Alternative Secondary Structures Research Articles

Template switching enables chemical probing of native RNA structures.

RNAs are often studied in non-native sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods - a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added post-probing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5' and 3' ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced non-native interactions in the full length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to non-native sequence elements.

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

DNA fragility at topologically associated domain boundaries is promoted by alternative DNA secondary structure and topoisomerase II activity.

CCCTC-binding factor (CTCF) binding sites are hotspots of genome instability. Although many factors have been associated with CTCF binding site fragility, no study has integrated all fragility-related factors to understand the mechanism(s) of how they work together. Using an unbiased, genome-wide approach, we found that DNA double-strand breaks (DSBs) are enriched at strong, but not weak, CTCF binding sites in five human cell types. Energetically favorable alternative DNA secondary structures underlie strong CTCF binding sites. These structures coincided with the location of topoisomerase II (TOP2) cleavage complex, suggesting that DNA secondary structure acts as a recognition sequence for TOP2 binding and cleavage at CTCF binding sites. Furthermore, CTCF knockdown significantly increased DSBs at strong CTCF binding sites and at CTCF sites that are located at topologically associated domain (TAD) boundaries. TAD boundary-associated CTCF sites that lost CTCF upon knockdown displayed increased DSBs when compared to the gained sites, and those lost sites are overrepresented with G-quadruplexes, suggesting that the structures act as boundary insulators in the absence of CTCF, and contribute to increased DSBs. These results model how alternative DNA secondary structures facilitate recruitment of TOP2 to CTCF binding sites, providing mechanistic insight into DNA fragility at CTCF binding sites.

Unraveling the complexity: Advanced methods in analyzing DNA, RNA, and protein interactions.

Exploring the intricate interplay within and between nucleic acids, as well as their interactions with proteins, holds pivotal significance in unraveling the molecular complexities steering cancer initiation and progression. To investigate these interactions, a diverse array of highly specific and sensitive molecular techniques has been developed. The selection of a particular technique depends on the specific nature of the interactions. Typically, researchers employ an amalgamation of these different techniques to obtain a comprehensive and holistic understanding of inter- and intramolecular interactions involving DNA-DNA, RNA-RNA, DNA-RNA, or protein-DNA/RNA. Examining nucleic acid conformation reveals alternative secondary structures beyond conventional ones that have implications for cancer pathways. Mutational hotspots in cancer often lie within sequences prone to adopting these alternative structures, highlighting the importance of investigating intra-genomic and intra-transcriptomic interactions, especially in the context of mutations, to deepen our understanding of oncology. Beyond these intramolecular interactions, the interplay between DNA and RNA leads to formations like DNA:RNA hybrids (known as R-loops) or even DNA:DNA:RNA triplex structures, both influencing biological processes that ultimately impact cancer. Protein-nucleic acid interactions are intrinsic cellular phenomena crucial in both normal and pathological conditions. In particular, genetic mutations or single amino acid variations can alter a protein's structure, function, and binding affinity, thus influencing cancer progression. It is thus, imperative to understand the differences between wild-type (WT) and mutated (MT) genes, transcripts, and proteins. The review aims to summarize the frequently employed methods and techniques for investigating interactions involving nucleic acids and proteins, highlighting recent advancements and diverse adaptations of each technique.

N-methyl mesoporphyrin IX (NMM) as electrochemical probe for detection of guanine quadruplexes

G-quadruplexes (G4) are stable alternative secondary structures of nucleic acids. With increasing understanding of their roles in biological processes and their application in bio- and nanotechnology, the exploration of novel methods for the analysis of these structures is becoming important. In this work, N-methyl mesoporphyrin IX (NMM) was used as a voltammetric probe for an easy electrochemical detection of G4s. Cyclic voltammetry on a hanging mercury drop electrode (HMDE) was used to detect NMM with a limit of detection (LOD) of 40 nM. Characteristic reduction signal of NMM was found to be substantially higher in the presence of G4 oligodeoxynucleotides (ODNs) than in the presence of single- or double-stranded ODNs and even ODNs susceptible to form G4s but in their unfolded, single-stranded forms. Gradual transition from unstructured single strand to G4, induced by increasing concentrations of the G4 stabilizing K+ ions, was detected by an electrochemical method for the first time. All obtained results were supported by circular dichroism spectroscopy. This work expands on the concept of electrochemical probes utilization in DNA secondary structure recognition and offers a proof of principle that can be potentially employed in the development of novel electroanalytical methods for nucleic acid structure studies.

Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches.

