Atomistic insights into the reentrant phase-transitions in polyuracil and polylysine mixtures.

The phase separation of protein and RNA mixtures underpins the assembly and regulation of numerous membraneless organelles in cells. The ubiquity of protein-RNA condensates in cellular regulatory processes is in part due to their sensitivity to RNA concentration, which affects their physical properties and stability. Recent experiments with poly-cationic peptide-RNA mixtures have revealed closed-loop phase diagrams featuring lower and upper critical solution temperatures. These diagrams indicate reentrant phase transitions shaped by biomolecular interactions and entropic forces such as solvent and ion reorganization. We employed atomistic simulations to study mixtures with various RNA-polylysine stoichiometries and temperatures to elucidate the microscopic driving forces behind reentrant phase transitions in protein-RNA mixtures. Our findings reveal an intricate interplay between hydration, ion condensation, and specific RNA-polylysine hydrogen bonding, resulting in distinct stoichiometry-dependent phase equilibria governing stabilities and structures of the condensate phase. Our simulations show that reentrant transitions are accompanied by desolvation around the phosphate groups of RNA, with increased contacts between phosphate and lysine side chains. In RNA-rich systems at lower temperatures, RNA molecules can form an extensive pi-stacking and hydrogen bond network, leading to percolation. In protein-rich systems, no such percolation-induced transitions are observed. Furthermore, we assessed the performance of three prominent water force fields-Optimal Point Charge (OPC), TIP4P-2005, and TIP4P-D-in capturing reentrant phase transitions. OPC provided a superior balance of interactions, enabling effective capture of reentrant transitions and accurate characterization of changes in solvent reorganization. This study offers atomistic insights into the nature of reentrant phase transitions using simple model peptide and nucleotide mixtures. We believe that our results are broadly applicable to larger classes of peptide-RNA mixtures exhibiting reentrant phase transitions.

Validation and classification of RNA binding proteins identified by mRNA interactome capture.

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

Binding and stabilizating effect of RNA triplex poly(U)⋠poly(A)*poly(U) by enantiomers of ruthenium(II) polypyridyl complex [Ru(bpy)2(dppx)]2.

Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. Spectrophotometric titrations and viscosity measurements suggested that enantiomers Λ-1 and Δ-1 bind with the triplex through intercalation, while the binding strengths of the two enantiomers toward the triplex differed only slightly from each other. Fluorescence titrations showed that although enantiomers Λ-1 and Δ-1 exhibited molecular "light switch" effects toward the triplex, the effect of Δ-1 was more marked. Furthermore, Furthermore, thermal denaturation showed that the two enantiomers have significantly different stabilizing effects on the triplex. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar to a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex. Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar as a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex.

Interactions of arene ruthenium(II) complexes [η6-(C6H6)Ru(pprip)Cl]+ and [η6-(C6H6)Ru(H2iiP)Cl]+ with RNA triplex poly(U)•poly(A)*poly(U).

Two arene ruthenium(II) complexes [η6-(C6H6)Ru(pprip)Cl]PF6 (Ru1; pprip = 2-(3-phenyl-1H-pyrazol-4-yl)-imidazolo[4,5-f][1,10]phenanthroline) and [η6-(C6H6)Ru(H2iiP)Cl]PF6 (Ru2; H2iiP = 2-(indole-3-yl)-imidazolo[4,5-f][1,10]phenanthroline) have been synthesized and characterized in this work. Binding properties of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)*poly(U) were investigated by spectrophotometry and spectrofluorometry as well as viscosimetry. Analysis of spectroscopic titrations and viscosity measurements show that the two complexes bind with the triplex through intercalation, while the binding affinity for Ru2 toward the triplex is stronger than that for Ru1. Melting experiments indicate that the stabilizing effects of Ru1 and Ru2 toward the triplex differ from each other. Under the conditions used herein, Ru1 only stabilizes the Hoogsteen base-paired strand (third strand) without affecting stabilization of the Watson-Crick base-paired strand (the template duplex) of the triplex, while Ru2 stabilizes both the template duplex and the third strand. Although the two complexes prefer to stabilizing the third strand rather than the template duplex, the third-strand stabilization effect of Ru2 is stronger than that of Ru1. The obtained results of this work reveal that the planarity of the intercalative ligands plays an important role in the triplex stabilization by arene Ru(II) complexes.

Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors.

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.

Interaction of double-stranded polynucleotide poly(A:U) with graphene/graphene oxide.

Hybrids formed by DNA/RNA and graphene family nanomaterials are considered as potentially useful multifunctional agents in biosensing and nanomedicine. In this work, we study the noncovalent interaction between double-stranded (ds) RNA, polyadenylic:polyuridylic acids (poly(A:U)) and graphene oxide/graphene (GO/Gr) using UV absorption spectroscopy and molecular dynamics (MD) simulations. RNA melting showed that relatively long ds-RNA is adsorbed onto GO (at an ionic strength of [Formula: see text]) at that a large fraction of RNA maintains the duplex structure. It was revealed that this fraction decreases over long time (during a few days), indicating a slow adsorption process of the long polymer. MD simulations showed that the adsorption of duplex (rA)[Formula: see text]: (rU)[Formula: see text] or (rA)[Formula: see text]: (rU)[Formula: see text] on graphene starts with the interaction between [Formula: see text]-systems of graphene and base pairs located at a duplex tail. In contrast to relatively long duplex (rA)[Formula: see text]: (rU)[Formula: see text] which keeps parallel arrangement along the graphene surface, the shorter one ((rA)[Formula: see text]: (rU)[Formula: see text]) always adopts a perpendicular orientation relative to graphene even in case of the initial parallel orientation. It was found out that (rA)[Formula: see text]: (rU)[Formula: see text] forms the stable hybrid with graphene keeping essential fraction of the duplex, while (rA)[Formula: see text]: (rU)[Formula: see text] demonstrates the duplex unzipping into two single strands with time. The interaction energies between adenine/uracil stacked with graphene as well between nucleotides in water environment were determined.

Single-stranded nucleic acid elasticity arises from internal electrostatic tension.

Understanding of the conformational ensemble of flexible polyelectrolytes, such as single-stranded nucleic acids (ssNAs), is complicated by the interplay of chain backbone entropy and salt-dependent electrostatic repulsions. Molecular elasticity measurements are sensitive probes of the statistical conformation of polymers and have elucidated ssNA conformation at low force, where electrostatic repulsion leads to a strong excluded volume effect, and at high force, where details of the backbone structure become important. Here, we report measurements of ssDNA and ssRNA elasticity in the intermediate-force regime, corresponding to 5- to 100-pN forces and 50-85% extension. These data are explained by a modified wormlike chain model incorporating an internal electrostatic tension. Fits to the elastic data show that the internal tension decreases with salt, from [Formula: see text]5 pN under 5 mM ionic strength to near zero at 1 M. This decrease is quantitatively described by an analytical model of electrostatic screening that ascribes to the polymer an effective charge density that is independent of force and salt. Our results thus connect microscopic chain physics to elasticity and structure at intermediate scales and provide a framework for understanding flexible polyelectrolyte elasticity across a broad range of relative extensions.

Flexibility and Preorganization of Fluorescent Nucleobase-Pyrene Conjugates Control DNA and RNA Recognition.

We synthesized a new amino acid-fluorescent nucleobase derivative (qAN1-AA) and from it two new fluorescent nucleobase-fluorophore (pyrene) conjugates, whereby only the analogue with the longer and more flexible linker (qAN1-pyr2) self-folded into intramolecularly stacked qAN1/pyrene conformation, yielding characteristic, 100 nm-red-shifted emission (λmax = 500 nm). On the contrary, the shorter and more rigid linker resulted in non-stacked conformation (qAN1-pyr1), characterized by the emission of free pyrene at λmax = 400 nm. Both fluorescent nucleobase-fluorophore (pyrene) conjugates strongly interacted with ds-DNA/RNA grooves with similar affinity but opposite fluorescence response (due to pre-organization), whereas the amino acid-fluorescent base derivative (qAN1-AA) was inactive. However, only intramolecularly self-folded qAN1-pyr2 showed strong fluorescence selectivity toward poly U (Watson-Crick complementary to qAN1 nucleobase) and poly A (reverse Hoogsteen complementary to qAN1 nucleobase), while an opposite emission change was observed for non-complementary poly G and poly C. Non-folded analogue (qAN1-pyr1) showed no ss-RNA selectivity, demonstrating the importance of nucleobase-fluorophore pre-organization.

