Synthesis and evaluation of 10-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives as selective telomeric G-quadruplex DNA ligands

A class of 9(10H)-acridone derivatives with terminal ammonium substituents at C2 (and C7) position(s) on the acridone ring were successfully synthesized. The relative affinities of the acridone compounds to G-quadruplex DNA have been investigated and the results showed that these compounds had a binding specificity for G-quadruplex over duplex sequences. The acridones with two terminal ammonium substituents had much more effects on the human telomeric G-quadruplex DNA than the corresponding acridone derivatives with one terminal ammonium substituent, and more positive charges introduced to the side chains can improve the formation and stabilization of the G-quadruplex.     

Formation of (3+1) G-quadruplexes with a long loop by human telomeric DNA spanning five or more repeats

Structural studies of human telomeric repeats represent an active field of research with potential applications toward the development of specific telomeric quadruplex-targeting drugs for anticancer treatment. To date, high-definition structures were limited to DNA sequences containing up to four GGGTTA repeats. Here we investigate the formation of G-quadruplexes in sequences spanning five to seven human telomeric repeats using NMR, UV, and CD spectroscopy. A (3+1) G-quadruplex with a long propeller loop was isolated from a five-repeat sequence utilizing a guanine-to-inosine substitution. A simple approach of selective site-specific labeling of guanine residues was devised to rigorously determine the folding topology of the oligonucleotide. The same scaffold could be extrapolated to six- and seven-repeat sequences. Our results suggest that long human telomeric sequences consisting of five or more GGGTTA repeats could adopt (3+1) G-quadruplex structures harboring one or more repeat(s) within a single loop. We report on the formation of a Watson-Crick duplex within the long propeller loop upon addition of the complementary strand, demonstrating that the long loop could serve as a new recognition motif.     

Multiplexed and amplified chemiluminescence resonance energy transfer (CRET) detection of genes and microRNAs using dye-loaded hemin/G-quadruplex-modified UiO-66 metal–organic framework nanoparticles

Dye-loaded UiO-66 metal–organic framework nanoparticles (NMOFs) modified with catalytic hemin/G-quadruplex DNAzyme labels act as functional hybrid modules for the chemiluminescence resonance energy transfer (CRET) analysis of miRNAs (miRNA-155 or miRNA-21) or genes (p53 or BRCA1). The dye-loaded NMOFs (dye = fluorescein (Fl) or rhodamine 6G (Rh 6G)) are modified with hairpin probes that are engineered to include in their loop domains recognition sequences for the miRNAs or genes, and in their stem regions caged G-quadruplex domains. In the presence of the analytes miRNAs or genes, the hairpin structures are opened, leading, in the presence of hemin, to the self-assembly of hemin/G-quadruplex DNAzyme labels linked to the dye-loaded NMOFs. In the presence of luminol and H₂O₂, the hemin/G-quadruplex DNAzyme labels catalyze the generation of chemiluminescence that provides radiative energy to stimulate the process of CRET to the dye loaded in the NMOFs, resulting in the luminescence of the loaded dye without external excitation. The resulting CRET signals relate to the concentrations of the miRNAs or the genes and allow the sensitive analysis of miRNAs and genes. In addition, the DNA hairpin-functionalized dye-loaded NMOF sensing modules were further applied to develop amplified miRNA or gene CRET-based sensing platforms. The dye-loaded NMOFs were modified with hairpin probes that include in their loop domain the recognition sequences for miRNA-155 or miRNA-21 or the recognition sequences for the p53 or BRCA1 genes. Subjecting the hairpin-modified NMOFs to the respective miRNAs or genes, in the presence of two hairpins H_i and H_j that include in their stem regions caged G-quadruplex subunit domains, results in the analyte-triggered opening of the probe hairpin linked to the NMOFs, and the opened hairpin tethers induce the cross-opening of the hairpins H_i and H_j by the hybridization chain reaction, HCR, resulting in the assembly of G-quadruplex wires tethered to the NMOFs. The binding of hemin to the HCR-generated chains yields hemin/G-quadruplex DNAzyme wires that enhance, in the presence of luminol/H₂O₂, the CRET processes in the hybrid nanostructures. These amplification platforms lead to the amplified sensing of miRNAs and genes. By mixing the Fl- and Rh 6G-loaded hairpin-functionalized UiO NMOFs, the multiplexed CRET detection of miRNA-155, miRNA-21 and the p53 and BRCA1 genes is demonstrated.