In addition to proteins, microbes can use structured RNAs such as riboswitches for the important task of regulating gene expression. Riboswitches control gene expression by changing their structure in response to binding a small molecule and are widespread among bacteria. Here we determine the mechanism of regulation in a riboswitch that responds to corrinoids-a family of coenzymes related to vitamin B12. We report the alternative RNA secondary structures that couple corrinoid sensing with response in a repressing and novel activating corrinoid riboswitch. We then applied this knowledge to flipping the regulatory sign by constructing synthetic riboswitches that activate expression to a higher level than the natural one. In the process, we observed patterns in which sequence, in addition to structure, impacts function in paired RNA regions. The synthetic riboswitches we describe here have potential applications as biosensors.

Cytosine epigenetic modifications and conformational changes in G‐quadruplex DNA: An ultraviolet resonance Raman spectroscopy study

Epigenetic modifications of DNA are known to play important regulatory roles in biological systems, especially in regulation of gene expression, and are associated with many types of human diseases, including cancer. Alternative DNA secondary structures, such as G‐quadruplexes, can also influence gene transcription, thus suggesting that such structures may represent a distinctive layer of epigenetic information. G‐quadruplex structures and DNA epigenetic modifications often go side by side, and recent evidence reveals that cytosine modifications within loops of G‐quadruplexes can play a role in modulating their stability and structural polymorphism. Therefore, the development and validation of experimental techniques that can easily and reliably analyse G‐quadruplex structures are highly desirable. In the present study, we propose to exploit the advantages of UV resonance Raman (UVRR) spectroscopy to investigate cytosine epigenetic modifications along with conformational changes in G‐quadruplex-forming DNA. Our findings show that clear and specific spectral changes occur when there is a change in a G‐quadruplex structure. Moreover, UVRR spectral analysis can indirectly distinguish the spectral variations occurring because of modifications in the guanine glycosidic conformations, as well as detect changes in the loops induced by H-bond formation or hydration of nitrogenous bases. The results further underscore the utility of UVRR spectroscopy for G‐quadruplex structure elucidation under biologically relevant solution conditions.

Genome-wide identification of A-to-I RNA editing events provides the functional implications in PDAC.

RNA editing, a wide-acknowledged post-transcriptional mechanism, has been reported to be involved in the occurrence and development of cancer, especially the abnormal alteration of adenosine to inosine. However, fewer studies focus on pancreaticcancer. Therefore, we aimed to explore the possible linkages between altered RNA editing events and the development of PDAC. We characterized the global A-to-I RNA editing spectrum from RNA and matched whole-genome sequencing data of 41 primary PDAC and adjacent normal tissues. The following analyses were performed: different editing level and RNA expression analysis,pathway analysis, motif analysis, RNA secondary structure analysis, alternative splicing events analysis, and survival analysis.The RNA editing of single-cell RNA public sequencing data was also characterized. A large number of adaptive RNA editing events with significant differences in editing levels were identified, which are mainly regulated by ADAR1. Moreover, RNA editing in tumors has a higher editing level and more abundant editing sites in general. 140genes were screened out since they were identified with significantly different RNA editing events and were significantly different in expression level between tumor and matched normal samples. Further analysis showed a preference that in the tumor-specific group, they are mainly enriched in cancer-related signal pathways, while in the normal tissue-specific group, they are mainly enriched in pancreatic secretion. At the same time, we also found positively selected differentially edited sites in a series of cancer immune genes, including EGF, IGF1R, and PIK3CD. RNA editing might participate in pathogenisis of PDAC through regulating the alternative splicing and RNA secondary structure of important genesto further regulate gene expression and protein synthesis, including RAB27B and CERS4. Furthermore, single cell sequencing results showed that type2 ductal cells contributed the most to RNA editing events in tumors. RNA editing is an epigenetic mechanism involved in the occurrence and development of pancreatic cancer, which has the potential to diagnose of PDAC and is closely related to the prognosis.

DNA Base Excision Repair Intermediates Influence Duplex-Quadruplex Equilibrium.

Although genomic DNA is predominantly duplex under physiological conditions, particular sequence motifs can favor the formation of alternative secondary structures, including the G-quadruplex. These structures can exist within gene promoters, telomeric DNA, and regions of the genome frequently found altered in human cancers. DNA is also subject to hydrolytic and oxidative damage, and its local structure can influence the type of damage and its magnitude. Although the repair of endogenous DNA damage by the base excision repair (BER) pathway has been extensively studied in duplex DNA, substantially less is known about repair in non-duplex DNA structures. Therefore, we wanted to better understand the effect of DNA damage and repair on quadruplex structure. We first examined the effect of placing pyrimidine damage products uracil, 5-hydroxymethyluracil, the chemotherapy agent 5-fluorouracil, and an abasic site into the loop region of a 22-base telomeric repeat sequence known to form a G-quadruplex. Quadruplex formation was unaffected by these analogs. However, the activity of the BER enzymes were negatively impacted. Uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1) were inhibited, and apurinic/apyrimidinic endonuclease 1 (APE1) activity was completely blocked. Interestingly, when we performed studies placing DNA repair intermediates into the strand opposite the quadruplex, we found that they destabilized the duplex and promoted quadruplex formation. We propose that while duplex is the preferred configuration, there is kinetic conversion between duplex and quadruplex. This is supported by our studies using a quadruplex stabilizing molecule, pyridostatin, that is able to promote quadruplex formation starting from duplex DNA. Our results suggest how DNA damage and repair intermediates can alter duplex-quadruplex equilibrium.

