Cross-linked DNA generated by "bis-click" reactions with bis-functional azides: site independent ligation of oligonucleotides via nucleobase alkynyl chains

Template-free cross-linking of single-stranded DNA bearing octadiynyl side chains at the 7-position of 8-aza-7-deazapurine moieties with bisfunctional azides is reported employing a Cu(I)-catalyzed azide-alkyne "bis-click" reaction. Bis-adducts were formed when the bis-azide:oligonucleotide ratio was 1:1; monofunctionalization occurred when the ratio was 15:1. Four-stranded DNA consisting of two cross-linked duplexes was obtained after hydridization. Cross-linked duplexes are as stable as individual duplexes when ligation was introduced at terminal positions; ligation at a central position led to a slight duplex destabilization.     

Cross-linked DNA: propargylated ribonucleosides as "click" ligation sites for bifunctional azides

2'-O or 3'-O-propargylated adenosines and ribothymidines were used as click targets for cross-linking of oligonucleotides with aliphatic and aromatic azides. The cross-link generates a sugar modification at the 2'-O-ligation site. Inexpensive ribonucleosides were used as starting materials. Cross-linking of oligonucleotides was performed at internal or terminal positions. Hybridization of homodimers with two complementary single strands resulted in stable ligated DNA duplexes.     

Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne "click" reaction

5-(Octa-1,7-diynyl)-2'-deoxyuridine was converted into the furano-dU derivative 7 by copper-catalyzed cyclization; the pyrolodC-derivative 3 was formed upon ammonolysis. The bicyclic nucleosides 3 and 7 as well as the corresponding non-cyclic precursors 4 and 6 all containing terminal C[triple bond]C bonds were conjugated with the non-fluorescent 3-azido-7-hydroxycoumarin 5 employing the copper(I)-catalyzed Huisgen-Sharpless-Meldal cycloaddition "click reaction". Strongly fluorescent 1H-1,2,3-triazole conjugates (30-33) are formed incorporating two fluorescent reporters-the pyrdC nucleoside and the coumarin moiety. Oligonucleotides incorporating 6-alkynyl and 6-alkyl 7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleosides (3 and 2f) have been prepared by solid-phase synthesis using the phosphoramidite building blocks 10 and 13 ; the pyrrolo-dC oligonucleotides are formed during ammonia treatment. The duplex stability of oligonucleotides containing 3 and related derivatives was studied. Oligonucleotides with terminal triple bonded nucleosides such as 3 are more stabilizing than those lacking a side chain with terminal unsaturation; open-chain derivatives (4) are even more efficient. The click reaction was also performed on oligonucleotides containing the pyrdC-derivative and the fluorescence properties of nucleosides, oligonucleotides and their coumarin conjugates were studied.     

7-Deazapurine and 8-aza-7-deazapurine nucleoside and oligonucleotide pyrene "click" conjugates: synthesis, nucleobase controlled fluorescence quenching, and duplex stability

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene "click" conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two "double clicked" pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6-12 °C for duplexes with a modified "adenine" base and 2-6 °C for a modified "guanine" base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching.     

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.     

Circular DNA by “Bis‐Click” Ligation: Template‐Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides

A highly effective and convenient “bis‐click” strategy was developed for the template‐independent circularization of single‐stranded oligonucleotides by employing copper(I)‐assisted azide–alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7‐deaza‐2′‐deoxyadenosine and 2′‐deoxyuridine residues with different side chains were used in solid‐phase synthesis with phosphoramidite chemistry. The bis‐click ligation of linear 9‐ to 36‐mer oligonucleotides with 1,4‐bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9‐mers. Compared with linear duplexes, circular bis‐click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis‐click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides.     

Stabilization by extra-helical thymines of a DNA duplex with Hoogsteen base pairs

We present the crystal structure of the DNA duplex formed by d(ATATATCT). The crystals contain seven stacked antiparallel duplexes in the asymmetric unit with A.T Hoogsteen base pairs. The terminal CT sequences bend over so that the thymines enter the minor groove and form a hydrogen bond with thymine 2 of the complementary strand in the Hoogsteen duplex. Cytosines occupy extra-helical positions; they contribute to the crystal lattice through various kinds of interactions, including a unique CAA triplet. The presence of thymine in the minor groove apparently contributes to the stability of the DNA duplex in the Hoogsteen conformation. These observations open the way toward finding under what conditions the Hoogsteen duplex may be stabilized in vivo. The present crystal structure also confirms the tendency of A.T-rich oligonucleotides to crystallize as long helical stacks of duplexes.     

