Three-way Junction Research Articles

Ni(II) Cylinders Damage DNA in Cancer Cells and Preferentially Bind Y-Shaped DNA Three-Way Junctions Blocking DNA Synthesis.

DNA three-way junctions are critical in various biological processes and hold significant potential for disease treatment and therapeutic applications. In this study, it is demonstrated that triple-stranded dinuclear [Ni2L3]4+ cylinders (L = C25H20N4) exhibit a preferential binding affinity for Y-shaped DNA three-way junctions (3WJs), even in the presence of an excess of competing DNA structures, including G-quadruplexes. Notably, the investigated Ni(II) cylinders are capable of halting DNA synthesis catalyzed by DNA polymerase by stabilizing the 3WJ on the template strand. Using an extended 1D nanoarchitecture model, it is further established the high affinity and selectivity of the cylinders for DNA 3WJs and explored their potential application in stabilizing short-armed 3WJs for constructing DNA nanomaterials. The combined use of Ni(II) cylinders and DNA damage response inhibitors also revealed that the cylinders promote DNA damage, leading to the formation of double-strand breaks. This effect is likely associated with i) the binding of cylinders to 3WJs and ii) the cytotoxic activity of the cylinders in cancer cells.

An ultrasensitive liquid crystal aptasensing chip assisted by three-way junction DNA pockets for acrylamide detection in food samples

Acrylamide, an unsaturated amide found in heat-processed foods, poses serious risks to human health due to its neurotoxicity, carcinogenicity, and genotoxicity. This highlights the importance of quantitative determination of acrylamide in foods and the environments to ensure public health safety. Therefore, there is an urgent need for simple, rapid, and highly sensitive methods to accurately quantify acrylamide. In the present study, a user-friendly aptasensor was designed to quantify ultra-low levels of acrylamide in nuts for the first time. This innovative approach utilizes chemical engineering of a glass slide as a portable sensing platform, which incorporates liquid crystal (LC) molecules and a three-way junction (TWJ) DNA pocket. The immobilized TWJ pocket can disrupt the vertical alignment of LCs, turning the dark polarized background of the aptasensor to a colorful state. The binding of the specific aptamer to acrylamide disrupts the TWJ structure, enabling the LCs to return to their homotropic alignment. This structural change restores the dark polarized view of the sensing platform. The TWJ-engineered LC aptasensor effectively detects ultra-low concentrations of acrylamide in the range of 0.0005 to 50 fmol/L, with a detection limit of 0.106 amol/L. The aptasensor was successfully applied to real roasted nut samples, including peanut, almond, pistachio, and hazelnut, achieving recovery values ranging from 96.84 % to 99.61 %. With its simplicity, portability, ease of use, and cost-effectiveness, this aptasensor is a powerful sensing device for food safety monitoring.

Combining isolated G-quadruplexes with CHA-3WJ/DNA: A triple signal amplification colorimetric biosensor for detecting Escherichia coli O157: H7

In this study, a colorimetric biosensor was developed based on the use of aptamer-functionalized magnetic beads and catalytic hairpin assembly (CHA), in conjunction with a three-way junction DNA (3WJ/DNA) and isolated G-quadruplex/hemin DNAzyme triple signal amplification. This approach enabled the label-free, visual and sensitive detection of Escherichia coli O157: H7. The target bacterium binds to the aptamer, which releases complementary DNA, thereby triggering the CHA to produce 3WJ/DNA. The three ends of 3WJ/DNA can each form a G-quadruplex/DNAzyme, thereby catalyzing the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system to turn blue. Notably, we provide a triple signal amplification method of G-quadruplex/hemin DNAzyme based on the triple end of CHA-3WJ/DNA by cleverly screening and designing an isolated G-quadruplex, significantly improving the sensitivity. The proposed method’s limit of detection (LOD) was 8 CFU/mL, exhibiting good linearity within the range of 100-108 CFU/mL. Impressively, the method successfully detected E. coli O157: H7 in ultrahigh-temperature-sterilized, pasteurized and raw milk, exhibiting good stability and reproducibility.