Hemin/G-quadruplex DNAzyme-modified metal–organic framework nanoparticles act as functional hybrids for the catalyzed oxidation of luminol by H₂O₂, causing chemiluminescence and activation of chemiluminescence resonance energy transfer to the dye loads.     

Not all G-quadruplexes exhibit ion-channel-like properties: NMR study of ammonium ion (non)movement within the d(G(3)T(4)G(4))(2) quadruplex

A solution-state NMR study on 15NH4(+) ion movement within d(G(3)T(4)G(4))(2), a dimeric G-quadruplex consisting of three G-quartets and two T(4) loops, rather unexpectedly demonstrated the absence of 15NH4(+) ion movement between the binding sites U and L along the central axis of the G-quadruplex. Distinct temperature dependences of autocorrelation signals for U and L binding sites have been observed in 15N-1H NzExHSQC spectra which correlate with the local stiffness of the G-quadruplex. The volumes of the cross-peaks, which are the result of 15NH4(+) ion movement, have been interpreted in terms of rate constants, T(1) relaxation, and proton exchange. 15NH4(+) ion movements from the binding sites U and L into the bulk solution are characterized by lifetimes of 139 ms and 1.7 s at 298 K, respectively. The 12 times faster movement from the binding site U demonstrates that 15NH4(+) ion movement is controlled by the structure of T4 loop residues, which through diagonal- vs edge-type orientations impose distinct steric restraints for cations to leave or enter the G-quadruplex. Arrhenius-type analysis has afforded an activation energy of 66 kJ mol(-)1 for the UB process, while it could not be determined for the LB process due to slow rates at temperatures below 298 K. We further the use of the 15NH4(+) ion as an NMR probe to gain insight into the occupancy of binding sites by cations and kinetics of ion movement which are intrinsically correlated with the structural details, dynamic fluctuations, and local flexibility of the DNA structure.     

Synthesis and binding studies of novel diethynyl-pyridine amides with genomic promoter DNA G-quadruplexes

Herein, we report the design, synthesis and biophysical evaluation of novel 1,2,3-triazole-linked diethynyl-pyridine amides and trisubstituted diethynyl-pyridine amides as promising G-quadruplex binding ligands. We have used a Cu(I)-catalysed azide-alkyne cycloaddition click reaction to prepare the 1,2,3-triazole-linked diethynyl-pyridine amides. The G-quadruplex DNA binding properties of the ligands have been examined by using a F?rster resonance energy transfer (FRET) melting assay and surface plasmon resonance (SPR) experiments. The investigated compounds are conformationally flexible, having free rotation around the triple bond, and exhibit enhanced G-quadruplex binding stabilisation and specificity between intramolecular promoter G-quadruplex DNA motifs compared to the first generation of diaryl-ethynyl amides (J. Am. Chem. Soc. 2008, 130, 15950-15956). The ligands show versatility in molecular recognition and promising G-quadruplex discrimination with 2-50-fold selectivity exhibited between different intramolecular promoter G-quadruplexes. Circular dichroism (CD) spectroscopic analysis suggested that at higher concentration these ligands disrupt the c-kit2 G-quadruplex structure. The studies validate the design concept of the 1,3-diethynyl-pyridine-based scaffold and demonstrate that these ligands exhibit not only significant selectivity over duplex DNA but also variation in G-quadruplex interaction properties based on small chemical changes in the scaffold, leading to unprecedented differential recognition of different DNA G-quadruplex sequences.     

