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.     

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.     

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.     

Novel bicyclic nucleoside analogue (1S,5S,6S)-6- hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo[3.2.1]octane: synthesis and incorporation into oligodeoxynucleotides

The novel bicyclic nucleoside (1S,5S,6S)-6-hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo[3.2.1]octane [2'-deoxy-1'-C,4'-C-(2-oxapropano)uridine] (15), expected to be restricted into an O4'-endo furanose conformation, was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 5. The phosphoramidite derivative of 15 was successfully incorporated into oligodeoxynucleotides using standard methods, and thermal denaturation studies showed moderate decreases in duplex stabilities of -2.1 and -1.5 degrees C per modification toward complementary DNA and RNA, respectively.     

Influence of the Type of Junction in DNA-3′-Peptide Nucleic Acid (PNA) Chimeras on Their Binding Affinity to DNA and RNA

The automated on-line synthesis of DNA-3′-PNA (PNA=Polyamide Nucleic Acids) chimeras 1 – 3 is described, in which the 3′-terminal part of the oligonucleotide is linked to the aminoterminal part of the PNA either via a N-(2-mercaptoethyl)- (X=S), a N-(2-hydroxyethyl)- (X=O), or a N-(2-aminoethyl)- (X=NH) N-[(thymin-1-yl)acetyl]glycine unit. Furthermore, the DNA-3′-PNA chimera 4 without a nucleobase at the linking unit was prepared. The binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA, RNA, or DNA containing a mismatch or abasic site opposite to the linker unit. We found that all investigated chimeras with a nucleobase at the junction form more stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. The influence of X on duplex stabilization was determined to be in the order O>S≈NH, rendering the phosphodiester bridge the most favored linkage at the DNA/PNA junction. The observed strong duplex-destabilizing effects, when base mismatches or non-basic sites were introduced opposite to the nucleobase at the DNA/PNA junction, suggest that the base at the linking unit contributes significantly to duplex stabilization.     

Global structure of a three-way junction in a phi29 packaging RNA dimer determined using site-directed spin labeling

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H(T) and H(L)) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H(T) and H(L) stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

Synthesis and conformational properties of oligonucleotides incorporating 2'-O-phosphorylated ribonucleotides as structural motifs of pre-tRNA splicing intermediates

To synthesize oligonucleotides containing 2'-O-phosphate groups, four kinds of ribonucleoside 3'-phosphoramidite building blocks 6a-d having the bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) group were prepared according to our previous phosphorylation procedure. These phosphoramidite units 6a-d were not contaminated with 3'-regioisomers and were successfully applied to solid-phase synthesis to give oligodeoxyuridylates 15, 16 and oligouridylates 21, 22. Self-complementary Drew-Dickerson DNA 12mers 24-28 replaced by a 2'-O-phosphorylated ribonucleotide at various positions were similarly synthesized. In these syntheses, it turned out that KI(3) was the most effective reagent for oxidative desulfurization of the initially generated thiophosphate group to the phosphate group on polymer supports. Without using this conversion step, a tridecadeoxyuridylate 17 incorporating a 2'-O-thiophosphorylated uridine derivative was also synthesized. To investigate the effect of the 2'-phosphate group on the thermal stability and 3D-structure of DNA(RNA) duplexes, T(m) measurement of the self-complementary oligonucleotides obtained and MD simulation of heptamer duplexes 33-36 were carried out. According to these analyses, it was suggested that the nucleoside ribose moiety phosphorylated at the 2'-hydroxyl function predominantly preferred C2'-endo to C3'-endo conformation in DNA duplexes so that it did not significantly affect the stability of the DNA duplex. On the other hand, the 2'-modified ribose moiety was expelled to give a C3'-endo conformation in RNA duplexes so that the RNA duplexes were extremely destabilized.     

Nucleic acid secondary structures containing the double-headed nucleoside 5'(S)-C-(2-(thymin-1-yl)ethyl)thymidine

Oligodeoxynucleotides containing the double-headed nucleoside 5'(S)-C-(2-(thymin-1-yl)ethyl)thymidine were prepared by standard solid phase synthesis. The synthetic building block for incorporating the double-headed moiety was prepared from thymidine, which was stereoselectively converted to a protected 5'(S)-C-hydroxyethyl derivative and used to alkylate the additional thymine by a Mitsunobu reaction. The oligodeoxynucleotides were studied in different nucleic acid secondary structures: duplexes, bulged duplexes, three-way junctions and artificial DNA zipper motifs. The thermal stability of these complexes was studied, demonstrating an almost uniform thermal penalty of incorporating one double-headed nucleoside moiety into a duplex or a bulged duplex, comparable to the effects of the previously reported double-headed nucleoside 5'(S)-C-(thymin-1-yl)methylthymidine. The additional base showed only very small effects when incorporated into DNA or RNA three-way junctions. The various DNA zipper arrangements indicated that extending the linker from methylene to ethylene almost completely removed the selective minor groove base-base stacking interactions observed for the methylene linker in a (-3)-zipper, whereas interactions, although somewhat smaller, were observed for the ethylene linker in a (-4)-zipper motif.     