Individual-portal support with a pre-tensioned canopy for a coal mine roadway junction

Abstract This paper presents an alternative model of individual-portal support structure with a pre-tensioned canopy for coal mine roadway junctions in the geological and mining conditions of underground mines in Vietnam. The aim of the research is to determine the bearing capacity of the individual-portal support structure with various configuration. For this purpose, an analysis of the rock mass behaviour around the three-way roadway junction was carried out using the difference element method-based programme (FLAC3D). Additionally, other analysis of the bearing capacity of the selected individual-portal support structure was also conducted using the finite element method-based programme (COSMOS/M). The geological and mining conditions of Vietnamese mines were taken into account during performing these analyses. Based on the results, the optimal individual-portal support structure was proposed for the roadway junctions driven in the geological and mining conditions of underground mines in Vietnam.

Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq.

Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features thought to be required for hTERT binding. The proportion of hTR CR4/5 that is folded into the primary functional conformation does not require hTERT expression and the fraction of hTR that assumes a misfolded CR4/5 domain is not refolded by overexpression of its hTERT binding partner. This result suggests a functional role for an RNA folding cofactor other than hTERT during telomerase biogenesis. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in telomerase RNP complexes purified from cells via an epitope tag on hTERT, supporting the hypothesis that only the major CR4/5 conformer is active. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.

Engineering acyclovir-induced RNA nanodevices for reversible and tunable control of aptamer function

Small molecule-regulated RNA devices have the potential to modulate diverse aspects of cellular function, but the small molecules used to date have potential toxicities limiting their use in cells. Here we describe a method for creating drug-regulated RNA nanodevices (RNs) using acyclovir, a biologically compatible small molecule with minimal toxicity. Our modular approach involves a scaffold comprising a central F30 three-way junction, an integrated acyclovir aptamer on the input arm, and a variable effector-binding aptamer on the output arm. This design allows for the rapid engineering of acyclovir-regulated RNs, facilitating temporal, tunable, and reversible control of intracellular aptamers. We demonstrate the control of the Broccoli aptamer and the iron-responsive element (IRE) by acyclovir. Regulating the IRE with acyclovir enables precise control over iron-regulatory protein (IRP) sequestration, consequently promoting the inhibition of ferroptosis. Overall, the method described here provides a platform for transforming aptamers into acyclovir-controllable antagonists against physiologic target proteins.

Organelle communication maintains mitochondrial and endosomal homeostasis during podocyte lipotoxicity.

Organelle stress exacerbates podocyte injury, contributing to perturbed lipid metabolism. Simultaneous organelle stresses can occur in the kidney in the diseased state; therefore, a thorough analysis of organelle communication is crucial for understanding the progression of kidney diseases. Although organelles closely interact with one another at membrane contact sites, limited studies have explored their involvement in kidney homeostasis. The endoplasmic reticulum (ER) protein, PDZ domain-containing 8 (PDZD8), is implicated in multiple-organelle-tethering processes and cellular lipid homeostasis. In this study, we aimed to elucidate the role of organelle communication in podocyte injury using podocyte-specific Pdzd8-knockout mice. Our findings demonstrated that Pdzd8 deletion exacerbated podocyte injury in an accelerated obesity-related kidney disease model. Proteomic analysis of isolated glomeruli revealed that Pdzd8 deletion exacerbated mitochondrial and endosomal dysfunction during podocyte lipotoxicity. Additionally, electron microscopy revealed the accumulation of abnormal, fatty endosomes in Pdzd8-deficient podocytes during obesity-related kidney diseases. Lipidomic analysis indicated that glucosylceramide accumulated in Pdzd8-deficient podocytes, owing to accelerated production and decelerated degradation. Thus, the organelle-tethering factor, PDZD8, plays a crucial role in maintaining mitochondrial and endosomal homeostasis during podocyte lipotoxicity. Collectively, our findings highlight the importance of organelle communication at the 3-way junction among the ER, mitochondria, and endosomes in preserving podocyte homeostasis.

