Kissing Loop Research Articles

Programmable Computational RNA Droplets Assembled via Kissing-Loop Interaction.

DNA droplets, artificial liquid-like condensates of well-engineered DNA sequences, allow the critical aspects of phase-separated biological condensates to be harnessed programmably, such as molecular sensing and phase-state regulation. In contrast, their RNA-based counterparts remain less explored despite more diverse molecular structures and functions ranging from DNA-like to protein-like features. Here, we design and demonstrate computational RNA droplets capable of two-input AND logic operations. We use a multibranched RNA nanostructure as a building block comprising multiple single-stranded RNAs. Its branches engaged in RNA-specific kissing-loop (KL) interaction enables the self-assembly into a network-like microstructure. Upon two inputs of target miRNAs, the nanostructure is programmed to break up into lower-valency structures that are interconnected in a chain-like manner. We optimize KL sequences adapted from viral sequences by numerically and experimentally studying the base-wise adjustability of the interaction strength. Only upon receiving cognate microRNAs, RNA droplets selectively show a drastic phase-state change from liquid to dispersed states due to dismantling of the network-like microstructure. This demonstration strongly suggests that the multistranded motif design offers a flexible means to bottom-up programming of condensate phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change provides a naked-eye-distinguishable readout of molecular sensing. Our computational RNA droplets can be applied to in situ programmable assembly of computational biomolecular devices and artificial cells from transcriptionally derived RNA within biological/artificial cells.

Cryo-EM structure and functional landscape of an RNA polymerase ribozyme.

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide triphosphates (triplets) as substrates - have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-Å resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70° angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs.

Structural Expansion of Catalytic RNA Nanostructures through Oligomerization of a Cyclic Trimer of Engineered Ribozymes.

The multimolecular assembly of three-dimensionally structured proteins forms their quaternary structures, some of which have high geometric symmetry. The size and complexity of protein quaternary structures often increase in a hierarchical manner, with simpler, smaller structures serving as units for larger quaternary structures. In this study, we exploited oligomerization of a ribozyme cyclic trimer to achieve larger ribozyme-based RNA assembly. By installing kissing loop (KL) interacting units to one-, two-, or three-unit RNA molecules in the ribozyme trimer, we constructed dimers, open-chain oligomers, and branched oligomers of ribozyme trimer units. One type of open-chain oligomer preferentially formed a closed tetramer containing 12 component RNAs to provide 12 ribozyme units. We also observed large assembly of ribozyme trimers, which reached 1000 nm in size.

Molecular Dynamics Simulations of RNA Motifs to Guide the Architectural Parameters and Design Principles of RNA Nanostructures.

Molecular dynamics (MD) simulations can be used to investigate the stability and conformational characteristics of RNA nanostructures. However, MD simulations of an RNA nanostructure is computationally expensive due to the size of nanostructure and the number of atoms. Alternatively, MD simulations of RNA motifs can be used to estimate the conformational stability of constructed RNA nanostructure due to their small sizes. In this chapter, we introduce the preparation and MD simulations of two RNA kissing loop (KL) motifs, a linear KL complex and a bent KL complex, and an RNA nanoring. The initial solvated system and topology files of each system will be prepared by two major force fields, AMBER and CHARMM force fields. MD simulations will be performed by NAMD simulation package, which can accept both force fields. In addition, we will introduce the use of the AMBER cpptraj program and visual molecular dynamics (VMD) for data analysis. We will also discuss how MD simulations of two KL motifs can be used to estimate the conformation and stability of RNA nanoring as well as to explain the vibrational characteristics of RNA nanoring.

An RNA Paranemic Crossover Triangle as A 3D Module for Cotranscriptional Nanoassembly.

RNA nanotechnology takes advantage of structural modularity to build self-assembling nano-architectures with applications in medicine and synthetic biology. The use of paranemic motifs, that form without unfolding existing secondary structure, allows for the creation of RNA nanostructures that are compatible with cotranscriptional folding in vitro and in vivo. In previous work, kissing-loop (KL) motifs have been widely used to design RNA nanostructures that fold cotranscriptionally. However, the paranemic crossover (PX) motif has not yet been explored for cotranscriptional RNA origami architectures and information about the structural geometry of the motif is unknown. Here, a six base pair-wide paranemic RNA interaction that arranges double helices in a perpendicular manner is introduced, allowing for the generation of a new and versatile building block: the paranemic-crossover triangle (PXT). The PXT is self-assembled by cotranscriptional folding and characterized by cryogenic electron microscopy, revealing for the first time an RNA PX interaction in high structural detail. The PXT is used as a building block for the construction of multimers that form filaments and rings and a duplicated PXT motif is used as a building block to self-assemble cubic structures, demonstrating the PXT as a rigid self-folding domain for the development of wireframe RNA origami architectures.

