Chemically Synthesized,Self-Assembling Small Interfering RNA-Prohead RNA Molecules Trigger Dicer-Independent Gene Silencing

RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo. An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer in vitro. However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5′ of the 3WJ (5′-siRNA-3WJ) relative to 3′ of the 3WJ (3′-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway.     

Mg2+‐Dependent High Mechanical Anisotropy of Three‐Way‐Junction pRNA as Revealed by Single‐Molecule Force Spectroscopy

Mechanical anisotropy is ubiquitous in biological tissues but is hard to reproduce in synthetic biomaterials. Developing molecular building blocks with anisotropic mechanical response is the key towards engineering anisotropic biomaterials. The three‐way‐junction (3WJ) pRNA, derived from ϕ 29 DNA packaging motor, shows strong mechanical anisotropy upon Mg²⁺ binding. In the absence of Mg²⁺, 3WJ‐pRNA is mechanically weak without noticeable mechanical anisotropy. In the presence of Mg²⁺, the unfolding forces can differ by more than 4‐fold along different pulling directions, ranging from about 47 pN to about 219 pN. Mechanical anisotropy of 3WJ‐pRNA stems from pulling direction dependent cooperativity for the rupture of two Mg²⁺ binding sites, which is a novel mechanism for the mechanical anisotropy of biomacromolecules. It is anticipated that 3WJ‐pRNA can be used as a key element for the construction of biomaterials with controllable mechanical anisotropy.     

Stochastic detection of motor protein-RNA complexes by single-channel current recording.

A label- and immobilization-free approach to detecting the reversible formation of complexes between nucleic acids and proteins at the single-molecule level is described. The voltage-driven translocation of individual oligoribonucleotides through a nanoscale protein pore is observed by single-channel current recordings. The oligoribonucleotide 5'-C25A(25)-3' gives rise to current blockades with an average duration of approximately 0.5 ms. In the presence of the RNA-binding ATPase P4, a viral packaging motor from bacteriophage phi8, longer events of tens to hundreds of milliseconds are observed. Upon addition of ATP the long events disappear, indicating the dissociation of the P4RNA complex. The frequency of events also depends on the concentration of P4 and the length of the oligoribonucleotide, thereby confirming the specificity of the P4RNA events. This study shows that single-channel current recordings can be used to monitor RNA-protein complex formation, thus opening up a new means to examine the motor activity of RNA- or DNA-processing enzymes.     

DNA three-way junction with a dinuclear iron(II) supramolecular helicate at the center: a NMR structural study

A tetracationic supramolecular helicate, [Fe2L3]4+ (L = C25H20N4), with a triple-helical architecture is found to induce the formation of a three-way junction (3WJ) of deoxyribonucleotides with the helicate located in the center of the junction. NMR spectroscopic studies of the interaction between the M enantiomer of the helicate and two different oligonucleotides, [5'-d(TATGGTACCATA)]2 and [5'-d(CGTACG)]2, show that, in each case, the 2-fold symmetry of the helicate is lifted, while the 3-fold symmetry around the helicate axis is retained. The 1:3 helicate/DNA stoichiometry estimated from 1D NMR spectra supports a molecular model of a three-way junction composed of three strands. Three separate double-helical arms of the three-way junction are chemically identical giving rise to one set of proton resonances. The NOE contacts between the helicate and DNA unambiguously show that the helicate is fitted into the center of the three-way junction experiencing a hydrophobic 3-fold symmetric environment. Close stacking interactions between the ligand phenyl groups and the nucleotide bases are demonstrated through unusually large downfield shifts (1-2 ppm) of the phenyl protons. The unprecedented 3WJ arrangement observed in solution has also been found to exist in the crystal structure of the helicate adduct of [d(CGTACG)2] (Angew. Chem., Int. Ed. 2006, 45, 1227).     

Stabilisation of a nucleic acid three-way junction by an oligonucleotide containing a single 2'-C to 3'-O-phosphate butylene linkage prepared by a tandem RCM-hydrogenation method

A cyclic dinucleotide with a butylene linker between the upper 2'-C position and the 3'-O-phosphate linkage was synthesised from simple nucleoside building blocks via a tandem ring-closing metathesis and hydrogenation procedure. The major of two phosphorus epimers was incorporated into an oligodeoxynucleotide, as well as into an LNA-DNA mixmer oligonucleotide. These were evaluated as parts in three different secondary structures, a duplex, a bulged duplex and a three-way junction, with both DNA and RNA complements. In the DNA:RNA hybrid molecule, the oligodeoxynucleotide containing this single 2'-C to 3'-O-phosphate butylene linkage was found to stabilise a three-way junction.     