Three-way junction-mediated three-letter coded SDA cascade CRISPR/Cas12a system for circRNA detection

Circular RNAs (circRNAs), a special type of non-coding RNAs with covalently closed circular structure, have emerged as promising liquid biopsy biomarker. However, the precise detection of circRNAs is severely hampered by interference from homologous linear RNAs, posing a significant obstacle for their applications in molecular diagnostics. Herein, a strategy named three-way junction (TWJ)-mediated three-letter coded strand displacement amplification (SDA) cascade CRISPR/Cas12a system (TTSC) has been devised to achieve precise detection of circRNAs. Three-letter coded SDA (TC-SDA) has been originally designed in this study for eliminating undesired interference from linear RNA and performing dual functionality in both discrimination and amplification. With this design, the proposed strategy elegantly distinguishes between circRNA and homologous linear RNA through proximity effect of TWJ structure and non-specific amplification blocking ability of the three-letter code. Ultimately, this strategy successfully identified circRNA down to 0.1 % abundance. In addition, benefiting from the synergistically accelerated multiple signal amplification in one step, TTSC strategy exhibited good linear relationship of 0.5–1000 pM with a detection limit of 80.6 fM. Notably, the recovery test indicated that the proposed strategy has good prospects for clinical applications. Therefore, TTSC is expected to provide an ideal platform for circRNA detection in biotechnology research and molecular diagnostics.

A three-way organelle junction controls PI(4)P metabolism and mitochondrial division.

Membrane contact sites (MCS) facilitate communication between organelles. Casler et al. (https://doi.org/10.1083/jcb.202308144) show that tripartite MCS between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM) regulate mitochondrial division and the distribution of phosphatidylinositol 4-phosphate [PI(4)P] on the PM.

A single CAA interrupt in a DNA three-way junction containing a CAG repeat hairpin results in parity-dependent trapping.

An increasing number of human disorders are attributed to genomic expansions of short tandem repeats (STRs). Secondary DNA structures formed by STRs are believed to play an important role in expansion, while the presence of nucleotide interruptions within the pure repeat sequence isknown to delay the onset and progression of disease. We have used two single-molecule fluorescence techniques to analyse the structure and dynamics of DNA three-way junctions (3WJs) containing CAG repeat hairpin slipouts, with and without a single CAA interrupt. For a 3WJ with a (CAG)10 slipout, the CAA interrupt is preferentially located in the hairpin loop, and the branch migration dynamics are 4-fold slower than for the 3WJ with a pure (CAG)10, and 3-fold slower than a 3WJ with a pure (CAG)40 repeat. The (CAG)11 3WJ with CAA interrupt adopts a conformation that places the interrupt in or near the hairpin loop, with similar dynamics to the pure (CAG)10 and (CAG)11 3WJs. We have shown that changing a single nucleotide (G to A)in a pure repeat can have a large impact on 3WJ structure and dynamics, which may be important for the protective role of interrupts in repeat expansion diseases.

Tuning phenolic hemicyanines for ratiometric DNA sensing and live cell nuclear bioimaging applications

Activatable fluorescent probes with turn-on emission in response to a biological target can reduce the background signal and improve the detection limit needed for biosensor and bioimaging resolution. Turn-on probes with dual emission (ratiometric probes) have further advantages, as they have self-calibration ability for more reliable detection. Herein, we demonstrate the tunability of the phenolic hemicyanine (HC) scaffold for both DNA biosensing and bioimaging applications using the quinine-binding DNA aptamer (MN4) as a host-guest biosensor platform and live ovarian cancer cells for nuclear imaging. The DNA aptamer MN4 contains a three-way junction (3WJ) consisting of a hydrophobic branch point connecting three double-stranded stems. Phenolic HCs bearing electron-withdrawing halogen substituents (FPhOBtz and Cl2PhOBTz) bind to the 3WJ of MN4 as the phenolate with strong turn-on emission. Subsequent displacement of the phenolate by the quinine target causes a turn-off emission response. In contrast, the parent phenolic HC (PhOBtz) and those bearing electron-donating groups (Me2PhOBtz and (MeO)2PhOBtz) exhibit dual (phenol and phenolate) fluorescence upon MN4 binding. Phenol excitation generates phenolate emission by an excited-state proton transfer (ESPT) process with DNA components serving as the proton acceptor. Quinine displacement of PhOBtz from the 3WJ of MN4 affords a turn-on ratiometric response with preferential light-up of phenol emission. The ability of the phenolic HCs to serve as cellular nuclear stains further highlights the tunability of the phenolic HC probes with the Cl2PhOBtz analog displaying superior staining ability. The simplicity and tunability of phenolic HCs make them attractive fluorescent probes for DNA sensing and bioimaging applications.

