Synthesis and application of stationary phase for DNA-affinity chromatographic analysis of unmodified and antisense oligonucleotide

The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses. The oligonucleotide-based stationary phase was applied for the retention studies. Unmodified oligonucleotides of different complementarity to the molecule attached as a stationary phase, as well as antisense oligonucleotides, were tested. The comparative study upon complex optimization of oligonucleotide analysis in different liquid chromatography modes was performed. Results have shown that this stationary phase may be applied for oligonucleotide analysis in hydrophilic interaction liquid chromatography and ion exchange chromatography, but no unique sequence-based selectivity was obtained. Contrary results were observed for affinity chromatography, which allowed for specific separation of the complementary strands based on hydrogen bonding and stacking interactions, where the temperature was the main factor influencing the selectivity of the separation. Furthermore, the oligonucleotide-based stationary phase may be applied for comparative antisense oligonucleotide hybridization studies to a specific RNA sequence. All of the results have shown that affinity chromatography with oligonucleotide-based stationary phases is a powerful technique for the specific base recognition of polynucleotides. Graphical abstract

Alpha Thalassemia Genotypes in Kuwait

Background: The frequency of the alpha thalassemia trait is approximately 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of patients who were referred for molecular diagnosis over a 20-year period.Methods: This is a retrospective study of the a-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was performed by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot blot hybridization (Vienna Lab Strip Assay).Results: Four hundred samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years from 283 unrelated families. Most (90.8%) were Kuwaiti nationals. The commonest genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3% of the samples, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. PA-1 was therefore the most frequent allele (0.59). The frequency of the α0 (--MED) allele was 0.017. Rare alleles that were found in very low frequencies included α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria.Conclusion: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but nondeletional PA-1 is by far the most common cause of the moderate to severe HbH (β4 tetramer) disease phenotype. The α0 (–MED) allele is also encountered, which has implications for premarital counseling, especially for the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

Alpha Thalassemia Genotypes in Kuwait

Background: The frequency of alpha thalassemia trait is about 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of the patients who were referred for molecular diagnosis over a 20-year period. Methods: This is a retrospective study of the a-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot-blot hybridization (Vienna Lab Strip Assay). Results: Four hundred samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years from 283 unrelated families.. Most (90.8%) were Kuwaiti nationals. The common genotype was homozygosity for the polyadenylation-1 mutation (α PA-1 α/α PA-1 α) in 33.3%, followed by heterozygosity (αα/α PA-1 α) for the same mutation in 32.3%. The PA-1 was therefore the most frequent allele (0.59). The frequency of the α 0 , – MED was 0.017. Rare alleles that were found in very low frequencies included the α 0 (-- FIL ) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria. Conclusion : There is a wide variety of alpha thalassemia alleles among Kuwaitis, but the nondeletional PA-1 is by far the most common cause of moderate to severe HbH (β4 tetramer), disease phenotype. The α 0 (– MED ) allele is also encountered, which has implications for pre-marital counselling especially the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

Alpha Thalassemia Genotypes in Kuwait

Background: The frequency of alpha thalassemia trait is about 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of the patients who were referred for molecular diagnosis over a 20-year period.Methods: This is a retrospective study of the a-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot-blot hybridization (Vienna Lab Strip Assay).Results: Four hundred samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years from 283 unrelated families.. Most (90.8%) were Kuwaiti nationals. The common genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3%, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. The PA-1 was therefore the most frequent allele (0.59). The frequency of the α0, –MED was 0.017. Rare alleles that were found in very low frequencies included the α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria.Conclusion: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but the nondeletional PA-1 is by far the most common cause of moderate to severe HbH (β4 tetramer), disease phenotype. The α0 (–MED) allele is also encountered, which has implications for pre-marital counselling especially the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

5′-fluoro(di)phosphate-labeled oligonucleotides are versatile molecular probes for studying nucleic acid secondary structure and interactions by 19F NMR

The high sensitivity of 19F nucleus to changes in the chemical environment has promoted the use of fluorine-labeled molecular probes to study structure and interactions of nucleic acids by 19F NMR. So far, most efforts have focused on incorporating the fluorine atom into nucleobase and ribose moieties using either monomer building blocks for solid-phase synthesis, or nucleoside triphosphates for enzymatic synthesis. Here, we report a simple and efficient synthesis of 5′-fluoromonophosphorylated and 5′-fluorodiphosphorylated oligodeoxyribonucleotides, which combines solid-phase and in-solution synthesis methods and requires only commercially available nucleoside phosphoramidites, followed by their evaluation as 19F NMR probes. We confirmed that the fluorine atom at the oligonucleotide 5′ end did not alter the secondary structure of DNA fragments. Moreover, at the same time, it enabled real-time 19F NMR monitoring of various DNA-related biophysical processes, such as oligonucleotide hybridization (including mismatch identification), G-quadruplex folding/unfolding and its interactions with thrombin, as well as formation of an i-motif structure and its interaction with small-molecule ligands.

Spectrum of Alpha Thalassemia Alleles in Kuwait

Background: The frequency of alpha thalassemia trait is about 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of the patients who were referred for molecular diagnosis over a 20-year period. Methods: Blood samples from suspected cases were sent to the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University. A retrospective study of the molecular characterization of samples from 1994 to 2015 was carried out. The alpha globin genotypes were determined by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot-blot hybridization (Vienna Lab Strip Assay). Results: 400 samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years (~60% children and adolescents). Most (90.8%) were Kuwaiti nationals. The common genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3%, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. The PA-1 was therefore the most frequent allele (0.733). The frequency of the α0, –MED was 0.19. Rare alleles that were found in very low frequencies included the α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana,and Hb Icaria. Conclusion: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but the nondeletional PA-1 is by far the most common cause of moderate to severe HbH disease phenotype. The α0 (–MED) allele is also encountered, which has implications for pre-marital counselling especially the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).