In vivo single-molecule analysis reveals COOLAIR RNA structural diversity

Cellular RNAs are heterogeneous with respect to their alternative processing and secondary structures, but the functional importance of this complexity is still poorly understood. A set of alternatively processed antisense non-coding transcripts, which are collectively called COOLAIR, are generated at the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC)1. Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions2–7. Here, to further investigate the function of COOLAIR, we developed an RNA structure-profiling method to determine the in vivo structure of single RNA molecules rather than the RNA population average. This revealed that individual isoforms of the COOLAIR transcript adopt multiple structures with different conformational dynamics. The major distally polyadenylated COOLAIR isoform in warm conditions adopts three predominant structural conformations, the proportions and conformations of which change after cold exposure. An alternatively spliced, strongly cold-upregulated distal COOLAIR isoform6 shows high structural diversity, in contrast to proximally polyadenylated COOLAIR. A hyper-variable COOLAIR structural element was identified that was complementary to the FLC transcription start site. Mutations altering the structure of this region changed FLC expression and flowering time, consistent with an important regulatory role of the COOLAIR structure in FLC transcription. Our work demonstrates that isoforms of non-coding RNA transcripts adopt multiple distinct and functionally relevant structural conformations, which change in abundance and shape in response to external conditions.

Splice site proximity influences alternative exon definition

ABSTRACT Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5′ and 3′ splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5′ and 3′ splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.

Pif1 Activity is Modulated by DNA Sequence and Structure.

The gene encoding the Pif1 helicase was first discovered in a Saccharomyces cerevisiae genetic screen as a mutant that reduces recombination between mitochondrial respiratory mutants and was subsequently rediscovered in a screen for genes affecting the telomere length in the nucleus. It is now known that Pif1 is involved in numerous aspects of DNA metabolism. All known functions of Pif1 rely on binding to DNA substrates followed by ATP hydrolysis, coupling the energy released to translocation along DNA to unwind duplex DNA or alternative DNA secondary structures. The interaction of Pif1 with higher-order DNA structures, like G-quadruplex DNA, as well as the length of single-stranded (ss)DNA necessary for Pif1 loading have been widely studied. Here, to test the effects of ssDNA length, sequence, and structure on Pif1's biochemical activities in vitro, we used a suite of oligonucleotide-based substrates to perform a basic characterization of Pif1 ssDNA binding, ATPase activity, and helicase activity. Using recombinant, untagged S. cerevisiae Pif1, we found that Pif1 preferentially binds to structured G-rich ssDNA, but the preferred binding substrates failed to maximally stimulate ATPase activity. In helicase assays, significant DNA unwinding activity was detected at Pif1 concentrations as low as 250 pM. Helicase assays also demonstrated that Pif1 most efficiently unwinds DNA fork substrates with unstructured ssDNA tails. As the chemical step size of Pif1 has been determined to be 1 ATP per translocation or unwinding event, this implies that the highly structured DNA inhibits conformational changes in Pif1 that couple ATP hydrolysis to DNA translocation and unwinding.

A transgenic cell line with inducible transcription for studying (CGG)n repeat expansion mechanisms.

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington’s disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully confirmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantification was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for finding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantification of exogenous (CGG)n repeat instability in the transgenic cell lines’ genome.

Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control.

Transcriptional riboswitches involve RNA aptamers that are typically found in the 5′ untranslated regions (UTRs) of bacterial mRNAs and form alternative secondary structures upon binding to cognate ligands. Alteration of the riboswitch's secondary structure results in perturbations of an adjacent expression platform that controls transcription elongation and termination, thus turning downstream gene expression “on” or “off.” Riboswitch ligands are typically small metabolites, divalent cations, anions, signaling molecules, or other RNAs, and can be part of larger signaling cascades. The interconnectedness of ligand binding, RNA folding, RNA transcription, and gene expression empowers riboswitches to integrate cellular processes and environmental conditions across multiple timescales. For a successful response to an environmental cue that may determine a bacterium's chance of survival, a coordinated coupling of timescales from microseconds to minutes must be achieved. This review focuses on recent advances in our understanding of how riboswitches affect such critical gene expression control across time.

