Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.     

Synthesis and biological activity of phosphonoacetate- and thiophosphonoacetate-modified 2'-O-methyl oligoribonucleotides

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3′-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5′-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents.     

Synthesis and properties of novel l-isonucleoside modified oligonucleotides and siRNAs

Antisense oligonucleotides and siRNAs are potential therapeutic agents and their chemical modifications play an important role to improve the properties and activities of oligonucleotides. Isonucleoside is a type of nucleoside analogue, in which the nucleobase is moved from C-1 to other positions of ribose. In this report, a novel isonucleoside containing a 5'-CH(2)-extended chain at the sugar moiety was synthesized, thus isoadenosine and isothymidine were incorporated into a DNA single strand and siRNA. It was found that isonucleoside modified oligonucleotides can form stable double helical structures with their complementary DNA and RNA and the stability towards nuclease and ability to activate RNase H are more promising compared with the unmodified, natural analogues. In siRNA, passenger strand modified with isonucleoside () at 3' or 5' terminal can retain the silencing activity and minimize the passenger strand specific off-target effect.     

Structural characterization and biological evaluation of small interfering RNAs containing cyclohexenyl nucleosides

CeNA is an oligonucleotide where the (deoxy)ribose sugars have been replaced by cyclohexenyl moieties. We have determined the NMR structure of a CeNA:RNA duplex and have modeled this duplex in the crystal structure of a PIWI protein. An N puckering of the ribose nucleosides, a 2H3 conformation of the cyclohexenyl nucleosides, and an A-like helix conformation of the backbone, which deviates from the standard A-type helix by a larger twist and a smaller slide, are observed. The model of the CeNA:RNA duplex bound to the PIWI protein does not show major differences in the interaction of the guide CeNA with the protein when compared with dsRNA, suggesting that CeNA modified oligonucleotides might be useful as siRNAs. Incorporation of one or two CeNA units in the sense or antisense strands of dsRNA led to similar or enhanced activity compared to unmodified siRNAs. This was tested by targeting inhibition of expression of the MDR1 gene with accompanying changes in P-glycoprotein expression, drug transport, and drug resistance.     

Synthesis and hybridization properties of 2'-O-methylated oligoribonucleotides incorporating 2'-O-naphthyluridines

2'-O-(1-Naphthyl)uridine and 2'-O-(2-naphthyl)uridine were synthesized by a microwave-mediated reaction of 2,2'-anhydrouridine with naphthols. Using the 3'-phosphoramidite building blocks, these 2'-O-aryluridine derivatives were incorporated into 2'-O-methylated oligoribonucleotides. Incorporation of five 2'-O-(2-naphthyl)uridines into a 2'-O-methylated RNA sense strand significantly increased the thermostability of the duplex with a 2'-O-methylated RNA antisense strand. Circular dichroism spectroscopy and molecular dynamic simulation of the duplexes formed between the modified RNAs and 2'-O-methyl RNAs suggested that there are π-π interactions between two neighboring naphthyl groups in a sequence of the five consecutively modified nucleosides.     

Incorporation of 4'-C-aminomethyl-2'-O-methylthymidine into DNA by thermophilic DNA polymerases

The dual modified nucleotide 4'-C-aminomethyl-2'-O-methylthymidine 5'-triphosphate was synthesized and enzymatically incorporated into DNA by the thermophilic DNA polymerases Pfu and Therminator III. The dual ribose modification imparted increased exonuclease resistance to DNA compared to the well-known 2'-O-methyl modification.     

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.     

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.     

Spermine moiety attached to the C-5 position of deoxyuridine enhances the duplex stability of the phosphorothioate DNA/complementary DNA and shows the susceptibility of the substrate to RNase H

Novel phosphorothioate-modified oligodeoxynucleotides (S-ODNs) containing a deoxyuridine derivative bearing a spermine moiety at the C-5 position were synthesized. The study of the thermal stability and the thermodynamic stability showed that the modified S-ODNs have been able to form the stable duplexes with the complementary DNA. It was also found that the duplex composed of the modified S-ODN and its complementary RNA strand is the substrate for Escherichia coli RNase H, and the cleavage of the RNA strand by the enzyme was almost similar as in the case of the unmodified one.     

Role of locked nucleic acid modified complementary strand in quadruplex/Watson-Crick duplex equilibrium

In the human genome, the G-rich sequences that form quadruplexes are present along with their C-rich complementary strands; this suggests the existence of equilibrium between a quadruplex and a Watson-Crick duplex which allows the execution of their respective biological functions. We have investigated the sensitivity of this equilibrium to pharmacological agents by employing locked nucleic acid (LNA) modified complementary strands, and demonstrated successful invasion of the stable telomeric quadruplex d[(G(3)TTA)(3)G(3)]. Fluorescence, UV, ITC, and SPR studies were performed to understand the binding process involving the preformed quadruplex and LNA-modified complementary strands compared with that involving the unmodified complementary strand. Our data indicate that LNA modifications in the complementary strand shift the equilibrium toward the duplex state. These modifications confer increased thermodynamic stability to the duplex and increase the magnitude of relative free energy (DeltaDeltaG degrees) difference between duplex and quadruplex, thus favoring the predominance of duplex population over quadruplex. This superior ability of LNA-modified complementary strand can be exploited to pave an exploratory approach in which it hybridizes to a telomeric quadruplex and drives duplex formation, and inhibits the recognition of 3' G-rich overhang by RNA template of telomerase which guides telomere extension.     

