots represent only a small subset of all possible motifs, implying that some of the ‘missing’ motifs may represent novel RNAs. To help pinpoint RNA-like motifs, we show that the motifs of existing functional RNAs are clustered in a narrow range of topological characteristics. We also illustrate the applications of our approach to the design of novel RNAs and automated comparison of RNA structures; we report several occurrences of RNA motifs within larger RNAs. Thus, our graph theory approach to RNA structures has implications for RNA genomics, structure analysis and design.     

Rational ligand design for RNA: the role of static structure and conformational flexibility in target recognition

The role of static structure and conformational flexibility in the recognition of RNA targets by small molecule ligands is discussed with emphasis on the natural aminoglycoside antibiotics and their promiscuity in RNA target binding. A brief overview is given of previous efforts to design simplified aminoglycoside derivatives targeted at the bacterial decoding site RNA.     

Synthetic metallonucleases for RNA cleavage

Synthetic metallonucleases are versatile metal ion catalysts that use multiple catalytic strategies for the cleavage of RNA. Recent work in the design of more active metallonucleases combines a single metal ion with functional groups that interact with RNA, including amino acid fragments or additional metal ions. Rate enhancements by multifunctional catalysts for cleavage of simple model substrates with good leaving groups are as high as 10(6) but somewhat lower (10(5)) for real RNA. However, cleavage of RNA substrates is complicated by different binding modes and steric interactions that can interfere with catalysis. Antisense oligonucleotides, peptides and small molecules that act as RNA recognition agents increase the strength of substrate binding, but not necessarily the catalytic rate constant. In general, catalytic strategies used by synthetic metallonucleases are probably not optimized. A better grasp of the mechanism of RNA cleavage by metal ions and more effort on positioning the metal ion complex with respect to the cleavage site may lead to improved catalysts.     

A comparison of viral RNA-dependent RNA polymerases

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. These viruses also use a small protein primer, named VPg, to initiate RNA replication. The recent explosion of structural information on picornaviral 3D polymerases has provided insights into the initiation of RNA synthesis and chain elongation. Comparing these data with results from previous structural analyses of viral RNA-dependent RNA polymerases that catalyze de novo RNA synthesis sheds light on the different strategies that these viruses use to initiate replication.     

Comparison of test systems for RNAinterference

RNAinterference (RNAi) has developed within a short time from an area of basic research occupied by a few experts to a widely used technical tool for reverse genetics, which is expected to have a broad utility not only in research, but also in medical and diagnostic applications. Despite its widespread use, the application of RNAi is often hampered because a difference of only a few nucleotides in the sequence of the target RNA can change the efficiency of a small interfering RNA (siRNA) from high to zero, and publicly available design tools for siRNAs are not yet perfect. We therefore developed and compared RNAi test systems based on different promoters, reporters, and target sequences. Here, we show that fluorescence-based test systems have obvious disadvantages compared to luciferase-based test systems and that some combinations of promoter, reporter, and target sequences, although currently in use, are not well suited for testing RNAi effects.     

New RNAi strategy for selective suppression of a mutant allele in polyglutamine disease

In gene therapy of dominantly inherited diseases with small interfering RNA (siRNA), mutant allele specific suppression may be necessary for diseases in which the defective gene normally has an important role. It is difficult, however, to design a mutant allele-specific siRNA for trinucleotide repeat diseases in which the difference of sequences is only repeat length. To overcome this problem, we use a new RNA interference (RNAi) strategy for selective suppression of mutant alleles. Both mutant and wild-type alleles are inhibited by the most effective siRNA, and wild-type protein is restored using the wild-type mRNA modified to be resistant to the siRNA. Here, we applied this method to spinocerebellar ataxia type 6 (SCA6). We discuss its feasibility and problems for future gene therapy.     

Improved silencing properties using small internally segmented interfering RNAs

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo.     

Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2'-O-methylation of the 3'-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2'-O-methyl modification of small RNA 3'-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.     

Biochemical and thermodynamic characterization of compounds that bind to RNA hairpin loops: toward an understanding of selectivity

Elucidation of the molecular forces governing small molecule-RNA binding is paramount to the progress of rational design strategies. The extensive characterization of the aminoglycoside-16S rRNA A-site interaction has deepened our understanding of how aminoglycosides bind to their target and exert their antimicrobial effects. However, to date no other RNA binding compounds have undergone such rigorous evaluation, and in general the origins of small molecule-RNA binding remain a mystery. We recently reported the identification of small molecules, dimers of 2-deoxystreptamine, which are able to bind selectively to RNA tetraloops and octaloops, respectively [Thomas, Liu, and Hergenrother (2005) J. Am. Chem. Soc. 127, 12434-12435]. Described herein is the biochemical and biophysical characterization of the RNA binding properties of the most selective compound, B-12, as well as closely related analogues. These studies further substantiate that B-12 is indeed selective for RNA octaloop sequences and indicate that the origin of this selectivity may lie in B-12's unusual binding mode, in which entropic factors are major contributors to the overall binding energy. In fact, isothermal titration calorimetry (ITC) experiments indicate that the binding of B-12 and most of its analogues is associated with a strong entropic contribution to the total binding energy. This is in stark contrast to the aminoglycosides, for which favorable enthalpy typically provides the driving force for binding. These studies are the first to examine small molecule-RNA hairpin loop binding in detail and are a necessary step toward the design of compounds that are specific binders for a given RNA sequence.     

Improved siRNA/shRNA functionality by mismatched duplex

siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and the miRNAs are generally 22 nt in length and contain multiple internal mismatches, we tested if the functionality can be increased by designing the si/shRNAs to mimic a miRNA structure. We systematically investigated the effect of single or multiple mismatches introduced in the passenger strand at different positions on siRNA functionality. Mismatches at certain positions could significantly increase the functionality of siRNAs and also, in some cases decreased the unwanted passenger strand functionality. The same strategy could also be used to design shRNAs. Finally, we showed that both si and miRNA structured oligos (siRNA with or without mismatches in the passenger strand) can repress targets in all individual Ago containing cells, suggesting that the Ago proteins do not differentiate between si/miRNA-based structure for silencing activity.