Recognition of DNA three-way junctions by metallosupramolecular cylinders: gel electrophoresis studies

The interaction of metallosupramolecular cylinders with DNA three-way junctions has been studied by gel electrophoresis. A recent X-ray crystal structure of a palindromic oligonucleotide forming part of a complex with such a cylinder revealed binding at the heart of a three-way junction structure. The studies reported herein confirm that this is not solely an artefact of crystallisation and reveal that this is a potentially very powerful new mode of DNA recognition with wide scope. The cylinders are much more effective at stabilizing three-way junctions than simple magnesium di-cations or organic or metallo-organic tetra-cations, with the M cylinder enantiomer being more effective than P. The recognition is not restricted to three-way junctions formed from palindromic DNA with a central AT step at the junction; non-palindromic three-way junctions and those with GC steps are also stabilised. The cylinder is also revealed to stabilise other Y-shaped junctions, such as that formed at a fraying point in duplex DNA (for example, a replication fork), and other DNA three-way junction structures, such as those containing unpaired nucleotides, perhaps by opening up this structure to access the central cavity.     

Solution structure of a HNA-RNA hybrid

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.     

Automated prediction of three-way junction topological families in RNA secondary structures

We present an algorithm for automatically predicting the topological family of any RNA three-way junction, given only the information from the secondary structure: the sequence and the Watson–Crick pairings. The parameters of the algorithm have been determined on a data set of 33 three-way junctions whose 3D conformation is known. We applied the algorithm on 53 other junctions and compared the predictions to the real shape of those junctions. We show that the correct answer is selected out of nine possible configurations 64% of the time. Additionally, these results are noticeably improved if homology information is used. The resulting software, Cartaj, is available online and downloadable (with source) at: http://cartaj.lri.fr.     

Conformational Control of a Polyether-Linked Porphyrin Dimer Induced by Complexation with a Sodium Cation

Abstract

A porphyrin dimer with a polyether linkage was newly synthesized and its conformational change triggered by complexation with an sodium cation was examined. It was confirmed by ¹H NMR titration experiments that the dimer bearing a pentaoxyehtylene linkage (1b) was effectively formed a 1:1 complex with a sodium perchlorate. A detailed investigation of the CIS values for the protons confirmed that the complex of 1b?Na⁺ adopts a slipped face-to-face conformation.     

Mapping the backbone dihedral free-energy surfaces in small peptides in solution using adiabatic free-energy dynamics

Ramachandran surfaces for the alanine di- and tripeptides in gas phase and solution are mapped out using the recently introduced adiabatic free-energy dynamics (AFED) approach introduced by Rosso et al. (J. Chem. Phys. 2002, 116, 4389) as applied to the CHARMM22 force field. It is shown that complete surfaces can be mapped out with an order of magnitude of greater efficiency with the AFED approach than they can using the popular umbrella sampling method. In the alanine dipeptide, it is found, in agreement with numerous other studies using the CHARMM22 force field, that the lowest free-energy structure is the extended beta conformation, (phi, psi) = (-81, 81), while in solution, the extended beta, (phi, psi) = (-81, 153) and right-handed alpha-helical, (phi, psi) = (-81, 63) conformations are nearly isoenergetic. In solution, a secondary minimum at (phi, psi) = (63, -81), corresponding to a C(7)ax conformation, occurs approximately 2.3 kcal/mol above the global free-energy minimum. The alanine tripeptide, a system that has received considerably less attention in the literature, is found to exhibit a similar structure to the alanine dipeptide with the extended beta conformation being the free-energy minimum in the gas phase and the beta and right-handed alpha-helical conformations being isoenergetic in solution. These studies indicate that the AFED method can be a powerful tool for studying multidimensional free-energy surfaces in complex systems.     

Redox‐induced configuration conversion for thioacetamide dimer can function as a molecular switch

The electronic switching properties of thioacetamide dimer (TAD) were investigated using the nonequilibrium Green's function method combined with density functional theory for design of a novel molecular switch. The H‐bonded TAD can be converted upon hole‐trapping to a three‐electron (3e)‐bonded configuration with a S∴S linkage which could provide a more favorable channel for charge transfer than the before. The redox‐induced configuration conversion between the H‐bonded and the 3e‐bonded TADs could govern the charge migration through the molecular junction with a considerable difference in conduction currents. The calculated I–V characteristic curves of two configurations exhibit a switching behavior with an On‐Off ratio in a range of about 4.3–7.6 within the applied voltages. Clearly, this hypothetical scheme provides a potential way to explore the novel conformation‐dependent molecular switch. © 2010 Wiley Periodicals, Inc. J Comput Chem 2010     