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).     

O6-Alkylguanine DNA Alkyltransferase Mediated Disassembly of a DNA Tetrahedron

Tetrahedron DNA structures were formed by the assembly of three-way junction ( TWJ ) oligonucleotides containing O⁶-2′-deoxyguanosine-alkylene-O⁶-2′-deoxyguanosine (butylene and heptylene linked) intrastrand cross-links (IaCLs) lacking a phosphodiester group between the 2′-deoxyribose residues. The DNA tetrahedra containing TWJs were shown to undergo an unhooking reaction by the human DNA repair protein O⁶-alkylguanine DNA alkyltransferase (hAGT) resulting in structure disassembly. The unhooking reaction of hAGT towards the DNA tetrahedra was observed to be moderate to virtually complete depending on the protein equivalents. DNA tetrahedron structures have been explored as drug delivery platforms that release their payload in response to triggers, such as light, chemical agents or hybridization of release strands. The dismantling of DNA tetrahedron structures by a DNA repair protein contributes to the armamentarium of approaches for drug release employing DNA nanostructures.     

Automated solid-phase synthesis and photophysical properties of oligodeoxynucleotides labeled at 5'-aminothymidine with Ru(bpy)2(4-m-4'-cam-bpy)2+

A facile and automated procedure for the incorporation of a derivatized Ru(bpy)3(2+) in an oligodeoxynucleotide is reported. The Ru(bpy)3(2+)-thymidine phosphoramidite is synthesized, and then incorporated in DNA using a standard protocol on an automated DNA solid-phase synthesizer. The structure of the DNA duplex is not altered after labeling with Ru(bpy)3(2+). Photophysical studies of the novel ruthenium trisdiimine thymidine complex as well as the corresponding labeled oligodeoxynucleotides demonstrate that the favorable properties associated with the ruthenium complex are retained after covalent attachment to the nucleoside and oligodeoxynucleotide.     

Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5'-(CpG) motif: model of a malondialdehyde cross-link.

Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis. MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine. In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F. H. and Goldstein, B. D. Science 1976, 191, 868--869). The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines. An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions. The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link. The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation. This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.     

Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages

We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3'-5' linkages are more stable in a duplex than isomeric non-native 2'-5' linkages, this strategy is predicted to favor the formation of 3'-5' linkages. All of the new deoxyribozymes indeed create only linear 3'-5' RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with k(obs) values up to 0.5 h(-1) at 37 degrees C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which to date have been highly elusive via other selection approaches.     

STRUCTURE-SPECIFIC BINDING and PHOTOSENSITIZED CLEAVAGE OF BRANCHED DNA THREE-WAY JUNCTION COMPLEXES BY CATIONIC PORPHYRINS

Abstract— The interactions of cationic porphyrins with DNA oligonucleotides that form branched, three-way junction complexes (TWJ) were investigated using native gel electrophoresis, absorption spectroscopy and photochemical probing using DNA sequencing techniques. Meso-tetra(pa ra-N-trimethylaniliniumyrjporphine (TMAP), meso-tetra (4-JV-methylpyridiniumyl)porphine (T4MPyP) and meso-tetra(4- N -methylpyridiniumytyporphine (T3MPyP) were found to bind more tightly to DNA TWJ than to DNA duplexes. The binding to the junction DNA persists at high ionic strength, conditions that greatly decrease porphyrin binding affinity to duplex DNA. The TWJ DNA binding sites of TMAP and T4MPyP were localized to the junction region based on the observation of site- and structure-specific, porphyrin-sensitized photodamage to guanosine residues flanking the junction region.     