2'-O-Methylation can increase the abundance and lifetime of alternative RNA conformational states.

2′-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3′-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.

Representation of the conformational ensemble of peptides in coarse grained simulations.

In their native state, many proteins/peptides display an ensemble of conformations, rather than a unique tertiary structure. Novel experimental techniques have enabled a quantitative analysis of this structural heterogeneity. In molecular dynamics simulations, however, capturing this conformational ensemble quantitatively remains a major challenge even with all atom simulations. In coarse grained (CG) simulations, with fewer degrees of freedom, representation of the conformational ensemble becomes more problematic. Here, we revisit a CG model from our group, which was designed to address the conformational transferability problem by using the LKα14 peptide as a model system. The LKα14 peptide transitions from a random/unstructured state in dilute solution to a solely α-helical conformation upon aggregation as evidenced by circular dichroism. Here, we demonstrate that the structure/physics based approach, used in the original parameterization of our CG model, strongly depends on the reference system chosen and excluded volume interactions that are often considered to be of secondary importance. We first tune the excluded volume parameters by using both α-helix and β-sheet type structures as reference and then update the nonbonded interactions by using a goodness-of-fit metric for representation of the conformational ensemble of LKα14. We demonstrate that the updated model can recover the whole conformational ensemble quantitatively while maintaining the aggregation driven conformational transition. This balanced parametrization with regard to alternative secondary structures opens the door for the generalization of the CG model to other sequences, which we demonstrate on a β-sheet forming triblock peptide.

Helix-Based RNA Landscape Partition and Alternative Secondary Structure Determination.

RNA is a versatile macromolecule with the ability to fold into and interconvert between multiple functional conformations. The elucidation of the RNA folding landscape, especially the knowledge of alternative structures, is critical to uncover the physical mechanism of RNA functions. Here, we introduce a helix-based strategy for RNA folding landscape partition and alternative secondary structure determination. The benchmark test of 27 RNAs involving alternative stable structures shows that the model has the ability to divide the whole landscape into distinct partitions at the secondary structure level and predict the representative structures for each partition. Furthermore, the predicted structures and equilibrium populations of metastable conformations for the 2′dG-sensing riboswitch reveal the allosteric conformational switch on transcript length, which is consistent with the experimental study, indicating the importance of metastable states for RNA-based gene regulation. The model delivers a starting point for the landscape-based strategy toward the RNA folding mechanism and functions.

Coarse-grained dynamic RNA titration simulations.

Electrostatic interactions play a pivotal role in many biomolecular processes. The molecular organization and function in biological systems are largely determined by these interactions. Owing to the highly negative charge of RNA, the effect is expected to be more pronounced in this system. Moreover, RNA base pairing is dependent on the charge of the base, giving rise to alternative secondary and tertiary structures. The equilibrium between uncharged and charged bases is regulated by the solution pH, which is therefore a key environmental condition influencing the molecule's structure and behaviour. By means of constant-pH Monte Carlo simulations based on a fast proton titration scheme, coupled with the coarse-grained model HiRE-RNA, molecular dynamic simulations of RNA molecules at constant pH enable us to explore the RNA conformational plasticity at different pH values as well as to compute electrostatic properties as local pK a values for each nucleotide.

MiR-122 and Ago interactions with the HCV genome alter the structure of the viral 5' terminus.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5′ untranslated region (UTR) and this interaction promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5′ UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1–117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5′ terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5′ UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5′ terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES–40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5′ terminus and promote formation of the HCV IRES.

Mimicking Co-Transcriptional RNA Folding Using a Superhelicase.

Vectorial folding of RNA during transcription can produce intermediates with distinct biochemical activities. Here, we design an artificial minimal system to mimic cotranscriptional RNA folding in vitro. In this system, a presynthesized RNA molecule begins to fold from its 5'-end, as it is released from a heteroduplex by an engineered helicase that translocates on the complementary DNA strand in the 3'-to-5' direction. This chemically stabilized "superhelicase" Rep-X processively unwinds thousands of base pairs of DNA. The presynthesized RNA enables us to flexibly position fluorescent labels on the RNA for single-molecule fluorescence resonance energy transfer analysis and allows us to study real-time conformational dynamics during the vectorial folding process. We observed distinct signatures of the maiden secondary and tertiary folding of the Oryza sativa twister ribozyme. The maiden vectorial tertiary folding transitions occurred faster than Mg2+-induced refolding, but were also more prone to misfolding, likely due to sequential formation of alternative secondary structures. This novel assay can be applied to studying other kinetically controlled processes, such as riboswitch control and RNA-protein assembly.