A new reactive nucleoside analogue for highly reactive and selective cross-linking reaction to cytidine under neutral conditions

We have already demonstrated that the oligonucleotides DNA (ODNs) bearing a 2-amino-6-vinylpurine derivative (1) exhibited efficient interstrand cross-linking to cytidine selectively. In this study, a new reactive nucleoside analogue, 2-amino-6-(1-ethylsulfoxy)vinylpurine derivative (7), was designed based on a computational method to achieve high and selective alkylation with cytidine under neutral conditions. It has been demonstrated that the ODN (13) bearing 2-amino-6-(1-ethylsulfoxy)vinylpurine achieved highly selective and efficient cross-linking to cytidine under neutral conditions.     

Fast DNA Interstrand Cross-Linking Reaction by 6-Vinylpurine

Oligonucleotides that incorporate a reactive moiety to form an interstrand cross-link have been widely studied for their potential toward inhibiting gene expression or as basic tools for chemical biology studies. The 6-vinylpurine (2) newly designed in the current study serves well as a new purine-base moiety for increasing cross-link reactivity to target cytosine. Thus, oligonucleotides containing 6-vinylpurine exhibit a more selective and much smoother DNA cross-linking ability to cytosine than the oligonucleotide analogs derived from 2-amino-6-vinylpurine (1) previously explored.     

Incorporation of an inducible nucleotide analog into DNA by DNA polymerases

Non-natural nucleotides with diverse functionalities are highly useful in many areas of basic research and practical applications. We have previously developed an efficient method for post-synthetic modifications of 2-amino-6-vinylpurine (AVP)-containing oligonucleotides, which permits conjugations of a variety of useful functional appendages to the AVP moiety in DNA. Here we report an investigation on the ability of various DNA polymerases to use 5′-triphosphate of 2′-deoxyribosyl-2-amino-6-(2-methylthioethyl)purine (a stable precursor of AVP) as the substrate for templated DNA synthesis.     

Site-directed alkylation to cytidine within duplex by the oligonucleotides containing functional nucleobases

We have previously described that oligonucleotides (ODN) containing phenylsulfoxide derivative of 2-amino-6-vinylpurine nucleoside analog (1) are activated within duplex to form cross-link toward cytidine selectively at the target site. In this paper, we wish to report the search for more stable precursor susceptible for activation within duplex.     

SITE-DIRECTED ALKYLATION TO CYTIDINE WITHIN DUPLEX BY THE OLIGONUCLEOTIDES CONTAINING FUNCTIONAL NUCLEOBASES

We have previously described that oligonucleotides (ODN) containing phenylsulfoxide derivative of 2-amino-6-vinylpurine nucleoside analog (1) are activated within duplex to form cross-link toward cytidine selectively at the target site. In this paper, we wish to report the search for more stable precursor susceptible for activation within duplex.     

Efficient cross-linking to cytidine by functional nucleobases capable of in situ activation.

We have previously demonstrated that the ODNs with 2-amino-6-(2-phenylsulfoxyethyl)purine nucleoside derivative were capable of efficient interstrand cross-linking with cytidine selectively. In this new strategy, less reactive precursor was auto-activated within a duplex to generate 2-amino-6-vinylpurine derivative. However, it turned out that 2-amino-6-(2-phenylsulfinyl)-ethylpurine nucleoside was not applicable as the precursor for the synthesis of DNA oligomers with G-rich sequences. In this report, 2-amino-6-(2-methylsulfinylethyl)purine nucleoside has been proven to be more suitable as a precursor for DNA synthesis. In addition, the ODNs incorporating either 2-amino-6-(2-phenylsulfoxy ethyl)purine or 2-amino-6-vinylpurine showed high reactivity toward the cytidine at the target site but quite less reactivity was observed for it at non-target site, demonstrating high site-selectivity.     

Development of a 3′-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.     

Alteration of cross-linking selectivity with the 2′-OMe analogue of 2-amino-6-vinylpurine and evaluation of antisense effects

We previously reported that oligodeoxynucleotides containing 2-amino-6-vinylpurine (2-AVP: 1) exhibit efficient selective cross-linking to cytosine. In this study, the 2′-OMe nucleoside analogue (2) of 2-AVP was designed in order to increase its affinity to RNA and enhance metabolic stability. It has been demonstrated that 2′-OMe oligonucleotides bearing 2 achieve highly selective cross-linking to the thymine base in DNA and show higher antisense effect on luciferase production in cell lysate.     