RNA Homopolymers Form Higher-Curvature Virus-like Particles Than Do Normal-Composition RNAs.

Unlike double-stranded DNA, single-stranded RNA can be spontaneously packaged into spherical capsids by viral capsid protein (CP) because it is a more compact and flexible polymer. Many systematic investigations of this self-assembly process have been carried out using CP from cowpea chlorotic mottle virus, with a wide range of sequences and lengths of single-stranded RNA. Among these studies are measurements of the relative packaging efficiencies of these RNAs into spherical capsids. In this work, we address a fundamental issue that has received very little attention, namely the question of the preferred curvature of the capsid formed around different RNA molecules. We show in particular that homopolymers of RNA-polyribouridylic acid and polyriboadenylic acid-form exclusively T = 2-sized (∼22-nm diameter) virus-like particles (VLPs) when mixed with cowpea chlorotic mottle virus CP, independent of their length, ranging from 500 to more than 4000 nucleotides. This is in contrast to "normal-composition" RNAs (i.e., molecules with comparable numbers of each of the four nucleotides and hence capable of developing a large amount of secondary structure because of intramolecular complementarity/basepairing); a curvature corresponding to T = 3-size (∼28 nm in diameter) is preferred for the VLPs formed with such RNAs. Our work is consistent with the preferred curvature of VLPs being a consequence of interaction of CP with RNA-in particular, the presence or absence of short RNA duplexes-and suggests that the equilibrium size of the capsid results from a trade-off between this optimum size and the cost of confinement.

Role of hydroxyl groups in the B-ring of flavonoids in stabilization of the Hoogsteen paired third strand of Poly(U).Poly(A)*Poly(U) triplex.

We have reported the interaction of two flavonoids namely quercetin (Q) and morin (M) with double stranded poly(A).poly(U) (herein after A.U) and triple stranded poly(U).poly(A)*poly(U) (herein after U.A*U, dot represents the Watson-Crick and asterisk represents Hoogsteen base pairing respectively) in this article. It has been observed that relative positions of hydroxyl groups on the B-ring of the flavonoids affect the stabilization of RNA. The double strand as well as the triple strand of RNA-polymers become more stabilized in presence of Q, however both the duplex and triplex remain unaffected in presence of M. The presence of catechol moiety on the B-ring of Q is supposed to be responsible for the stabilization. Moreover, after exploiting a series of biophysical experiments, it has been found that, triple helical RNA becomes more stabilized over its parent duplex in presence of Q. Fluorescence quenching, viscosity measurement and helix melting results establish the fact that Q binds with both forms of RNA through the mode of intercalation while M does not bind at all to either forms of RNA.

Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization.

We report the effect of neutral macromolecular crowders poly(ethylene glycol) (PEG) (8 kDa) and Ficoll (70 kDa) on liquid-liquid phase separation in a polyuridylic acid (polyU)/spermine complex coacervate system. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature (TC). We interpret these effects on phase behavior as arising due to excluded volume and preferential interactions on both the secondary structure/condensation of spermine-associated polyU molecules and on the association of soluble polyU/spermine polyelectrolyte complexes to form coacervate droplets. Examination of coacervates formed in the presence of fluorescently-labeled PEG or Ficoll crowders indicated that Ficoll is accumulated while PEG is excluded from the coacervate phase, which provides further insight into the differences in phase behavior. Crowding agents impact distribution of a biomolecular solute: partitioning of a fluorescently-labeled U15 RNA oligomer into the polyU/spermine coacervates was increased approximately two-fold by 20 wt% Ficoll 70 kDa and by more than two orders of magnitude by 20 wt% PEG 8 kDa. The volume of the coacervate phase decreased in the presence of crowder relative to a dilute buffer solution. These findings indicate that potential impacts of macromolecular crowding on phase behavior and solute partitioning should be considered in model systems for intracellular membraneless organelles.