双卟啉化合物的构象平衡及π-π作用研究

制备并表征了一系列以柔韧烷氧化相连的自由双卟啉及其锌配合物,以＾１Ｈ－ＮＭＲ考察了烷氧链长度及锌离子对双卟啉构象平衡的影响。结果表明,双卟啉存在开放式及闭合式构象平衡,随烷氧链的增长,构象平衡由开放式向闭合式移动,当链上碳原子数为４时最有利于双卟啉形成闭合式构象。     

Molecular‐Dynamics Studies of Single‐Stranded Hexitol,Altritol, Mannitol,and Ribose Nucleic Acids (HNA,MNA, ANA,and RNA,Resp.) and of the Stability of HNA⋅RNA,ANA⋅RNA,and MNA⋅RNA Duplexes

The influence of the orientation of a 3′‐OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular‐dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long‐range electrostatic interactions. An equatorial orientation of the 3′‐OH group in the single‐stranded D ‐mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H‐bonds between the 3′‐OH group and the O−C(6′) of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA⋅RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA⋅RNA complexes. An axial orientation of the 3′‐OH group in the D ‐altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single‐stranded ANA chain. The averaged conformation of the ANA⋅RNA complex is similar to that of A‐RNA, with only minor changes in groove width, helical curvature, and H‐bonding pattern. The relative stabilities of ANA⋅RNA vs. HNA⋅RNA (HNA=D ‐hexitol nucleic acid without 3′‐OH group) can be explained by differences in restricted movements, H‐bonds, and solvation effects.     

Stabilisation of nucleic acid secondary structures by oligonucleotides with an additional nucleobase; synthesis and incorporation of 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine

A nucleoside with two nucleobases is incorporated into oligonucleotides. The synthetic building block, 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine, 2, is prepared from uridine via 5',3'-TIPDS-protected 2'-deoxy-2'-C-allyluridine by an oxidative cleavage of the allyl group, a Mitsunobu reaction for the introduction of thymine and appropriate deprotection reactions. This compound is converted into a DMT-protected phosphoramidite and incorporated once into a 13-mer oligodeoxynucleotide sequence, once in an isosequential LNA-modified oligodeoxynucleotide and four times in the middle of a 12-mer oligodeoxynucleotide. These sequences are mixed with different complementary DNA and RNA sequences in order to study the effect of the additional nucleobase in duplexes, in bulged duplexes and in three-way junctions. The first additional thymine is found to be well-accommodated in a DNA-RNA duplex, whereas a DNA-DNA duplex was slightly destabilised. A three-way junction with the additional thymine in the branching point is found to be stabilised in both a DNA-DNA and a DNA-RNA context but destabilised where the modified LNA-sequence is used. In a Mg2+-containing buffer, however, the relative stability of the three-way junctions is found to be opposite with especially the LNA-modified DNA-DNA complex being significantly stabilised by the additional nucleobase.     

A Large Conformational Change of a Bridged β-Cyclodextrin Dimer in Aqueous Solution

A novel bridged β-CD dimer in which two β-cyclodextrins were linked by a naphthalene at positions 2 and 7 has been synthesized. 1H and 13CNMR measurements showed that a large change in the conformation of the dimer occurred in aqueous solution. The dimer interacted with methyl and ethyl orange to form stable inclusion complexes via "induced fit" mechanism.     

Branchpoint expansion in a fully complementary three-way DNA junction

Branched nucleic acid molecules serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resolution single-molecule FRET, time-resolved spectroscopy, and molecular modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in solution. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the molecule. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramolecular receptors and nanosystems.     

Synthesis and properties of oligonucleotides having a phosphorus chiral center by incorporation of conformationally rigid 5'-cyclouridylic acid derivatives

This paper describes the design and synthesis of a conformationally rigid dimer building block Umpc3Um as a chiral center at the phosphate group with the S/N junction where c3 refers to a propylene bridge linked between the uracil 5-position and 5'-phosphate group of pUm. The extensive H1 NMR analysis of Umpc3Um suggests that the 5'-upstream Um has predominantly a C2'-endo conformation and the pc3Um moiety exists almost exclusively in a C3'-endo conformation. The absolute configuration of the diastereomers Umpc3Um(fast) (8a) and Umpc3Um(slow) (8b) was determined by CD spectroscopy as well as computer simulations. The oligonucleotides U4[Umpc3Um(fast)]U4 (13a) and U4[Umpc3Um(slow)]U4 (13b) incorporating 8a and 8b were synthesized by use of the phosphoramidite building blocks 11a and 11b, respectively. The Tm experiments of the duplexes formed between these modified oligomers and the complementary oligomers imply that the modified oligomer 13a having Umpc3Um(fast) has the Sp configuration at the chiral phosphoryl group.     