The alkyl-connected 2-amino-6-vinylpurine (AVP) crosslinking agent for improved selectivity to the cytosine base in RNA

We have previously reported that the 2-amino-6-vinylpurine (AVP) nucleoside exhibits a highly efficient and selective crosslinking reaction toward cytosine and displayed an improved antisense inhibition in cultured cells. In this study, we further investigated the alkyl-connected AVP nucleoside analogs for more efficient crosslinking to the cytosine base (rC) of the target RNA. We synthesized three AVP analogs which connect the 2-amino-6-vinylpurine unit to the 2′-deoxyribose through a methylene, an ethylene, or a butylene linker. The ODN incorporating the AVP analog with the methylene or the butylene linker showed a slightly higher crosslinking to the target rC of RNA than the original AVP with no linker. In contrast, the AVP with the ethylene linker formed a selective and efficient crosslink to the rC of the target RNA.     

Efficient synthesis of oligonucleotide conjugates on solid-support using an (aminoethoxycarbonyl)aminohexyl group for 5′-terminal modification

Solid-support conjugation at the 5′-terminal primary amine of oligonucleotides is a convenient and powerful method for introducing various functional groups. However, conventional aliphatic amines do not necessarily provide conjugates with sufficient yields. To improve the modification efficacy, we used the amino-linker (aminoethoxycarbonyl)aminohexyl group (ssH-linker), for solid-support conjugation. In the ssH-linker terminal modification, reactive free amino group could be easily presented onto a solid-support due to rapid removal of the amino-protecting group, and activated amino acids or cholesterol molecules could be covalently connected more efficiently than to typical 6-aminohexyl-linkers. Based on these results, the ssH-linker can be a useful terminal modification for the solid-support conjugation of functional molecules.     

2'-Functionalized nucleic acids as structural tools in molecular biology

Modified oligonucleotides bearing 2'-reactive groups or 2'-conjugated molecules have found wide application as structural tools in molecular biology. Of principal interest has been the use of 2'-reactive oligonucleotides for cross-linking with biomolecules and of 2'-conjugated oligonucleotides in hybridization assays. In this review we compare a range of electrophilic, nucleophilic and photoreactive groups for cross-linking and conjugation studies.     

Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog

The specific recognition of homopurine–homo pyrimidine regions in duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Alkylation of nucleobases with functionalized TFOs would have the potential for site-directed mutagenesis. Recently, we demonstrated that a TFO bearing 2-amino-6-vinylpurine derivative, 1, achieves triplex-mediated reaction with high selectivity toward the cytosine of the G-C target site. In this report, we have investigated the use of this reagent to target mutations to a specific site in a shuttle vector plasmid, which replicates in mammalian cells. TFOs bearing 1 produced adducts at the complementary position of 1 and thereby introduced mutations at that site during replication/repair of the plasmid in mammalian cells. Reagents that produce covalent cytosine modifications are relatively rare. These TFOs enable the preparation of templates carrying targeted cytosine adducts for in vitro and in vivo studies. The ability to target mutations may prove useful as a tool for studying DNA repair, and as a technique for gene therapy and genetic engineering.     

Development of the novel drug releasing system triggered by hybridization with target sequence

We have already established the strategy of synchronous activation by hybridization, in which the highly reactive cross-linking agent, 2-amino-6-vinylpurine nucleoside analog, can be generated from its stable precursors, the phenylsulfide derivatives, by a hybridization-promoted activation process with selectivity to cytosine. In this study, this in situ activation system was applied to the method for the drug releasing system triggered by hybridization with the target sequence.     

Selective cross-linking to the adenine of the TA interrupting site within the triple helix

The triplex-forming oligonucleotide incorporating the new nucleoside derivative (2) that connects the 2-amino-6-vinylpurine moiety to the 2-deoxyribose unit with an ethyl spacer has exhibited highly selective cross-linking reaction to the adenine of the TA interrupting site within the triple helix.     

Development Of The Novel Drug Releasing System Triggered By Hybridization With Target Sequence

We have already established the strategy of synchronous activation by hybridization, in which the highly reactive cross-linking agent, 2-amino-6-vinylpurine nucleoside analog, can be generated from its stable precursors, the phenylsulfide derivatives, by a hybridization-promoted activation process with selectivity to cytosine. In this study, this in situ activation system was applied to the method for the drug releasing system triggered by hybridization with the target sequence.     