Facile and Sensitive Method for Protein Kinase A Activity Assay Based on Fluorescent Off-On PolyU-peptide Assembly.

Phosphorylation mediated by protein kinases plays a pivotal role in metabolic and cell-signaling processes, and the dysfunction of protein kinases such as protein kinase A (PKA) may induce several human diseases. Therefore, it is of great significance to develop a facile and effective method for PKA activity assay and high-throughput screening of inhibitors. Herein, we develop a new fluorescent off-on method for PKA assay based on the assembly of anionic polyuridylic acid (polyU) and cationic fluorescent peptide. The phosphorylation of the peptide disrupts its electrostatic binding with polyU, suppresses the concentration quenching effect of polyU, and thus causes fluorescence recovery. The recovered fluorescence intensity at 585 nm is directly proportional to the PKA activity in the range of 0.1-3.2 U/mL with a detection limit of 0.05 U/mL. Using our method, the PKA activity in HeLa cell lysate is determined to be 58.2 ± 5.1 U/mg protein. The method has also been employed to evaluate the inhibitory effect of PKA inhibitors with satisfactory results and may be expected to be a promising candidate for facile and cost-effective assay of kinase activity and high-throughput inhibitor screening.

Removal of protein S1 from Escherichia coli ribosomes without the use of affinity chromatography.

The paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion. To avoid co-sedimentation of the S1-bound polypyrimidine with the ribosomes, its length should not exceed several hundred nucleotides. Unlike popular affinity chromatography through a poly(U) Sepharose or poly(U) cellulose column, the method tolerates limited polyribonucleotide degradation by eventual traces of ribonucleases, and can readily be incorporated into standard protocols for the isolation of ribosomes by centrifugation.

Binding Differences of Two Homochiral [Ru(bpy)2dppz]2+ Complexes with poly(U)·poly(A)*poly(U) Triplex RNA.

The first investigation of chiral ruthenium(II) complexes Δ- and Λ-[Ru(bpy)2dppz]2+ and triplex RNA poly(U)·poly(A)*poly(U) was carried out, which showed that Δ enantiomer displayed significant ability in stabilizing model triplex RNA.

Structure of the Yeast DEAD box protein Mss116p reveals two wedges that crimp RNA.

The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF(3)(-), or ADP-AlF(4)(-). The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge alpha helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

Structures of the T. brucei kRNA editing factor MRB1590 reveal unique RNA-binding pore motif contained within an ABC-ATPase fold.

Kinetoplastid RNA (kRNA) editing is a process that creates translatable mitochondrial mRNA transcripts from cryptogene encoded RNAs and is unique for kinetoplastids, such as Trypanosoma brucei. In addition to the catalytic 20S editosome, multiple accessory proteins are required for this conversion. Recently, the multiprotein mitochondrial RNA binding complex 1 (MRB1) has emerged as a key player in this process. MRB1 consists of six core proteins but makes dynamic interactions with additional accessory proteins. Here we describe the characterization of one such factor, the 72 kDa MRB1590 protein. In vivo experiments indicate a role for MRB1590 in editing mitochondrial mRNA transcripts, in particular the transcript encoding the ATP synthase subunit 6 (A6). Structural studies show that MRB1590 is dimeric and contains a central ABC-ATPase fold embedded between novel N- and C-terminal regions. The N-terminal domains combine to create a basic pore and biochemical studies indicate residues in this region participate in RNA binding. Structures capturing distinct MRB1590 conformations reveal that the RNA binding pore adopts closed and open states, with the latter able to accommodate RNA. Based on these findings, implications for MRB1590 function are discussed.

Effects of Water on the Single-Chain Elasticity of Poly(U) RNA.