Structural Landscape of the Transition from an ssDNA Dumbbell Plus Its Complementary Hairpin to a dsDNA Microcircle Via a Kissing Loop Intermediate.

The experimental construction of a double-stranded DNA microcircle of only 42 base pairs entailed a great deal of ingenuity and hard work. However, figuring out the three-dimensional structures of intermediates and the final product can be particularly baffling. Using a combination of model building and unrestrained molecular dynamics simulations in explicit solvent we have characterized the different DNA structures involved along the process. Our 3D models of the single-stranded DNA molecules provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in the single-stranded loops of the dumbbell. We propose that a kissing loop involving six base pairs makes up the core of the nascent dsDNA microcircle. We also suggest a feasible pathway for the hybridization of the remaining complementary bases and characterize the final covalently closed dsDNA microcircle as possessing two well-defined U-turns. Additional models of the pre-ligation complex of T4 DNA ligase with the DNA dumbbell and the post-ligation pre-release complex involving the same enzyme and the covalently closed DNA microcircle are shown to be compatible with enzyme recognition and gap ligation.

Thermodynamic investigation of kissing-loop interactions

Kissing loop interactions (KLIs) are a common motif that is critical in retroviral dimerization, viroid replication, mRNA, and riboswitches. In addition, KLIs are currently used in a variety of biotechnology applications, such as in aptamer sensors, RNA scaffolds and to stabilize vaccines for therapeutics. Here we describe the thermodynamics of a basic intramolecular DNA capable of engaging in a KLI, consisting of two hairpins connected by a flexible linker. Each hairpin loop has a five-nucleotide complementary sequence theoretically capable of engaging in a KLI. On either side of each loop is two thymines which will not engage in kissing but are present to provide more flexibility and optimal KLI positioning. Our results suggest that the KLI occurs even at physiological salt levels, and that the KLI does not alter the thermodynamics and stability of the two stem structures. The KLI does not involve all five nucleotides, or at least each base-pair stack is not making full contact. Adding a second strand complementary to the bottom of the kissing complex removes flexibility and causes destabilization of the stems. The KLI of this less flexible complex is maintained but the TM is reduced, indicating an entopic penalty to its formation.

Eukaryotic Hibernating Ribosome Dimers are Maintained by a Kissing Loop Formed by Ribosomal RNA

Eukaryotic Hibernating Ribosome Dimers are Maintained by a Kissing Loop Formed by Ribosomal RNA

An unexpected RNA distal interaction mode found in an essential region of the hepatitis C virus genome.

The 3’X tail is a functionally essential 98-nt sequence located at the 3′-end of the hepatitis C virus (HCV) RNA genome. The domain contains two absolutely conserved dimer linkage sequence (DLS) and k nucleotide segments involved in viral RNA dimerization and in a distal base-pairing interaction with stem-loop 5BSL3.2, respectively. We have previously shown that domain 3’X forms an elongated structure comprising two coaxially stacked SL1’ and SL2’ stem-loops. This conformation favors RNA dimerization by exposing a palindromic DLS segment in an apical loop, but buries in the upper stem of hairpin SL2’ the k nucleotides involved in the distal contact with 5BSL3.2. Using nuclear magnetic resonance spectroscopy and gel electrophoresis experiments, here we show that the establishment of the complex between domain 3’X and stem-loop 5BSL3.2 only requires a rearrangement of the nucleotides forming the upper region of subdomain SL2’. The results indicate that the interaction does not occur through a canonical kissing loop mechanism involving the unpaired nucleotides of two terminal loops, but rather involves a base-paired stem and an apical loop and may result in a kissing three-way junction. On the basis of this information we suggest how the 3’X tail switches between monomer, homodimer and heterodimer states to regulate the HCV viral cycle.