Structure of human telomeric DNA in crowded solution

G-quadruplex structures formed by DNA at the human telomeres are attractive anticancer targets. Human telomeric sequences can adopt a diverse range of intramolecular G-quadruplex conformations: a parallel-stranded conformation was observed in the crystalline state, while at least four other forms were seen in K(+) solution, raising the question of which conformation is favored in crowded cellular environment. Here, we report the first NMR structure of a human telomeric G-quadruplex in crowded solution. We show that four different G-quadruplex conformations are converted to a propeller-type parallel-stranded G-quadruplex in K(+)-containing crowded solution due to water depletion. This study also reveals the formation of a new higher-order G-quadruplex structure under molecular crowding conditions. Our molecular dynamics simulations of solvent distribution provide insights at molecular level on the formation of parallel-stranded G-quadruplex in environment depleted of water. These results regarding human telomeric DNA can be extended to oncogenic promoters and other genomic G-rich sequences.     

(S)-stereoisomer of telomestatin as a potent G-quadruplex binder and telomerase inhibitor

Total synthesis of the (S)-stereoisomer of telomestatin (1) was accomplished. (S)-Telomestatin exhibited potency four times that of the natural product, (R)-telomestatin, which was the most potent telomerase inhibitor previously reported. In the circular dichroism spectral analysis of the complexes possessing randomly structured single-stranded d[TTAGGG](4) oligonucleotide, (S)-telomestatin, like (R)-telomestatin, induced an antiparallel G-quadruplex structure. The melting temperature (T(m)) value of the (S)-isomer complex was greater than that of the (R)-telomestatin complex. Therefore, it is concluded that the stereochemistry of the thiazoline of telomestatin is important to the binding ability of a G-quadruplex binder, and (S)-telomestatin as a G-quadruplex binder is more potent than the natural product.     

Chiral metallo-supramolecular complexes selectively induce human telomeric G-quadruplex formation under salt-deficient conditions

Chiral molecular recognition of human telomeric DNA is important for rational drug design and developing structural probes of G-quadruplexes. Here we report that a chiral supramolecular complex can selectively induce human telomeric G-quadruplex formation and discriminate different G-quadruplex sequences under salt-deficient conditions studied by circular dichroism (CD), UV meltings, stopped-flow spectroscopy, fluorescence resonance energy transfer, enzyme cleavage, and gel electrophoresis. P-enantiomer induced G-quadruplex formation is fast and does not require a large excess of P enantiomer. More importantly, this chiral compound induces loop sequence-dependent G-quadruplex formation.     

Photo-Oxidizing Ruthenium(II) Complexes with Enhanced Visible-Light Absorption and G-quadruplex DNA Binding Abilities

Photosensitizers that gather high photo-oxidizing power and strong visible-light absorption are of great interest in the development of new photo-chemotherapeutics. Indeed, such compounds constitute attractive candidates for the design of type I photosensitizers that are not dependent on the presence of oxygen. In this paper, we report on the synthesis and studies of new ruthenium(II) complexes that display strong visible-light absorption and can oxidize guanine residues under visible-light irradiation, as evidenced by nanosecond transient absorption spectroscopy. The reported compounds also tightly bind to G-quadruplex DNA structures from the human telomeric sequence (TTAGGG repeat). The kinetic and thermodynamic parameters of the interaction of these Ru(II) complexes with G-quadruplex and duplex DNA were studied thanks to luminescence titrations and bio-layer interferometry measurements, which revealed higher affinities towards the non-canonical G-quadruplex architecture. Docking experiments and non-covalent ionic analysis allowed us to gain information on the mode and the strength of the interaction of the compounds towards G-quadruplex and duplex DNA. The different studies emphasize the substantial influence of the position and the number of non-chelating nitrogen atoms on the interaction with both types of DNA secondary structures.     