2'-modified-2-thiothymidine oligonucleotides

[reaction: see text] Oligonucleotides with novel modifications, 2'-O-[2-(methoxy)ethyl]-2-thiothymidine and 2'-deoxy-2'-fluoro-2-thiothymidine, have been synthesized. These modified oligonucleotides exhibited very high thermal stability when hybridized with complementary RNA. 2-O-(2-Methoxy)ethyl-2-thiothymidine modified oligonucleotide phosphodiesters showed enhanced resistance toward nucleases (t(1/2) > 24 h), but 2'-deoxy-2'-fluoro-2-thiothymidine modified oligonucleotide phosphodiesters showed limited stability to nucleotytic degradation.     

Psoralen-conjugated oligonucleotide with hairpin structure as a novel photo-sensitive antisense molecule

Hairpin type psoralen-cojugated oligonucleotides cross-linked with RNA when they hybridized with a perfectly complementary RNA.     

Preferentially linear connection of gold nanoparticles in derivatization with phosphorothioate oligonucleotides

A gold nanosphere in water is considered to attain special stability in derivatization like an artificial atom when the octet rule is satisfied by forming four covalent bonds with two 5'-phosphorothioate-modified oligonucleotide molecules. Owing to this, the hybridization of two mutually complementary gold-bound oligonucleotides makes gold nanospheres preferentially connected linearly by duplexes to produce strands like linear artificial molecules. We have then fixated the linear strands of DNA-linked gold nanospheres by reducing Ag(+) ion clusters immobilized around duplexes to show the absorption spectrum of silver-coated artificial-molecular nanorods.     

Bicyclo[3.2.1]amide-DNA: a chiral,nonchiroselective base-pairing system

The design, synthesis, and base-pairing properties of bicyclo[3.2.1]amide-DNA (bca-DNA), a novel phosphodiester-based DNA analogue, are reported. This analogue consists of a conformationally constrained backbone entity, which emulates a B-DNA geometry, to which the nucleo-bases were attached through an extended, acyclic amide linker. Homobasic adenine-containing bca decamers form duplexes with complementary oligonucleotides containing bca, DNA, RNA, and, surprisingly, also L-RNA backbones. UV and CD spectroscopic investigations revealed the duplexes with D- or L-complements to be of similar stability and enantiomorphic in structure. Bca oligonucleotides that contain all four bases form strictly antiparallel, left-handed complementary duplexes with themselves and with complementary DNA, but not with RNA. Base-mismatch discrimination is comparable to that of DNA, while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior to those of DNA or RNA. A detailed molecular modeling study of left- and right-handed bca-DNA-containing duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA association.     

Enhanced recognition of non-complementary hybridization by single-LNA-modified oligonucleotide probes

Locked nucleic acid (LNA) is a deoxyribonucleotide analogue with an unusual ‘locked’ furanose conformation. LNA-modified oligonucleotide probes have demonstrated an enhanced binding affinity towards their complementary strands; however, their potential to discriminate non-complementary hybridization of mismatches has not been explored. In this study, we investigated the effect of the chemical nature of LNA nucleobases on the hybridization stability and the capability of LNA-modified oligonucleotides to discriminate the LNA:DNA mismatched base pairs. It was observed that LNA modification indeed improves the discrimination capability of oligonucleotides by increasing the melting temperature differences between the complementary duplexes and hybrids containing mismatches. Particularly, LNA purines offer a greater potential to recognize the mismatches than LNA pyrimidines and DNA purines. Real-time PCR experiments further confirmed that LNA modifications at the 3′-end are more effective. The results and conclusions in this study provide useful information for hybridization-based nucleic acid analysis where designing sound oligonucleotide probes is crucial to the success of the analyses.   Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA polymorphism as an origin of adenine-thymine tract length-dependent threading intercalation rate

Binuclear ruthenium complexes that bind DNA by threading intercalation have recently been found to exhibit an exceptional kinetic selectivity for long polymeric adenine-thymine (AT) DNA. A series of oligonucleotide hairpin duplexes containing a central tract of 6-44 alternating AT base pairs have here been used to investigate the nature of the recognition mechanism. We find that, above a threshold AT tract length corresponding to one helix turn of B-DNA, a dramatic increase in threading intercalation rate occurs. In contrast, such length dependence is not observed for rates of unthreading. Intercalation by any mechanism that depends on the open end of the hairpin was found not to be important in the series of oligonucleotides used, as verified by including in the study a hairpin duplex cyclized by a copper-catalyzed "click" reaction. Our observations are interpreted in terms of a conformational pre-equilibrium, determined by the length of the AT tract. We finally find that mismatches or loops in the oligonucleotide facilitate the threading process, of interest for the development of mismatch-recognizing probes.     

