NON-CHROMATOGRAPHIC OLIGONUCLEOTIDE PURIFICATION AND AUTOMATED POLYETHYLENEGLYCOL SYNTHESIS

Synthetic oligodeoxynucleotides (ODN) have various applications in many areas such as, synthetic biology, chemical biology, antisense drug development, and data storage. As a result, there is a high demand for synthetic ODNs. Many advances have been made with ODN synthesis the purification still remains a bottleneck. The non-chromatographic purification method is developed to address this problem. Similarly, polyethylene glycols (PEGs) have been used in many areas including pharmaceutical applications, surface science, and nanomedicine due to its unique properties. However, monodispersed PEG synthesis is expensive with existing methods. A novel automated solid phase synthesis method is developed to obtain monodispersed PEGs. The first two chapters are an introduction to synthetic oligodeoxynucleotide purification. These chapters describe oligonucleotides and applications, chemical synthesis of oligodeoxynucleotides, impurities in synthetic oligonucleotides, and current purification methods. Even though many chromatographic purification methods are available for synthetic ODN purification, there is no method that is capable of high throughput purification, large scale ODN purification, and long ODN purification. To address this problem, we developed two non-chromatographic purification methods which are based on polymerization, to purify synthetic ODNs. One is catching failure sequences by polymerization, and the second method is catching full-length sequences by polymerization. The second method is less expensive for large-scale and long ODN purification compared to the first method. It is demonstrated that this new non-chromatographic method is suitable for high throughput, large scale, and long ODN purification. The final chapters describe applications of polyethylene glycol and current PEG synthesis methods. For many applications monodispersed PEGs are preferred. However, PEGs synthesized using current synthesis methods are polydispersed and excessive purification is required to obtain monodispersed PEGs. Therefore, it is expensive to obtain monodispersed PEGs using these existing methods. An automated solid-phase stepwise synthesis method is developed for monodispersed PEG synthesis. This solid-phase xxii synthesis method is cost effective because the purification is not required to obtain monodispersed PEGs using this method. Monodispersed PEGs with eight, twelve, and sixteen ethylene glycol units and their derivatives were synthesized using automated stepwise addition of tetra ethylene glycol monomer without chromatographic purification. The monomer consists of a 4,4′ dimethoxy trityl group at one end and the tosyl group at the other hydroxyl group. Wang resin was used as the solid support for the automated synthesis. The automated synthesis cycle consists of three steps, deprotonation, coupling, and detritylation. PEGs were cleaved off from the solid support and analyzed with ESI-MS. After PEG12 synthesis, the coupling step with DMTr monomer had some complications and therefore a new monomer with a smaller protecting group was designed. Using this new monomer monodispersed PEGs with ten, fifteen, and twenty ethylene glycol units were synthesized. The synthesis was done using automated solid-phase stepwise addition of penta ethylene glycol units without chromatographic purification. The penta ethylene glycol monomer consists of a 2-phenethyl group at one end, and the tosyl group at the other hydroxyl group was used for the synthesis. The first step is deprotection and deprotonation, and the second step is coupling. These two steps were performed alternatively until the PEG with the desired length was obtained. After the synthesis, PEGs were cleaved from the solid support and analyzed using ESI-MS

Global Environmental Flow Information for the Sustainable Development Goals

Environmental flows (EF) are an important component of Goal 6 (the ‘water goal’) of the Sustainable Development Goals (SDGs). Yet, many countries still do not have well-defined criteria on how to define EF. In this study, we bring together the International Water Management Institute’s (IWMI’s) expertise and previous research in this area to develop a new methodology to quantify EF at a global scale. EF are developed for grids (0.1 degree spatial resolution) for different levels of health (defined as environmental management classes [EMCs]) of river sections. Additionally, EF have been separated into surface water and groundwater components, which also helps in developing sustainable groundwater abstraction (SGWA) limits. An online tool has been developed to calculate EF and SGWA in any area of interest

Electrophilic oligodeoxynucleotide synthesis using dM-Dmoc for amino protection

Solid-phase synthesis of electrophilic oligodeoxynucleotides (ODNs) was achieved using dimethyl-Dmoc (dM-Dmoc) as amino protecting group. Due to the high steric hindrance of the 2-(propan-2-ylidene)-1,3-dithiane side product from deprotection, the use of excess nucleophilic scavengers such as aniline to prevent Michael addition of the side product to the deprotected ODN during ODN cleavage and deprotection was no longer needed. The improved technology was demonstrated by the synthesis and characterization of five ODNs including three modified ones. The modified ODNs contained the electrophilic groups ethyl ester, α-chloroamide, and thioester. Using the technology, the sensitive groups can be installed at any location within the ODN sequences without using any sequence- or functionality-specific conditions and procedures