Insight into G-DNA structural polymorphism and folding from sequence and loop connectivity through free energy analysis

The lengths of G-tracts and their connecting loop sequences determine G-quadruplex folding and stability. Complete understanding of the sequence-structure relationships remains elusive. Here, single-loop G-quadruplexes were investigated using explicit solvent molecular dynamics (MD) simulations to characterize the effect of loop length, loop sequence, and G-tract length on the folding topologies and stability of G-quadruplexes. Eight loop types, including different variants of lateral, diagonal, and propeller loops, and six different loop sequences [d0 (i.e., no intervening residues in the loop), dT, dT(2), dT(3), dTTA, and dT(4)] were considered through MD simulation and free energy analysis. In most cases the free energetic estimates agree well with the experimental observations. The work also provides new insight into G-quadruplex folding and stability. This includes reporting the observed instability of the left propeller loop, which extends the rules for G-quadruplex folding. We also suggest a plausible explanation why human telomere sequences predominantly form hybrid-I and hybrid-II type structures in K(+) solution. Overall, our calculation results indicate that short loops generally are less stable than longer loops, and we hypothesize that the extreme stability of sequences with very short loops could possibly derive from the formation of parallel multimers. The results suggest that free energy differences, estimated from MD and free energy analysis with current force fields and simulation protocols, are able to complement experiment and to help dissect and explain loop sequence, loop length, and G-tract length and orientation influences on G-quadruplex structure.     

Methods for investigating G-quadruplex DNA/ligand interactions

DNA is considered an important target for drug design and development. Until recently, the focus was on double-stranded (duplex) DNA structures. However, it has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplex structures. The more interesting G-quadruplex DNA structures comprise four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds. The DNA sequences with potential to form G-quadruplex structures are found at the chromosomal extremities (i.e. the telomeres) and also at the intra-chromosomal region (i.e. oncogenic promoters) in several important oncogenes. The formation of G-quadruplex structures is considered to have important consequences at the cellular level and such structures have been evoked in the control of expression of certain genes involved in carcinogenesis (c-myc, c-kit, K-ras etc.) as well as in the perturbation of telomeric organization. It has been shown that the formation of quadruplexes inhibits the telomere extension by the telomerase enzyme, which is up-regulated in cancer cells. Therefore, G-quadruplex structures are an important target for drug design and development and there is a huge interest in design and development of small molecules (ligands) to target these structures. A large number of so-called G-quadruplex ligands, displaying varying degrees of affinity and more importantly selectivity (i.e. the ability to interact only with quadruplex-DNA and not duplex-DNA), have been reported. Access to efficient and robust in vitro assays is needed to effectively monitor and quantify the G-quadruplex DNA/ligand interactions. This tutorial review provides an overview of G-quadruplex ligands and biophysical techniques available to monitor such interactions.     

Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.     

钾离子浓度依赖的铅离子稳定G-四链体构型转化

已有研究普遍认为铅离子(Pb²⁺)诱导富G适体链形成的G-四链体(Pb²⁺-G4)比钾离子(K⁺)诱导富G适体链形成的G-四链体(K⁺-G4)更为稳定,因而Pb²⁺可以置换K⁺-G4中的K⁺,而且K⁺的存在不影响Pb²⁺-G4的稳定性。有趣的是本研究发现K⁺ (20 μmol∙L⁻¹–1 mmol∙L⁻¹)不仅可以诱导10 µmol∙L⁻¹ Pb²⁺稳定的T2TT(Pb²⁺-T2TT,杂合G4结构)发生构型转换,甚至还可取代Pb²⁺-T2TT中的Pb²⁺,形成K⁺稳定的T2TT (K⁺-T2TT,平行G4结构),最终转化形成的K⁺-G4结构与单独K⁺诱导富G适体链形成K⁺-G4的构型基本一致。随后,进一步考察了另外7条富G适体链,发现这一转化过程具有一定的普适性。该研究结果为理解G4构型转化以及内嵌离子交换提供了新的视角,也为拓展G4在生化分析和生物领域的应用提供了新的理论基础。     