Nucleoside and oligonucleotide pyrene conjugates with 1,2,3-triazolyl or ethynyl linkers: synthesis,duplex stability,and fluorescence changes generated by the DNA-dye connector

Fluorescent nucleosides and oligonucleotides functionalized with pyrene were synthesized using ‘click’ chemistry or the Sonogashira cross-coupling reaction. The dye was connected to position-7 of 7-deaza-2′-deoxyguanosine or to the 2′-deoxyribofuranose moiety. Four different DNA-dye connectors with 1,2,3-triazolyl residues or triple bonds were constructed. Phosphoramidites of the pyrene conjugates (9, 14, 25) were prepared and used in solid-phase synthesis. Short linkers (2, 4) destabilize DNA, while long linkers (1) increased duplex stability. Nucleosides and oligonucleotides with single dye incorporations show linker dependent fluorescence. Linker dependent excimer emission with pyrenes in proximal positions was also observed. A ‘superchromophore’ formed by the 7-deaza-2′-deoxyguanosine ethynylpyrene conjugate shows strong red shifted fluorescence emission at 495 nm.     

Metal-enhanced fluorescent probes based on silver nanoparticles and its application in IgE detection

In this paper, a novel metal plasmon coupled with an aptamer–nucleotide hybridized probe was fabricated and applied for protein detection. The specific aptamer and single-strand oligonucleotide were chemically bound to silver nanoparticles (AgNPs), and Cy5-labeled, complementary single-strand oligonucleotides were hybridized with the particle-bound oligonucleotides. The hybridized DNA duplexes were regarded as rigid rods that separated the fluorophore Cy5 and the surface of AgNPs to reduce the competitive quenching. Using a model system comprising human immunoglobulin E (IgE) as the analyte and goat antihuman IgE as immobilized capture antibody on glass slides, we demonstrate that the detection performance of the synthetic probe was superior to the aptamer-based fluorescent probes. The results showed a good linear correlation for human IgE in the range from 10 ng/ml to 6.25 μg/ml. The detection limit obtained was 1 ng/ml, which was 50 times lower than that using Cy5 oligonucleotide/aptamer hybrid duplex (Probe2) due to the metal-enhanced fluorescence effect. This new strategy opens the possibility for the preparation of high-sensitivity detection probes based on metal nanoparticles.     

Still Elusive – Pd(II)-Mediated Base Pairing by an Acetophenone Oxime Palladacycle within 15N-Labelled Double-Helical Oligonucleotides

Both α and β anomers of an acetophenone C-nucleoside were synthesized and incorporated in the middle of short oligodeoxynucleotides. The ketone oligonucleotides were converted to ¹⁵N-labelled oxime oligonucleotides by treatment with ¹⁵N-hydroxylamine and, finally, cyclopalladated by treatment with lithium tetrachloropalladate. Comparison of the UV melting profiles of duplexes bearing the β anomer of either the palladacyclic or the metal-free oxime C-nucleoside suggested formation of a stable Pd(II)-mediated base pair, especially with adenine or thymine as the base pairing partner. Melting profiles of the corresponding duplexes bearing the α anomer were much more convoluted, precluding meaningful comparison. ¹⁵N NMR spectra were obtained for the β anomeric oxime oligonucleotide as well as its palladacyclic derivative but the signals unfortunately diminished below detection limit when the latter was hybridized with a complementary strand placing a ¹⁵N3-labelled thymine opposite to the palladacyclic residue.     

2′-Lipid-modified oligonucleotides via a ‘Staudinger-Vilarrasa’ reaction

We report a new access to 2′-amido-2′-deoxyuridine via a Staudinger-Vilarrasa coupling reaction for the preparation of lipid-modified oligonucleotides. One or two lipidic moieties were inserted within the oligonucleotidic sequence (LONs) leading to a repertoire of original antagomir-like molecules targeting micro RNA (miRNA or miR). Melting temperature (Tm) experiments revealed that the stability of the duplexes depends on the lipid position and the number of lipid moieties inserted within the oligonucleotide sequence. Single lipid conjugations of positions 11 and 19 of LONs targeting miR-122 do not destabilize the duplexes.     

