New Strategies for the Chemical Synthesis of Biologically Important Nucleic Acid Derivatives

Abstract

This paper describes general methods for the synthesis of N-phosphorylated ribonucleosides and oligonucleotides containing a 2′-O-phosphorylated or 2′-O-thiophosphorylated ribonucleoside. The NMR-based conformational analysis and computational molecular dynamics simulation of the 2′-O-phosphorylated ribonucleoside residue in such modified oligonucleotides suggested that the ribose residue existed preferentially in a C2′-endo conformation. It was also found that simple heating of 2′-O-phosphorylated oligonucleotides resulted in rapid dethiophosphorylation.     

A strong association between human earwax-type and apocrine colostrum secretion from the mammary gland

Here we provided the first genetic evidence for an association between the degree of apocrine colostrum secretion and human earwax type. Genotyping at the earwax-type locus, rs17822931 within the ABCC11 gene, revealed that 155 of 225 Japanese women were dry-type and 70 wet-type. Frequency of women without colostrum among dry-type women was significantly higher than that among wet-type women (P < 0.0002), and the measurable colostrum volume in dry-type women was significantly smaller than in wet-type women (P = 0.0341).     

Uniparental disomy analysis in trios using genome-wide SNP array and whole-genome sequencing data imply segmental uniparental isodisomy in general populations

Whole chromosomal and segmental uniparental disomy (UPD) is one of the causes of imprinting disorder and other recessive disorders. Most investigations of UPD were performed only using cases with relevant phenotypic features and included few markers. However, the diagnosis of cases with segmental UPD requires a large number of molecular investigations. Currently, the accurate frequency of whole chromosomal and segmental UPD in a normal developing embryo is not well understood. Here, we present whole chromosome and segmental UPD analysis using single nucleotide polymorphism (SNP) microarray data of 173 mother-father-child trios (519 individuals) from six populations (including 170 HapMap trios). For two of these trios, we also investigated the possibility of shorter segmental UPD as a consequence of homologous recombination repair (HR) for DNA double strand breaks (DSBs) during the early developing stage using high-coverage whole-genome sequencing (WGS) data from 1000 Genomes Project. This could be overlooked by SNP microarray. We identified one obvious segmental paternal uniparental isodisomy (iUPD) (8.2 mega bases) in one HapMap sample from 173 trios using Genome-Wide Human SNP Array 6.0 (SNP6.0 array) data. However, we could not identify shorter segmental iUPD in two trios using WGS data. Finally, we estimated the rate of segmental UPD to be one per 173 births (0.578%) based on the UPD screening for 173 trios in general populations. Based on the autosomal chromosome pairs investigated, we estimate the rate of segmental UPD to be one per 3806 chromosome pairs (0.026%). These data imply the possibility of hidden segmental UPD in normal individuals.     

Copy number alteration and uniparental disomy analysis categorizes Japanese papillary thyroid carcinomas into distinct groups

The aim of the present study was to investigate chromosomal aberrations in sporadic Japanese papillary thyroid carcinomas (PTCs), concomitant with the analysis of oncogene mutational status. Twenty-five PTCs (11 with BRAF(V600E), 4 with RET/PTC1, and 10 without mutation in HRAS, KRAS, NRAS, BRAF, RET/PTC1, or RET/PTC3) were analyzed using Genome-Wide Human SNP Array 6.0 which allows us to detect copy number alteration (CNA) and uniparental disomy (UPD), also referred to as copy neutral loss of heterozygosity, in a single experiment. The Japanese PTCs showed relatively stable karyotypes. Seven cases (28%) showed CNA(s), and 6 (24%) showed UPD(s). Interestingly, CNA and UPD were rarely overlapped in the same tumor; the only one advanced case showed both CNA and UPD with a highly complex karyotype. Thirteen (52%) showed neither CNA nor UPD. Regarding CNA, deletions tended to be more frequent than amplifications. The most frequent and recurrent region was the deletion in chromosome 22; however, it was found in only 4 cases (16%). The degree of genomic instability did not depend on the oncogene status. However, in oncogene-positive cases (BRAF(V600E) and RET/PTC1), tumors with CNA/UPD were less frequent (5/15, 33%), whereas tumors with CNA/UPD were more frequent in oncogene-negative cases (7/10, 70%), suggesting that chromosomal aberrations may play a role in the development of PTC, especially in oncogene-negative tumors. These data suggest that Japanese PTCs may be classified into three distinct groups: CNA(+), UPD(+), and no chromosomal aberrations. BRAF(V600E) mutational status did not correlate with any parameters of chromosomal defects.     