Computational studies on G-quadruplex DNA-stabilizing property of novel Wittig-based Schiff-Base ligands and their copper(II) complexes

A series of mono imine (C=N) group that contained Wittig-based Schiff-Base ligands was optimized using the DFT-based computational method and Gaussian 09 program package. These optimized molecules were docked with Quadruplex DNA (PDB ID: 1KF1) and duplex DNA (PDB ID: 1BNA) using AutoDock Vina program along with the reference molecules. Schiff-Base ligands derived from fused aromatic rings contained amines and precursor aldehyde (PA-5 both Z and E isomers) showed lower binding energy for G-quadruplex DNA among all and N-5 category both Z and E isomer Schiff-Base ligands have shown high selectivity for G-quadruplex DNA over duplex DNA which is a very important phenomenon to develop the G-quadruplex DNA stabilizers. For a few Schiff-Base molecules like Ligand-6 (1-{[2-Hydroxy-5-(2-pyren-1-yl-vinyl)-benzylidene]-amino}-naphthalen-2-ol) of N-5 category both Z and E isomers with groove binding and end stacking modes, molecular dynamic calculations were carried out. The study revealed that Ligand-6 of N-5 category E isomer with groove binding mode has higher stabilizing effect on G-quadruplex DNA in spite of having the higher binding energy value. Among Schiff-Base copper(II) complexes, Complex-3 (E-(1-{[2-Hydroxy-5-(2-pyren-1-yl-vinyl)-benzylidene]-amino}-naphthalen-2-ol)Cu) is having high binding affinity for G-quadruplex DNA as compared to others.

     

Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes

Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33 nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins.     

Two-repeat human telomeric d(TAGGGTTAGGGT) sequence forms interconverting parallel and antiparallel G-quadruplexes in solution: distinct topologies, thermodynamic properties, and folding/unfolding kinetics

We demonstrate by NMR that the two-repeat human telomeric sequence d(TAGGGTTAGGGT) can form both parallel and antiparallel G-quadruplex structures in K(+)-containing solution. Both structures are dimeric G-quadruplexes involving three stacked G-tetrads. The sequence d(TAGGGUTAGGGT), containing a single thymine-to-uracil substitution at position 6, formed a predominantly parallel dimeric G-quadruplex with double-chain-reversal loops; the structure was symmetric, and all guanines were anti. Another modified sequence, d(UAGGGT(Br)UAGGGT), formed a predominantly antiparallel dimeric G-quadruplex with edgewise loops; the structure was asymmetric with six syn guanines and six anti guanines. The two structures can coexist and interconvert in solution. For the latter sequence, the antiparallel form is more favorable at low temperatures (<50 degrees C), while the parallel form is more favorable at higher temperatures; at temperatures lower than 40 degrees C, the antiparallel G-quadruplex folds faster but unfolds slower than the parallel G-quadruplex.     

Inducement and stabilization of G-quadruplex DNA by a thiophene-containing dinuclear ruthenium(II) complex