Oligonucleotides Containing 7‐Deaza‐2′‐deoxyinosine as Universal Nucleoside: Synthesis of 7‐Halogenated and 7‐Alkynylated Derivatives,Ambiguous Base Pairing,and Dye Functionalization by the Alkyne–Azide ‘Click’ Reaction

Oligonucleotides containing 7‐deaza‐2′‐deoxyinosine derivatives bearing 7‐halogen substituents or 7‐alkynyl groups were prepared. For this, the phosphoramidites 2b – 2g containing 7‐substituted 7‐deaza‐2′‐deoxyinosine analogues 1b – 1g were synthesized (Scheme 2). Hybridization experiments with modified oligonucleotides demonstrate that all 2′‐deoxyinosine derivatives show ambiguous base pairing, as 2′‐deoxyinosine does. The duplex stability decreases in the order C_d>A_d>T_d>G_d when 2b – 2g pair with these canonical nucleosides (Table 6). The self‐complementary duplexes 5′‐d(F⁷c⁷I‐C)₆, d(Br⁷c⁷I‐C)₆, and d(I⁷c⁷I‐C)₆ are more stable than the parent duplex d(c⁷I‐C)₆ (Table 7). An oligonucleotide containing the octa‐1,7‐diyn‐1‐yl derivative 1g , i.e., 27 , was functionalized with the nonfluorescent 3‐azido‐7‐hydroxycoumarin ( 28 ) by the Huisgen–Sharpless–Meldal cycloaddition ‘click’ reaction to afford the highly fluorescent oligonucleotide conjugate 29 (Scheme 3). Consequently, oligonucleotides incorporating the derivative 1g bearing a terminal C?C bond show a number of favorable properties: i) it is possible to activate them by labeling with reporter molecules employing the ‘click’ chemistry. ii) Space demanding residues introduced in the 7‐position of the 7‐deazapurine base does not interfere with duplex structure and stability (Table 8). iii) The ambiguous pairing character of the nucleobase makes them universal probes for numerous applications in oligonucleotide chemistry, molecular biology, and nanobiotechnology.     

Site-selective DNA hydrolysis by combining Ce(IV)/EDTA with monophosphate-bearing oligonucleotides and enzymatic ligation of the scission fragments

By using two oligonucleotide additives that bear a monophosphate group at the termini through various linkers, gap structures were formed at predetermined positions in substrate DNA, and the monophosphate groups were placed at both edges of these gaps. At pH 7.0 and 37 degrees C, the phosphodiester linkages in the gap sites were efficiently and selectively hydrolyzed by Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The linkages in the middle of the gaps were predominantly hydrolyzed. Compared with DNA scission using oligonucleotide additives that bear no terminal monophosphate, the present scission was much faster (22-fold for a 3-base gap and 14-fold for a 5-base gap) and more site selective. Introduction of one monophosphate group to either edge of the gaps was also effective for promotion of both site selectivity and scission rate. The monophosphate group(s) at the gap site recruits the Ce(IV) to the target site and magnifies the difference in intrinsic reactivity between the target site and the others. Even at higher reaction temperatures, the site selectivity remained satisfactorily high. Furthermore, the fragments formed by the site-selective scission were connected with various oligonucleotides by using DNA ligase, producing desired recombinant DNAs.     

Stable and selective formation of hoogsteen-type triplexes and duplexes using twisted intercalating nucleic acids (TINA) prepared via postsynthetic Sonogashira solid-phase coupling reactions

Bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (5) into the middle of homopyrimidine oligodeoxynucleotides (twisted intercalating nucleic acids, TINA) obtained via postsynthetic Sonogashira coupling reaction led to extraordinary high thermal stability of Hoogsteen-type triplexes and duplexes, whereas Watson-Crick-type duplexes of the same nucleotide content were destabilized. Modified oligonucleotides were synthesized using the phosphoramidite of (S)-1-(4,4'-dimethoxytriphenylmethyloxy)-3-(4-iodo-benzyloxy)-propan-2-ol followed by treatment of the oligonucleotide on a CPG-support with the Sonogashira-coupling reaction mixture containing different ethynylaryls. Bulged insertion of the pyrene derivative 5 into oligonucleotides was found to be the best among the tested modifications for binding to the Hoogsteen-type triplexes and duplexes. Thus, at pH 7.2 an oligonucleotide with cytidine content of 36% possessing two bulged insertions of 5 separated by three bases formed a stable triplex (T(m) = 43.0 degrees C), whereas the native oligonucleotide was unable to bind to the target duplex. The corresponding Watson-Crick-type duplex with the same oligonucleotide had T(m) of 38.0 degrees C at pH 7.2, while the T(m) of unmodified dsDNA was 47.0 degrees C. Experiments with mismatched oligonucleotides, luminescent properties, and potential applications of TINA technology is discussed.