Oligonucleotides containing 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine as suitable precursors of 2'-aldehyde oligonucleotides for chemoselective ligation

2'-O-[2-(2,3-Diacetoxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite was prepared and used in solid-phase synthesis to obtain oligonucleotides containing a 1,2-diol group, which may then be converted into a 2'-aldehyde group. The oligonucleotides were conjugated efficiently to various molecules by chemoselective ligation that involves an addition-elimination reaction between the 2'-aldehyde group and a suitable nucleophile, such as a hydrazine, a O-alkylhydroxylamine or an 1,2-aminothiol. The method was applied successfully to the conjugation of peptides to oligonucleotides at the 2'-position.     

Synthesis and fluorescence characterization of pteridine adenosine nucleoside analogs for DNA incorporation.

Two fluorescent adenosine analogs, 4-amino-6-methyl-8-(2-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (6MAP) and 4-amino-2,6-dimethyl-8-(2'-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (DMAP), have been synthesized as phosphoramidites. These probes are site-selectively incorporated into oligonucleotides using automated DNA synthesis. Relative quantum yields are 0.39 for 6MAP and 0.48 for DMAP as monomers and range from >0.01 to 0.11 in oligonucleotides. Excitation maxima are 310 (6MAP) and 330 nm (DMAP) and the emission maximum for each is 430 nm. Fluorescence decay curves of each are monoexponential exhibiting lifetimes of 3.8 and 4.8 ns for 6MAP and DMAP, respectively. When these probes are incorporated into oligonucleotides they display quenching of fluorescence intensity, increases in the complexity of decay curves, and decreases in mean lifetimes. Because these changes are apparently mediated by interactions with neighboring bases, spectral changes that occur as probe-containing oligonucleotides meet and react with other molecules provide a means of monitoring these interactions in real time. These probes are minimally disruptive to DNA structure as evidenced by melting temperatures of probe-containing oligonucleotides that are very similar to those of controls. Digestion of probe-containing oligonucleotides with P1 nuclease confirms probe stability as fluorescence levels are restored to those expected for each monomer. These adenosine analog probes are capable of providing information on DNA structure as it responds to binding or catalysis through interaction with other molecules.     

Selective reaction to a flipping cytidine of the duplex DNA mediated by triple helix formation

A new nucleoside derivative (2) with a butyl spacer between the sugar part and the 2-amino-6-vinylpurine motif has been synthesized. The triplex-forming oligodeoxynucleotide incorporating 2 has achieved strand- and cytidine-selective cross-linking reaction to the G-C target site mediated by triple helix formation. It has been suggested that 2 reacts with a flipping cytidine at the target site.     

Disulfide bridge as a linker in nucleic acids’ bioconjugation. Part I: An overview of synthetic strategies

This 2-part article reviews methods of oligonucleotides functionalization with thiol tethers and their consecutive use in conjugation with other (macro)molecules via a disulfide bridge. This relatively inexpensive, robust and reversible method of conjugation of DNAs, RNAs and their analogs holds a prominent position in a modern biochemistry toolbox and therefore there is a wealth of literature on the subject. In part I methods of thiol/disulfide groups introduction into oligonucleotide strands have been systematized and discussed. A digest of conjugation methods is presented as well.     

N-terminal α-amino group modification of antibodies using a site-selective click chemistry method

Site-specific conjugation of small molecules to antibody molecules is a promising strategy for generation of antibody-drug conjugates. In this report, we describe the successful synthesis of a novel bifunctional molecule, 6-(azidomethyl)-2-pyridinecarboxyaldehyde (6-AM-2-PCA), which was used for conjugation of small molecules to peptides and antibodies. We demonstrated that 6-AM-2-PCA selectively reacted with N-terminal amino groups of peptides and antibodies. In addition, the azide group of 6-AM-2-PCA enabled copper-free click chemistry coupling with dibenzocyclooctyne-containing reagents. Bifunctional 6-AM-2-PCA mediated site-specific conjugation without requiring genetic engineering of peptides or antibodies. A key advantage of 6-AM-2-PCA as a conjugation reagent is its ability to modify proteins in a single step under physiological conditions that are sufficiently moderate to retain protein function. Therefore, this new click chemistry-based method could be a useful complement to other conjugation methods.