G-quadruplex structures are attractive targets for the development of anticancer drugs, as their formation in human telomere could impair telomerase activity, thus inducing apoptosis in cancer cells. In this work, a thiophene-containing dinuclear ruthenium(II) complex, [Ru₂(bpy)₄(H₂bipt)]⁴⁺ {bpy = 2,2′-bipyridine, H₂bipt = 2,5-bis[1,10]phenanthrolin[4,5-f]-(imidazol-2-yl)thiophene}, was prepared and the interaction between the complex and human telomeric DNA oligomers 5′-G₃(T₂AG₃)₃-3′ (HTG21) has been investigated by UV-Vis, fluorescence and circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, fluorescent intercalator displacement (FID) titrations, Job plot and color reaction studies. The results indicate that the complex can well induce and stabilize the formation of antiparallel G-quadruplex of telomeric DNA in the presence or absence of metal cations, and the ΔT_m value of the G-quadruplex DNA treated with the complex was obtained to be 12.8 °C even at levels of 50-fold molar of duplex DNA (calf-thymus DNA), suggesting that the complex exhibits higher G-quadruplex DNA selectivity over duplex DNA. The complex shows high interaction ability with G-quadruplex DNA at (1.17 ± 0.12) × 10⁷ M^?1 binding affinity using a 2:1 [complex]/[quadruplex] binding mode ratio. A novel visual method has been developed here for making a distinction between G-quadruplex DNA and duplex DNA by our ruthenium complex binding hemin to form the hemin-G-quadruplex DNAzyme.     

Synthesis and Structural Characterization of Stable Branched DNA G-Quadruplexes Using the Trebler Phosphoramidite

Guanine (G)-rich sequences can form a noncanonical four-stranded structure known as the G-quadruplex. G-quadruplex structures are interesting because of their potential biological properties and use in nanosciences. Here, we describe a method to prepare highly stable G-quadruplexes by linking four G-rich DNA strands to form a monomolecular G-quadruplex. In this method, one strand is synthesized first, and then a trebler molecule is added to simultaneously assemble the remaining three strands. This approach allows the introduction of specific modifications in only one of the strands. As a proof of concept, we prepared a quadruplex where one of the chains includes a change in polarity. A hybrid quadruplex is observed in ammonium acetate solutions, whereas in the presence of sodium or potassium, a parallel G-quadruplex structure is formed. In addition to the expected monomolecular quadruplexes, we observed the presence of dimeric G-quadruplex structures. We also applied the method to prepare G-quadruplexes containing a single 8-aminoguanine substitution and found that this single base stabilizes the G-quadruplex structure when located at an internal position.     

Fluorescent amplifying recognition for DNA G-quadruplex folding with a cationic conjugated polymer: a platform for homogeneous potassium detection

Single-stranded DNA with G-rich sequences can fold into secondary structures, G-quadruplexes, via intramolecular hydrogen-bonding interactions. This conformational change can be detected by a homogeneous assay method based on fluorescence resonance energy transfer (FRET) from a water-soluble cationic conjugated polymer (CCP) to a fluorescein chromophore labeled at the terminus of the G-quadruplex DNA. The space charge density around the DNA controls the efficiency of FRET from the CCP to the fluorescein. The higher FRET efficiency for the CCP/G-quadruplex pair is correlated to the stronger electrostatic interactions between the more condensed G-quadruplex and the CCP in comparison to the CCP/ssDNA pair. Since the potassium ion can specifically bind to the G-quadruplex DNA, the G-quartet-DNA/CCPs assembly can also be used as a platform to sense the potassium ion in water with high selectivity and sensitivity.     

Inhibition of translation by small RNA-stabilized mRNA structures in human cells

RNA-mediated gene regulation and expression are critically dependent on both nucleic acid architecture and recognition. We present a novel mechanism for the regulation of gene expression through direct RNA-RNA interactions between small RNA and mRNA in human cells. Using mRNA reporters containing G-rich sequences in the 5'-untranslated region (5'-UTR), in the coding region, or both, we showed that G-rich small RNAs bind to the reporter mRNAs and form an intermolecular RNA G-quadruplex that can inhibit gene translation in living cells. Using a combination of circular dichroism (CD) and RNase footprinting in vitro, we found that the intermolecular G-quadruplexes show a parallel G-quadruplex structure. We next investigated whether the intermolecular G-quadruplex is present in living cells. Employing the fluorophore-labeled probes, we found that two G-rich RNA molecules form an intermolecular G-quadruplex structure in living cells. These results extend the concept of small RNA-mediated expression and suggest an important role for such RNA structures in the inhibition of mRNA translation.