Identification of Non-tuberculous mycobacteria isolated from patients at Teaching Hospitals, Kandy and Peradeniya

Introduction Non-tuberculous mycobacteria (NTM) are known to cause opportunistic nosocomial infections. The aim of the study was to identify NTM using culture characteristics, biochemical and molecular methods (multiplex PCR) from stored mycobacteria isolated from patients presenting with pulmonary and extra-pulmonary tuberculosis at Teaching Hospitals, Kandy and Peradeniya during a 4-year period from 2004 to 2009.Methods Forty mycobacterial isolates obtained from 48 samples of sputum, urine, broncho-alveolar lavage and peritoneal fluid were analyzed initially by growth characteristics, followed by biochemical and molecular methods for the presence of NTM. Five main growth characteristics were analyzed, which included rate of growth, growth-temperature relationship, production of pigment, colony morphology and growth on McConkey agar. Species identification of these NTM isolates was by performing biochemical tests. Molecular identification was performed by multiplex-PCR technique on mycobacterial clinical isolates for the rapid identification of NTM.Results Of the 40 mycobacterial isolates, 10 were identified as NTM by using culture characteristics and multiplex PCR. All the NTM isolates were identified to species level by biochemical methods and eight species of NTM were identified. These species, obtained from sterile as well as non-sterile clinical samples were identified as M. scrofulaceum, M. haemophilum, M. chelonae, M. kansasii, M. phlei, M. flavescens, M. gastri and M. vaccae. Conclusion Non-tuberculous mycobacteria were isolated from a wide range of sterile and non-sterile clinical samples and contributed to 25% of the mycobacterial isolates in this study. The results of culture characteristics were compatible with the molecular identification (multiplex PCR) differentiating Mycobacterium tuberculosis from non-tuberculous mycobacterial strains. M. kansasii and M. gastri were the most common NTM isolated from clinical isolates.</p

Special Article - HUMAN ANTI-RHINOSPORIDIAL ANTIBODY DOES NOT CAUSE METABOLIC INACTIVATION OR MORPHOLOGICAL DAMAGE IN ENDOSPORES OF RHINOSPORIDIUM SEEBERI, IN VITRO

This report describes the use of the MTT-reduction and Evan′s blue-staining tests for the assessment of the viability and morphological integrity, respectively, of rhinosporidial endospores after exposure to sera from rhinosporidial patients with high titres of anti-rhinosporidial antibody. Sera from three patients, with nasal, ocular and disseminated rhinosporidiosis respectively were used, with human serum without anti-rhinosporidial antibody for comparison, with or without added fresh guinea pig serum as a source of complement. All four sera tested, with or without guinea-pig serum, had no effect on the morphological integrity or the viability of the endospores and it is suggested that anti-rhinosporidial antibody has no direct protective role against the endospores, the infective stage, in rhinosporidiosis. This finding is compatible with the occurrence of chronicity, recurrence and dissemination that are characteristic of rhinosporidiosis despite the presence of high titres of anti-rhinosporidial antibody in patients with these clinical characteristics. The possible occurrence of humoral mechanisms of immunity that involve anti-rhinosporidial antibody with cells such as leucocytes and NK cells, in vivo, cannot yet be discounted, although the presence of high titres of anti-rhinosporidial antibody in patients with chronic, recurrent and disseminated lesions might indicate that such antibody is non-protective in vivo

For Catching-by-Polymerization Oligo Purification: Scalable Synthesis of the Precursors to the Polymerizable Tagging Phosphoramidites

The catching-by-polymerization (CBP) oligodeoxynucleotide (oligo or ODN) purification method has been demonstrated suitable for large-scale, parallel, and long oligo purification. The authenticity of the oligos has been verified via DNA sequencing. Gene construction and expression have been demonstrated. A remaining obstacle to the practical utility of the CBP method is affordable polymerizable tagging phosphoramidites (PTPs) that are needed for the method. In this article, we report scalable synthesis of the four nucleoside (dA, dC, dG and dT) precursors to the PTPs using a route having six steps from inexpensive starting materials. The overall yields ranged from 21% to 35%. The scales of the synthesis presented here are up to 2.1 grams of the precursors. Because the syntheses are chromatography-free, they are predicted to be readily scalable. With the precursors, the PTPs can be synthesized in one step using standard methods involving a chromatography purification