Synthetic nucleic acid secondary structures containing the four stereoisomers of 1,4-bis(thymine-1-yl)butane-2,3-diol

The four stereoisomers of the double-headed acyclic nucleoside 1,4-bis(thymine-1-yl)butane-2,3-diol were incorporated in the central position of four 13-mer oligonucleotides. The phosphoramidite building blocks were synthesized in four or six steps from either D- or L-2,3-O-isopropylidenethreitol. Two epimeric and fully deprotected double-headed nucleosides were analyzed by X-ray crystallography. The incorporation into oligonucleotides was hampered by steric hindrance and formation of a cyclic phosphate. The use of pyridinium chloride as the activator and a kinetic analysis based on 31P NMR of the coupling and detritylation processes led to improved yields of the oligonucleotides. In comparison with the (S)-GNA monomer, one of the four stereoisomers was found to show a similar destabilization of a DNA duplex, indicating that the additional base can be introduced without a thermal penalty. Another stereoisomer was found to induce a thermal stabilization of a DNA:RNA three-way junction. Thus, the stereochemistry of this acyclic double-headed nucleoside motif is important, indicating potential for the design of artificial nucleic acid secondary structures.     

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.     

Solution structure of xDNA: a paired genetic helix with increased diameter

We describe the structure in aqueous solution of an extended-size DNA-like duplex with base pairs that are approximately 2.4 A longer than those of DNA. Deoxy-lin-benzoadenosine (dxA) was employed as a dA analogue to form hydrogen-bonded base pairs with dT. The 10mer self-complementary extended oligodeoxynucleotide 5'-d(xATxAxATxATTxAT) forms a much more thermodynamically stable duplex than the corresponding DNA sequence, 5'-d(ATAATATTAT). NMR studies show that this extended DNA (xDNA) retains many features of natural B-form DNA, but with a few structural alterations due to its increased helical diameter. The results give insight into the structural plasticity of the natural DNA backbone and lend insight into the evolutionary origins of the natural base pairs. Finally, this structural study confirms the hypothesis that extended nucleobase analogues can form stable DNA-like structures, suggesting that alternative genetic systems might be viable for storage and transfer of genetic information.     

Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands

To understand the influences of the ribonucleotide on a duplex conformation and its stability, we systematically studied the CD spectra and the thermodynamics of nucleic acid duplexes formed by the chimeric RNA-DNA strand in which ribonucleotides and deoxyribonucleotides were covalently attached. It was found that the duplex stability was context-dependent and independent of the number of ribonucleotides in the chimeric strand, whereas the CD spectra showed less overall structural perturbation by the chimeric junctions. Combining the results of the CD and the thermodynamic data revealed a stability-structure relationship for the duplexes. Importantly, DeltaG(o)37 values estimated for the chimeric junction formation in the RNA-DNA/DNA and the RNA-DNA/RNA duplexes were close to those of RNA/DNA and RNA/RNA interactions, respectively. Furthermore, DeltaG(o)37s of the DNA-RNA/DNA and DNA-RNA/DNA-RNA junctions were similar to those of the DNA duplex, and the values of DNA-RNA/RNA-DNA were similar to those of the DNA/RNA. The thermodynamic analyses suggest that the 5'-nucleotide may be the crucial factor that determines the stability at the chimeric junction. Our results not only suggest influences of the ribonucleotide on a duplex conformation and its stability but also are useful for the design of RNA-DNA chimeric strands applicable to biotechnology.     

Structural Basis for Recognition of Guanosine by a Synthetic Tricyclic Cytosine Analogue: Guanidinium G‐Clamp

An oligonucleotide analogue containing a novel heterocyclic analogue, the guanidinium G‐clamp, was designed to allow formation of five H‐bonds to guanosine. The guanidinium group was introduced postsynthetically by treatment of the deprotected oligonucleotide containing a free amino group with a solution of 1H‐pyrazole‐1‐carboxamidine and purified by a combination of size‐exclusion chromatography and reversed‐phase HPLC. A single incorporation of this modification into an oligodeoxynucleotide sequence was found to increase duplex stability by 13° and 16° per modification to RNA and DNA, respectively. Crystals of a self‐complementary decamer sequence containing this modification were grown and diffracted to 1‐Å resolution. The structure was solved by molecular replacement and revealed that the modification forms additional H‐bonds to O(6) and N(7) of guanosine through the amino and imino N‐atoms, respectively. The origins of enhanced duplex stability are also attributed to increased stacking interactions mediated by the phenoxazine moiety of the G‐clamp and formation of H‐bond networks between the positively charged guanidinium group, H₂O molecules, and negatively charged O‐atoms from phosphates on the adjacent strand.