Computational evaluation of intermolecular interactions of a universal base 3-nitropyrrole in stacked dimers and DNA duplexes

The stacking interactions between a universal base of 3-nitropyrrole (3NP) and four canonical nucleobases were studied by means of ab initio molecular orbital calculations. The stabilities of the complexes are comparable to those of the stacked dimers of canonical bases reported previously. The detailed analysis of the interaction energies revealed the importance of the dipole-dipole interaction included in the Hartree-Fock terms to determine the geometry dependence of the stacking energies. It was also clarified that the dispersion energies included in the electron-correlation terms were essential to obtain adequate stabilities. The contribution of the nitro group was evaluated by the comparative studies of pyrrole and 3NP. The increased molecular dipole moment and surface are expected to account for the enhancement of the stability of the stacked dimers containing 3NP. The force field parameters required for calculation of the molecular mechanics of 3NP were obtained for 3NP on the basis of these molecular orbital calculations. The energy-minimized structures obtained by the molecular mechanics calculations of 3NP accorded with those obtained by the molecular orbital calculations described above. A DNA duplex structure containing 3NP-A, 3NP-T, or 3NP-C was calculated by use of these force field parameters. In the case of 3NP-A, the computationally calculated structure was in good agreement with that previously determined by use of (1)H-NMR except for the orientation of the nitro group.     

A strong association of axillary osmidrosis with the wet earwax type determined by genotyping of the ABCC11 gene

Background

Lactase non-persistence is a condition where lactase activity is decreased in the intestinal wall after weaning. In European derived populations a single nucleotide polymorphism (SNP) C/T_-13910 residing 13.9 kb upstream from the lactase gene has been shown to define lactase activity, and several other single nucleotide polymorphisms (G/C_-14010 T/G_-13915, C/G_-13907 and T/C_-13913) in the same region have been identified in African and Middle East populations.

Results

The T_-13910 allele most common in European populations was present in 21.8% mixed ancestry (N = 62) individuals and it was absent in the Xhosa (N = 109) and Ghana (N = 196) subjects. Five other substitutions were also found in the region covering the previously reported variants in African and Middle East populations. These included the G/C_-14010 variant common in Kenyan and Tanzanian populations, which was present in 12.8% of Xhosa population and in 8.1% of mixed ancestry subjects. Two novel substitutions (C/T_-14091 and A/C_-14176) and one previously reported substitution G/A_-13937 (rs4988234) were less common and present only in the Xhosa population. One novel substitution G/A_-14107 was present in the Xhosa and Ghanaian populations. None of the other previously reported variants were identified.

Conclusion

Identification of the G/C_-14010 variant in the Xhosa population, further confirms their genetic relatedness to other nomadic populations members that belong to the Bantu linguistic group in Tanzania and Kenya. Further studies are needed to confirm the possible relationship of the novel substitutions to the lactase persistence trait.     

Computational Evaluation of Intermolecular Interactions of a Universal Base 3-Nitropyrrole in Stacked Dimers and DNA Duplexes

Abstract

The stacking interactions between a universal base of 3-nitropyrrole (3NP) and four canonical nucleobases were studied by means of ab initio molecular orbital calculations. The stabilities of the complexes are comparable to those of the stacked dimers of canonical bases reported previously. The detailed analysis of the interaction energies revealed the importance of the dipole-dipole interaction included in the Hartree-Fock terms to determine the geometry dependence of the stacking energies. It was also clarified that the dispersion energies included in the electron-correlation terms were essential to obtain adequate stabilities. The contribution of the nitro group was evaluated by the comparative studies of pyrrole and 3NP. The increased molecular dipole moment and surface are expected to account for the enhancement of the stability of the stacked dimers containing 3NP. The force field parameters required for calculation of the molecular mechanics of 3NP were obtained for 3NP on the basis of these molecular orbital calculations. The energy-minimized structures obtained by the molecular mechanics calculations of 3NP accorded with those obtained by the molecular orbital calculations described above. A DNA duplex structure containing 3NP-A, 3NP-T, or 3NP-C was calculated by use of these force field parameters. In the case of 3NP-A, the computationally calculated structure was in good agreement with that previously determined by use of ¹H-NMR except for the orientation of the nitro group.     

The novel imprinted carboxypeptidase A4 gene ( CPA4) in the 7q32 imprinting domain

By a search for novel human imprinted genes in the vicinity of the imprinted gene MEST, at chromosome 7q32, we identified the carboxypeptidase A4 gene ( CPA4) in a gene cluster of the carboxypeptidase family, 200 kb centromeric to MEST. Because CPA4 was originally identified as a protein induced in a prostate cancer cell line (PC-3) by histone deacetylase inhibitors, and was located at the putative prostate cancer-aggressiveness locus at 7q32, we investigated its imprinting status in fetal tissues and in adult benign hypertrophic prostate (BPH). RT-PCR using four intragenic polymorphisms as markers showed that CPA4 was expressed preferentially from the maternal allele in the fetal heart, lung, liver, intestine, kidney, adrenal gland, and spleen, but not in the fetal brain. It was also preferentially expressed in the BPH. These findings support that CPA4 is imprinted and may become a strong candidate gene for prostate cancer-aggressiveness. As a Silver-Russell syndrome (SRS) locus has been proposed to be located to a region near MEST and to be involved in imprinting, CPA4 would have been a candidate gene for SRS. However, analysis of ten SRS patients revealed no mutations in CPA4.