Construction of an aminooxy derivative for RNA and DNA labeling.

We previously reported that the reactivity of a primary amine is increased by connecting the amine to an aromatic residue with an alkyl linker. This strategy was applied to the construction of a reagent that could form a covalent bond with an aldehyde group. We synthesized a new reagent that consisted of aminooxy and aromatic groups and biotin. The new reagent showed a higher reaction rate to apurinic/apyrimidinic (AP) sites in DNA than a conventional aldehyde-reactive probe (ARP). This reagent also efficiently reacted with 2', 3'-dialdehyde groups, which were generated by the oxidation of the 2', 3'-terminal vicinal hydroxy groups of RNA. The reaction efficiency of the reagent closely related to the structure of the target molecules. We describe the synthesis and chemical properties of the new reagent.

Comparison of the chemical properties of a novel amino-linker with various amino modifications.

We previously reported a series of new amino-linkers, consisting of an aminoethyl carbamate structure (Komatsu, 2008). We have now examined the chemical properties of oligonucleotides modified with an ssH-linker, which is the simplest and most cost-effective derivative of the series. Although it was previously shown that monomethoxytrityl protection on a primary amine of the ssH-linker was cleaved under weakly acidic conditions (1% acetic acid), we found that the deprotection also proceeded in aqueous buffer solutions (pH 6.0, 7.0). The MMT group was removed much faster than other commercially available amino-linkers, and this property enabled the ssH-modified oligonucleotides to be conveniently purified with a cartridge column. Furthermore, the ssH-modified oligonucleotides were utilized in on-support labeling reactions. As compared with other amino-linkers, the ssH-linker was superior in terms of its purification and reaction efficiencies.

Novel amino linkers enabling efficient labeling and convenient purification of amino-modified oligonucleotides.

We developed new amino linker reagents for an oligonucleotide (ONT) terminus. These reagents consist of an aminoethyl carbamate main linkage and a side-chain residue, which was a naphthylmethoxymethyl, methoxymethyl, or methyl group or a hydrogen atom. The primary amine was protected with a monomethoxytrityl (MMT) group. The chemical properties of ONTs containing these amino-modifications were investigated. The MMT group of these amino-modifications could be quite rapidly removed from the amine under very mild acidic conditions, which are not strong enough for the deprotection of a conventional aliphatic amine. This significant feature enabled the amino-modified ONTs to be conveniently purified with a reverse phase column. Furthermore, the amino-modifications efficiently reacted to active esters, as compared with other amino-modifications. We also found that the pK(a) values of the amino-modifications were lower than that of the aliphatic amine. All of the experimental results showed that these chemical properties are closely related to their structures. We report here the chemical properties and the availability of the new amino linker reagents.

Enhanced reactivity of amino-modified oligonucleotides by insertion of aromatic residue.

We developed novel amino-modifying reagents, of which an amino group was connected with an aromatic residue by aliphatic linker. It was proved that the insertion of the aromatic residue could increase the reactivity of the amino group on oligonucleotides in comparison with conventional amino-modification.

Psychophysiological stress-regulated gene expression in mice.

Eight genes showed significant changes in expression in mice under psychophysiological stress provided by cage-restraint and water-immersion. The transcription level of most of these genes was affected in all the tissues analyzed, and some of them were responsive genes in several different stress systems. Peculiarly, the expression level of one gene, cdc2-like kinase 1 (CLK1), was reduced only in the brain, while the balance of partially- and alternatively-spliced CLK1 mRNA species changed in all the tissues including the brain. These results suggest that some stress-response mechanisms, including transcriptional and post-transcriptional events, are coordinated in the whole body in mice under psychophysiological stress.

Generation of an abasic site in an oligonucleotide by using acid-labile 1-deaza-2'-deoxyguanosine and its application to postsynthetic modification.

We developed a new convenient method for generation of an abasic site at the 3'-terminus of an oligonucleotide. This method uses a 1-deaza-2'-deoxyguanosine residue, which easily undergoes depurination under acidic conditions. The abasic site of the oligonucleotide can be further modified with external functional groups. We report herein the chemical stability of 1-deaza-2'-deoxyguanosine in the oligodeoxynucleotide and the application to the postsynthetic modification of an oligonucleotide by utilizing the chemical property of 1-deaza-2'-deoxyguanosine. [Structure: see text]

A serine protease inhibitor (serpin) from Haemaphysalis longicornis as an anti-tick vaccine.

The application of anti-tick vaccine has been shown to be the most promising alternative tick control strategy compared to the current use of acaricides that suffer from a number of limitations. The success of this strategy is dependent on the cloning, and characterization of tick molecules involved in the mediation of tick central physiological roles. Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a cDNA with high similarity in the reactive center loop (RCL) to representative serpin, heparin cofactor II. We have named this novel gene as Haemaphysalis longicornis serpin-2 (HLS2). RT-PCR analysis showed that HLS2 mRNA transcripts are not expressed in salivary glands but in hemolymph by feeding ticks. HLS2 was not introduced into the bite site as measured by Western blot analysis. The activated partial thromboplastin time (APTT) and the thrombin inhibitory assay using recombinant HLS2 (rHLS2) demonstrated prolonged coagulation time and inhibition of thrombin activity. These results suggested that HLS2 is present only in hemolymph of the feeding ticks and the function of HLS2 is homeostasis in tick physiological compartment. Vaccination of rabbits with rHLS2 conferred protective immunity against ticks, resulting in 44.6 and 43.0% mortality in nymphal and adult ticks, respectively. These results show that rHLS2 could be an important candidate as a component of a cocktail anti-tick vaccine.

High throughput purification of amino-modified oligonucleotides and its application to novel detection system of gene expression.

We report here a new amino-modifier reagent, which enable high-throughput purification of amino-modified oligonucleotides. Either monomethoxytrityl (MMT) or trifluoroacetyl (TFA) group has been used as the protecting group for the primary amine when amino-terminal oligonucleotides are prepared. Generally, the removal of MMT requires the stringent acidic treatment after the oligonucleotide synthesis. We chemically synthesized a novel amino-modifier with the MMT protection. It was found that the new amino-modifier released MMT group under mild acidic condition, and then rapid purification of diverse amino-modified oligonucleotides could be achieved with cartridge column of reverse phase. Furthermore, we tried to construct the new detection system to study gene expression using the amino-modified oligonucleotides. The new amino-modifier will be useful for molecular biology by facilitating the construction of oligonucleotide library and improving the chemical reactivity.

Synthesis and application of new amino modification analogues for functional oligonucleotide.

We synthesized new analogues to functionalize oligonucleotides. These analogues included a primary amino group tethering to an aromatic ring, and we introduced them into the 5'-end of each oligonucleotide. The oligonucleotides with the aromatic amino group (OAA) were easily purified from impurities by using their hydrophobic property of the aromatic residue. These OAA probes reacted with activated ester groups more efficiently than the conventional probe, which was modified with 6-aminohexyl group. Furthermore, we applied these OAAs to oligonucleotide array probes. The OAA probes were efficiently immobilized on the array surface, and the hybridization intensity on these probes increased as compared with the conventional probes. These new probes can be a new nucleic acid tool for biological assay systems.

A serine proteinase inhibitor (serpin) from ixodid tick Haemaphysalis longicornis; cloning and preliminary assessment of its suitability as a candidate for a tick vaccine.

Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins. We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1). Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24-48 h post-attachment) only in the tick midguts. Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits. Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding. rHLS1 could be a potential candidate for a cocktail anti-tick vaccine.