Steric effects in RNA interference: probing the influence of nucleobase size and shape

Nonpolar nucleosides with varying size and shape have been used to study the hydrogen-bonding stabilization and steric effects on RNA interference. The uracil and adenine residues of siRNA guide strands have been replaced by nonpolar isosteres of uracil and adenine and by steric variants. RNAi experiments targeting Renilla luciferase mRNA have shown close correlation between siRNA thermal stability and gene suppression. Interestingly, siRNA modified at position 7 on the guide strand does not follow this correlation, having substantial RNAi activity despite low thermal stability. Sequence-selectivity studies were carried out at this position with mutated target mRNAs and nucleobase analogues with varied size (2,4-difluoro- and 2,4-dichlorobenzene) and different shape (2,3-dichlorobenzene, 4-methylbenzimidazole). The results point out the importance of nucleobase shape and steric effects in RNA interference.     

Platinum interference with siRNA non-seed regions fine-tunes silencing capacity

Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ～20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.     

Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA. To acquire a detailed understanding of the structural features of LNA giving rise to its remarkable properties, we have conducted structural studies by use of NMR spectroscopy and now report high-resolution structures of two LNA:RNA hybrids, the LNA strands being d(5'-CTGAT(L)ATGC-3') and d(5'-CT(L)GAT(L)AT(L)GC-3'), respectively, T(L) denoting a modified LNA monomer with a thymine base, along with the unmodified DNA:RNA hybrid. In the structures, the LNA nucleotides are positioned as to partake in base stacking and Watson-Crick base pairing, and with the inclusion of LNA nucleotides, we observe a progressive change in duplex geometry toward an A-like duplex structure. As such, with the inclusion of three LNA nucleotides, the hybrid adopts an almost canonical A-type duplex geometry, and thus it appears that the number of modifications has reached a saturation level with respect to structural changes, and that further incorporations would furnish only minute changes in the duplex structure. We attempt to rationalize the conformational steering induced by the LNA nucleotides by suggesting that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.     

Oligonucleotide Analogues with a Nucleobase‐Including Backbone,Part 7, Molecular Dynamics Simulation of a DNA Duplex Containing a 2′‐Deoxyadenosine 8‐(Hydroxymethyl)‐Derived Nucleotide

The structure and stability of a 14‐mer DNA duplex containing a nucleotide analog with a hydroxymethyl substituent at the C(8) of 2′‐deoxyadenosine has been investigated by molecular‐dynamics simulation. The DNA duplex studied has the sequence 5′‐d(CGTAAGCTCGATAG)‐3′⋅5′‐d(CTATCGA*GCTTACG)‐3′, where the O(3′) of the dG₆ nucleotide in the second strand is linked through a phosphinato group with the O(10) of the dA 2′‐deoxyadenosine‐derived nucleotide. Previous experimental results showed that the stability of this duplex in aqueous solution of 0.1M NaCl at pH 7 and room temperature is significantly lower than that of the corresponding unmodified DNA duplex. Comparison of molecular‐dynamics trajectories of the unmodified and modified B‐DNA duplexes in aqueous solution, at similar conditions than the experiment, shows that the substitution of the dA nucleotide by the dA* nucleotide in the second strand induces stretching of the double helix, which results in opening of the grooves and consequent exposure of the double‐helix core to the solvent.     

The structure of a mixed LNA/DNA:RNA duplex is driven by conformational coupling between LNA and deoxyribose residues as determined from 13C relaxation measurements

A study of the internal dynamics of an LNA/DNA:RNA duplex has been performed to further characterize the conformational changes associated with the incorporation of locked nucleic acid (LNA) nucleotides in a DNA:RNA duplex. In general, it was demonstrated that the LNA/DNA:RNA duplex has a very high degree of order compared to dsDNA and dsRNA duplexes. The order parameters of the aromatic carbon atoms in the LNA/DNA strand are uniformly high, whereas a sharp drop in the degree of order was seen in the RNA strand in the beginning of the AUAU stretch in the middle of the strand. This can be related to a return to normal dsRNA dynamics for the central A:U base pair. The high order of the heteroduplex is consistent with preorganization of the chimera strand for an A-form duplex conformation. These results partly explain the dramatic increase in T(m) of the chimeric heteroduplex over dsDNA and DNA:RNA hybrids of the same sequence.     

Molecular dynamics simulation and binding energy calculation for estimation of oligonucleotide duplex thermostability in RNA-based therapeutics

For oligonucleotide-based therapeutics, a thorough understanding of the thermodynamic properties of duplex formation is critical to developing stable and potent drugs. For unmodified small interfering RNA (siRNA), DNA antisense oligonucleotide (AON) and locked nucleic acid (LNA), DNA/LNA modified oligonucleotides, nearest neighbor (NN) methods can be effectively used to quickly and accurately predict duplex thermodynamic properties such as melting point. Unfortunately, for chemically modified olignonucleotides, there has been no accurate prediction method available. Here we describe the potential of estimating melting temperature (T(m)) for nonstandard oligonucleotides by using the correlation of the experimental T(m) with the calculated duplex binding energy (BE) for oligonucleotides of a given length. This method has been automated into a standardized molecular dynamics (MD) protocol through Pipeline Pilot (PP) using the CHARMm component in Discovery Studio (DS). Results will be presented showing the correlation of the predicted data with experiment for both standard and chemically modified siRNA and AON.     

Application of high‐resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex

We investigated the application of a high‐resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high‐performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI‐Orbitrap or the MALDI‐TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI‐Orbitrap mass spectrometer enabled differentiation between two possible RNA‐based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In‐source decay (ISD) analysis using a MALDI‐TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4‐dihydroxyacetophenone (2,4‐DHAP) as a matrix. The ESI‐Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on‐line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI‐TOF mass spectrometer, in combination with 2,4‐DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds. Copyright © 2012 John Wiley & Sons, Ltd.