Structure-based investigation of a DNA aptamer targeting PTK7 reveals an intricate 3D fold guiding functional optimization

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.

Split T7 switch-mediated cell-free protein synthesis system for detecting target nucleic acids

Cell-free protein synthesis (CFPS) reactions can be used to detect nucleic acids. However, most CFPS systems rely on a toehold switch and exhibit the following critical limitations: (i) off-target signals due to leaky translation in the absence of target nucleic acids, (ii) a suboptimal detection limit of approximately 30 nM without pre-amplification, and (iii) labor-intensive screening processes due to sequence constraints for the target nucleic acids. To overcome these shortcomings, we developed a new split T7 switch-mediated CFPS system in which the split T7 promoter was applied to a three-way junction structure to selectively initiate transcription–translation only in the presence of target nucleic acids. Both fluorescence and colorimetric detection systems were constructed by employing different reporter proteins. Notably, we introduced the self-complementation of split fluorescent proteins to streamline preparation of the proposed system, enabling versatile applications. Operation of this one-pot approach under isothermal conditions enabled the detection of target nucleic acids at concentrations as low as 10 pM, representing more than a thousand times improvement over previous toehold switch-based approaches. Furthermore, the proposed system demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins encoded in the expression region. By eliminating issues associated with the previous toehold switch system, our split T7 switch-mediated CFPS system could become a core platform for detecting various target nucleic acids.

Retroviral PBS-segment sequence and structure: Orchestrating early and late replication events

An essential regulatory hub for retroviral replication events, the 5’ untranslated region (UTR) encodes an ensemble of cis-acting replication elements that overlap in a logical manner to carry out divergent RNA activities in cells and in virions. The primer binding site (PBS) and primer activation sequence initiate the reverse transcription process in virions, yet overlap with structural elements that regulate expression of the complex viral proteome. PBS-segment also encompasses the attachment site for Integrase to cut and paste the 3’ long terminal repeat into the host chromosome to form the provirus and purine residues necessary to execute the precise stoichiometry of genome-length transcripts and spliced viral RNAs. Recent genetic mapping, cofactor affinity experiments, NMR and SAXS have elucidated that the HIV-1 PBS-segment folds into a three-way junction structure. The three-way junction structure is recognized by the host’s nuclear RNA helicase A/DHX9 (RHA). RHA tethers host trimethyl guanosine synthase 1 to the Rev/Rev responsive element (RRE)-containing RNAs for m7-guanosine Cap hyper methylation that bolsters virion infectivity significantly. The HIV-1 trimethylated (TMG) Cap licenses specialized translation of virion proteins under conditions that repress translation of the regulatory proteins. Clearly host-adaption and RNA shapeshifting comprise the fundamental basis for PBS-segment orchestrating both reverse transcription of virion RNA and the nuclear modification of m7G-Cap for biphasic translation of the complex viral proteome. These recent observations, which have exposed even greater complexity of retroviral RNA biology than previously established, are the impetus for this article. Basic research to fully comprehend the marriage of PBS-segment structures and host RNA binding proteins that carry out retroviral early and late replication events is likely to expose an immutable virus-specific therapeutic target to attenuate retrovirus proliferation.Graphical abstract

Amodiaquine Nonspecifically Binds Double Stranded and Three-Way Junction DNA Structures.

The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of μM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy. These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.

Structural Optimization of Azacryptands for Targeting Three-Way DNA Junctions.