Mg2+ Binding Promotes SLV as a Scaffold in Varkud Satellite Ribozyme SLI-SLV Kissing Loop Junction

Though the structure of the substrate stem loop I (SLI)-stem loop V (SLV) kissing loop junction of the Varkud Satellite ribozyme has been experimentally characterized, the dynamics of this Mg2+-dependent loop-loop interaction have been elusive. Specifically, each hairpin loop contains a U-turn motif, but only SLV shows a conformational shift triggered by Mg2+ ion association. Here, we use molecular dynamics simulations to analyze the binding and dynamics of this kissing loop junction. We show that SLV acts as a scaffold, providing stability to the junction. Mg2+ ions associate with SLV when it is part of the junction in a manner similar to when it is unbound, but there is no specificity in Mg2+ binding for the SLI loop. This suggests that the entropic penalty of ordering the larger SLI is too high, allowing SLV to act as a scaffold for multiple substrate loop sequences.

A colorimetric nanosensor based on a selective target-responsive aptamer kissing complex.

Herein, we report a novel approach for the design of a colorimetric aptasensor based on functionalized gold nanoparticle probes. This approach relies on the conjugation of nanoparticles by two functional DNA and RNA hairpins that engage specific kissing (loop-loop) interactions in response to the addition of a small analyte ligand, leading to particle aggregation and then red-to-purple colour change of the colloidal solution.

A trifunctional, triangular RNA–protein complex

Multifunctional molecular complexes are valuable tools with a variety of applications. We have developed an RNA–protein complex (RNP) containing three different proteins attached to the tips of a triangular RNA scaffold. We designed and constructed three RNA strands that specifically bind a ribosomal protein, L7Ae, and that autonomously form a single triangular RNP via RNA kissing loop (KL) interactions. This RNP-based approach can be used as an alternative tool to produce unique, multifunctional molecules with customized dimensions, functions, and targets.

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.

Single-molecule conformational dynamics of a biologically functional hydroxocobalamin riboswitch.

Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression; commonly, this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain. Numerous studies have investigated the ligand binding process, but little is known about the structural changes in the regulatory element. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure.1 The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context. The results of this investigation reveal that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction is substantially decreased when the ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction directly sequesters the Shine-Dalgarno sequence (i.e., the ribosome binding site) via base-pairing, thus preventing translation initiation. These results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four-state kinetic model, whereby ligand binding promotes formation of the KL interaction. The results of complementary cell-based gene expression experiments conducted in Escherichia coli are highly correlated with the smFRET results, suggesting that KL formation is directly responsible for regulating gene expression.

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme.

Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.

Effect of AU Substitutions at the Stem on the Stability of Kissing Loop Complexes

Kissing loop complexes form when two RNA hairpins interact through their complementary loops. We have investigated the role of the helical stems adjacent to the loops on the overall stability of a model kissing loop complex.Our results show that a minimum length of 6 base pairs in the hairpin stems is required to form kissing loop complexes (in the absence of Mg2+) and that changing a GC base pair to an AU base pair near the loop facilitates the formation of kissing loop complexes. For instance, the melting temperature (Tm) of a kissing loop complex with a GC→AU substitution near the loop is 6 °C higher than the Tm of the parent complex. To determine if this stabilization is due to a decreased stability of the hairpin stems, we have tested the effect of AU substitutions that are further away from the loop. Our results show that these far away substitutions do not stabilize the kissing loop complex, suggesting that the facilitation observed with AU substitutions near the loop arises from specific effects at the stem-loop interface and not from an overall decrease in the stability of the stems. We are currently investigating whether AU substitutions near the loop eliminate a metal-ion binding site or affect the stacking interactions between the loops. Our overall results contribute to a better understanding of the rules governing the formation of kissing loop complexes, which are important to rationalize the principles of RNA-RNA recognition.

Effect of Helix Stability on the Formation of Loop–Loop Complexes

Kissing loop complexes are loop-loop complexes where two RNA hairpins interact through their complementary loops. In this work, we have investigated the role of the helical stems on the ability of hairpins derived from the ColE1 plasmid to associate as kissing loop complexes in the presence and absence of divalent cations. Our results show that although kissing loop complexes form more readily in the presence of Mg(2+), they are able to form in the presence of 850 mM NaCl, as long as their stems contain at least six base-pairs. Formation of the Na(+) loop-loop complexes is facilitated by changing the sequence at the stem-loop interface to include less stable AU base pairs. We suggest that the conformation at the stem-loop interface is critical in the formation of kissing loop complexes and that in the absence of Mg(2+) the conformation at the stem-loop interface is packed more loosely than with Mg(2+), to allow for a lower charge density. Consistent with this hypothesis, shortening the stems to five base pairs results in unfolding of the hairpins and formation of an extended duplex rather than a kissing loop complex because the short stems are not stable enough to tolerate the necessary conformation at the stem-loop interface to allow the formation of a kissing loop complex.

Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture

Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure–function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing–loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (kcat/KM) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing–loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

Understanding the effects of carbocyclic sugars constrained to north and south conformations on RNA nanodesign

Relatively new types of the modified nucleotides, namely carbocyclic sugars that are constrained to north or south (C2′ or C3′ exo) conformations, can be used for RNA nanoparticle design to control their structures and stability by rigidifying nucleotides and altering the helical properties of RNA duplexes. Two RNA structures, an RNA dodecamer and an HIV kissing loop complex where several nucleotides were replaced with north or south constrained sugars, were studied by molecular dynamics (MD) simulations. The substituted south constrained nucleotides in the dodecamer widened the major groove and narrowed and deepened the minor groove thus inducing local conformational changes that resemble a B-form DNA helix. In the HIV kissing loop complex, north and south constrained nucleotides were substituted into flanking bases and stems. The modified HIV kissing loop complex showed a lower RMSD value than the normal kissing loop complex. The overall twist angle was also changed and its standard deviation was reduced. In addition, the modified RNA dodecamer and HIV kissing loop complex were characterized by principal component analysis (PCA) and steered molecular dynamics (SMD). PCA results showed that the constrained sugars stabilized the overall motions. The results of the SMD simulations indicated that as the backbone δ angles were increased by elongation, more force was applied to the modified RNA due to the constrained sugar analogues.

Molecular Simulation Studies of Monovalent Counterion-Mediated Interactions in a Model RNA Kissing Loop

A kissing loop is a highly stable complex formed by loop-loop base-pairing between two RNA hairpins. This common structural motif is utilized in a wide variety of RNA-mediated processes, including antisense recognition, substrate recognition in riboswitches, and viral replication. Recent work has shown that the Tar-Tar(*) complex, an archetypal kissing loop, can form without Mg(2+), so long as high concentrations of alkali chloride salts are present. Interestingly, the stability of the complex is found to decrease with increasing cation size. In this work, we used molecular simulations to develop a molecular-level understanding of the origins of the observed counterion specificity. The ionic atmosphere of the Tar-Tar(*) complex was examined in the presence of 800 mm (where m denotes molality) NaCl, KCl, or CsCl. We used spatial free-energy density profiles to analyze differences in counterion accumulation at different spatial extents from the RNA molecule. We found that the lowest free-energy levels, which are situated in the vicinity of the loop-loop interface, can accommodate roughly two counterions, irrespective of counterion type. However, as we moved into higher free-energy levels, which are farther from the loop-loop interface, we observed increased differences in the numbers of accumulated counterions, with this number being largest for Na(+) and smallest for Cs(+). We analyzed the source of these differences and were able to attribute these to two distinct features: The extent of partial dehydration varies based on cation type; the smaller the cation, the greater the degree of dehydration. While smaller ions bind their first-hydration-shell water molecules more tightly than larger ions, they are also able to shed these water molecules for stronger electrostatic interactions with the RNA molecule. Secondly, we observed a distinct asymmetry in the numbers of accumulated cations around each hairpin in the Tar-Tar(*) complex. We were able to ascribe this asymmetry to the presence of a guanine tract in the Tar hairpin, which facilitates partial dehydration of the counterions. However, the smaller ions compensate for this asymmetry by forming a belt around the loop-loop interface in intermediate free-energy levels. As a result, the degree of asymmetry in counterion accumulation around individual hairpins shows an inverse correlation with the experimentally observed cation specificity for the stability of Tar-Tar(*) (i.e., the smaller the asymmetry, the greater the experimentally observed stability). This in turn provides a plausible explanation for why the smaller cations help stabilize the Tar-Tar(*) complex better than the larger cations. These findings suggest that the specific sequence and structural features of the Tar-Tar(*) complex may be the source of the experimentally observed cation specificity in Tar-Tar(*) stability. Our results lead to testable predictions for how changes in sequence might alter the observed counterion specificity in kissing loop stability.