Folding of 8-17 deoxyribozyme studied by three-color alternating-laser excitation of single molecules

The folding of 8-17 deoxyribozyme was investigated by three-color alternating-laser excitation (3c-ALEX), a new single-molecule fluorescence resonance energy transfer (FRET) method we recently developed. Since 3c-ALEX has the capability of simultaneously sorting fluorescent molecules based on their labeling status and monitoring three interprobe distances of a biomolecule by employing three-color FRET, it is an ideal tool to study folding of multibranched molecules. The 8-17 deoxyribozyme, a DNA enzyme that cleaves a specific RNA substrate, is a good model system for a multibranched molecule, since it has the structure of a three-way DNA junction with a bulge. Labeling all three branches of the 8-17 with different fluorescent probes, we studied its [Mg2+]-dependent folding in a Na+ buffer solution. With the stoichiometric sorting capability of 3c-ALEX, we first selected only the triply labeled 8-17 in a solution of all heterogeneous mixtures and then simultaneously measured all three interprobe distances of the selected species. Our results show that the 8-17 folds into a pyramidal form upon increasing [Mg2+], in a similar way with [Zn2+] as found in an earlier study conducted at the ensemble level. The apparent dissociation constant of Mg2+ was more than 100 times larger than that of Zn2+ and showed considerable variance with buffer concentration. No clear sign of two-step folding was observed for Mg2+, in contrast to the case of Zn2+. Compared with the hammerhead ribozyme, the 8-17 was found to require 10 times higher [Mg2+] to undergo folding. By comparison with the folding of several inactive 8-17 analogues, we found that the two conserved sequences (A and G) of the triad loop of the shortest branch are critical elements for folding, especially for the folding at low [Mg2+]. Our results suggest that the role of the stem loop is to provide a scaffold for the two bases to be properly positioned for the necessary interaction and that the two bases are directly involved in the interaction that plays a critical role in folding. This work demonstrates that 3c-ALEX is a powerful single-molecule method to study the structure and folding of complex and multibranched biomolecules.     

Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn-protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.     

Photoinduced Charge Transfer in a Conformational Switching Chlorin Dimer–Azafulleroid in Polar and Nonpolar Media

In the present study, a biomimetic reaction center model, that is, a molecular triad consisting of a chlorin dimer and an azafulleroid, is synthesized and its photophysical properties are studied in comparison with the corresponding molecular dyad, which consists only of a chlorin monomer and an azafulleroid. As evidenced by ¹H NMR, UV/Vis, and fluorescence spectroscopy, the chlorin dimer–azafulleroid folds in nonpolar media into a C₂‐symmetric geometry through hydrogen bonding, resulting in appreciable electronic interactions between the chlorins, whereas in polar media the two chlorins diverge from contact. Femtosecond transient absorption spectroscopy studies reveal longer charge‐separated states for the chlorin dimer–azafulleroid; ≈1.6 ns in toluene, compared with the lifetime of ≈0.9 ns for the corresponding chlorin monomer–azafulleroid in toluene. In polar media, for example, benzonitrile, similar charge‐separated states are observed, but the lifetimes are inevitably shorter: 65 and 73 ps for the dimeric and monomeric chlorin–azafulleroids, respectively. Nanosecond transient absorption and singlet oxygen phosphorescence studies corroborate that in toluene, the charge‐separated state decays indirectly via the triplet excited state to the ground state, whereas in benzonitrile, direct recombination to the ground state is observed. Complementary DFT studies suggest two energy‐minima conformations, that is, a folded chlorin dimer–azafulleroid, which is present in nonpolar media, and another conformation in polar media, in which the two hydrophobic chlorins wrap the azafulleroid. Inspection of the frontier molecular orbitals shows that in the folded conformation, the HOMO on each chlorin is equivalent and is shared owing to partial π–π overlap, resulting in delocalization of the conjugated π electrons, whereas the wrapped conformation lacks this stabilization. As such, the longer charge‐separated lifetime for the dimer is rationalized by both the electron donor–acceptor separation distance and the stabilization of the radical cation through delocalization. The chlorin folding seems to change the photophysical properties in a manner similar to that observed in the chlorophyll dimer in natural photosynthetic reaction centers.