Transient melting of the duplex-DNA (B-DNA) during DNA transactions allows repeated sequences to fold into non-B-DNA structures, including DNA junctions and G-quadruplexes. These noncanonical structures can act as impediments to DNA polymerase progression along the duplex, thereby triggering DNA damage and ultimately jeopardizing genomic stability. Their stabilization by ad hoc ligands is currently being explored as a putative anticancer strategy since it might represent an efficient way to inflict toxic DNA damage specifically to rapidly dividing cancer cells. The relevance of this strategy is only emerging for three-way DNA junctions (TWJs) and, to date, no molecule has been recognized as a reference TWJ ligand, featuring both high affinity and selectivity. Herein, we characterize such reference ligands through a combination of in vitro techniques comprising affinity and selectivity assays (competitive FRET-melting and TWJ Screen assays), functional tests (qPCR and Taq stop assays) and structural analyses (molecular dynamics and NMR investigations). We identify novel azacryptands TrisNP-amphi and TrisNP-ana as the most promising ligands, interacting with TWJs with high affinity and selectivity. These ligands represent new molecular tools to investigate the cellular roles of TWJs and explore how they can be exploited in innovative anticancer therapies.

Atlastin-1 regulates endosomal tubulation and lysosomal proteolysis in human cortical neurons

Mutation of the ATL1 gene is one of the most common causes of hereditary spastic paraplegia (HSP), a group of genetic neurodegenerative conditions characterised by distal axonal degeneration of the corticospinal tract axons. Atlastin-1, the protein encoded by ATL1, is one of three mammalian atlastins, which are homologous dynamin-like GTPases that control endoplasmic reticulum (ER) morphology by fusing tubules to form the three-way junctions that characterise ER networks. However, it is not clear whether atlastin-1 is required for correct ER morphology in human neurons and if so what the functional consequences of lack of atlastin-1 are. Using CRISPR-inhibition we generated human cortical neurons lacking atlastin-1. We demonstrate that ER morphology was altered in these neurons, with a reduced number of three-way junctions. Neurons lacking atlastin-1 had longer endosomal tubules, suggestive of defective tubule fission. This was accompanied by reduced lysosomal proteolytic capacity. As well as demonstrating that atlastin-1 is required for correct ER morphology in human neurons, our results indicate that lack of a classical ER-shaping protein such as atlastin-1 may cause altered endosomal tubulation and lysosomal proteolytic dysfunction. Furthermore, they strengthen the idea that defective lysosome function contributes to the pathogenesis of a broad group of HSPs, including those where the primary localisation of the protein involved is not at the endolysosomal system.

Dual-Recognition-Mediated Autocatalytic Amplification Assay for the Subpopulations of PD-L1 Positive Extracellular Vesicle.

The PD-L1 protein on extracellular vesicles (EVs) is a promising biomarker for tumor immunotherapy. However, PD-L1+ EVs have various cell origins, so further analysis of the subpopulations is essential to help understand better their relationship with tumor immunotherapy. Different from the previous work which focus on the level of total PD-L1+ EVs expression, we, herein, report a dual-recognition mediated autocatalytic amplification (DRMAA) assay to detect the PD-L1 derived from tumors (EpCAM+), immune T cells (CD3+), and total (Lipids) EVs, respectively. The DRMAA assay employed proximity hybridization to construct a complete trigger sequence and then catalyzed the cross-hybridization of three hairpin probes, producing a three-way DNA junction (3-WJ) structure carrying the newly exposed trigger sequence. The 3-WJ complex subsequently initiated an autocatalytic amplification reaction and higher sensitivity than the traditional catalytic hairpin assembly assay was obtained. It was found that the EpCAM+ and PD-L1+ EVs were more effective than others in distinguishing lung cancer patients from healthy people. Surprisingly, the CD3+ and PD-L1+ EVs in lung cancer patients were also upregulated, indicating that immune cell-derived PD-L1+ EVs are also non-negligible marker in a tumor microenvironment. Our results suggested that the DRMAA assay would improve the study of subpopulations of PD-L1+ EVs to provide new insights for cancer immunotherapies.

Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements.

Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg2+). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg2+ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 μsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg2+ are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg2+ remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2'-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg2+ ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg2+ and ligand also facilitate a more compact structure in the three-way junction region.

Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.