Water, the dominant component under the physiological condition, is a complicated solvent which greatly affects the properties of solute molecules. Here, we utilize atomic force microscope-based single-molecule force spectroscopy to study the influence of water on the single-molecule elasticity of an unstructured single-stranded RNA (poly(U)). In nonpolar solvents, RNA presents its inherent elasticity, which is consistent with the theoretical single-chain elasticity calculated by quantum mechanics calculations. In aqueous buffers, however, an additional energy of 1.88 kJ/mol·base is needed for the stretching of the ssRNA chain. This energy is consumed by the bound water rearrangement (Ew) during chain elongation. Further experimental results indicate that the Ew value is uncorrelated to the salt concentrations and stretching velocity. The results obtained in an 8 M guanidine·HCl solution provide more evidence that the bound water molecules around RNA give rise to the observed deviation between aqueous and nonaqueous environments. Compared to synthetic water-soluble polymers, the value of Ew of RNA is much lower. The weak interference of water is supposed to be the precondition for the RNA secondary structure to exist in aqueous solution.

Unequal effect of ethanol-water on the stability of ct-DNA, poly[(dA-dT)]2 and poly(rA)·poly(rU). Thermophysical properties.

Ethanol affects unequally the thermal stability of DNA and RNA. It stabilizes RNA, while destabilizing DNA. The variation of the relative viscosity (η/η0) of [poly(dA-dT)]2 with temperature unveils transitions close to the respective denaturation temperature, calculated spectrophotometrically and calorimetrically. From the raw data densities and speeds of sound, the volumetric observables were calculated. In all cases studied, a change in sign from low to high ethanol content occurred for both partial molar volume (ϕV) and partial molar adiabatic compressibility (ϕK(S)). The minima, close to 10%, should correspond to the highest solvation and the maxima, close to 30%, to the lowest solvation. For 40-50% ethanol, the solvation increases again. The complex structure of ethanol-water, for which changes are observed in regions close to such critical concentrations, justifies the observed behaviour. The variation of ϕV and ϕK(S) was sharper for RNA compared with respect to DNA, indicating that the solvation sequence is poly(rA)·poly(rU) < ct-DNA < [poly(dA-dT)]2.

Structural and mechanistic insights into poly(uridine) tract recognition by the hnRNP C RNA recognition motif.

HnRNP C is a ubiquitous RNA regulatory factor and the principal constituent of the nuclear hnRNP core particle. The protein contains one amino-terminal RNA recognition motif (RRM) known to bind uridine (U)-rich sequences. This work provides a molecular and mechanistic understanding of this interaction. We solved the solution structures of the RRM in complex with poly(U) oligomers of five and seven nucleotides. The five binding pockets of RRM recognize uridines with an unusual 5'-to-3' gradient of base selectivity. The target recognition is therefore strongly sensitive to base clustering, explaining the preference for contiguous uridine tracts. Using a novel approach integrating the structurally derived recognition consensus of the RRM with a thermodynamic description of its multi-register binding, we modeled the saturation of cellular uridine tracts by this protein. The binding pattern is remarkably consistent with the experimentally observed transcriptome-wide cross-link distribution of the full-length hnRNP C on short uridine tracts. This result re-establishes the RRM as the primary RNA-binding domain of the hnRNP C tetramer and provides a proof of concept for interpreting high-throughput interaction data using structural approaches.

The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3' poly(U) tail.

Hfq-dependent sRNAs contain, at least, an mRNA base-pairing region, an Hfq-binding site, and a Rho-independent terminator. Recently, we found that the terminator poly(U) of Escherichia coli sRNAs is essential for Hfq binding and therefore for riboregulation. In this study, we tried to identify additional components within Hfq-binding sRNAs required for efficient Hfq binding by using SgrS as a model. We demonstrate by mutational and biochemical studies that an internal hairpin and an immediately upstream U-rich sequence also are required for efficient Hfq binding. We propose that the functional Hfq-binding module of SgrS consists of an internal hairpin preceded by a U-rich sequence and a Rho-independent terminator with a long poly(U) tail. We also show that the Rho-independent terminator alone can act as a functional Hfq-binding module when it is preceded by an internal U-rich sequence. The 3' region of most known sRNAs share the features corresponding to either a double- or single-hairpin-type Hfq-binding module. We also demonstrate that increasing the spacing between the base-pairing region and the Hfq-binding module reduces or impairs the silencing ability. These findings allowed us to design synthetic Hfq-binding sRNAs to target desired mRNAs.