The generation and evolutionary study of novel aptamers for environmental contaminants

<p>The contamination of waterways by environmental pollutants is of growing global concern. The bio-accumulative properties of these contaminants suggest long-term impacts on many species, even those not directly exposed. There is ample evidence of the presence of environmental contaminants within biological fluids of humans, but their effects on health are largely unknown. Understanding the extent of this problem is hampered by labour-intensive extraction techniques that require expensive instrumentation and highly specialised technical expertise. Due to the prohibitive nature of routine analysis, the occurrence of many of these compounds in New Zealand waterways is unknown. Thus, a robust, portable and sensitive biosensor is urgently needed to guide regulatory agencies worldwide. Aptamers are single-stranded nucleic acid molecules that can bind to a specific target molecule with high affinity. Whilst the use of aptamers presents a novel technology to monitor small molecule environmental contaminants, the generation of high affinity aptamers has been limited. The objectives of this PhD study were to: (1) measure key emerging organic contaminants (EOC’s) in a selection of New Zealand waterways covering different land-use types; (2) generate and characterise aptamers that bind three key EOC’s, namely glyphosate, nonylphenol and oxybenzone, and; (3) explore the evolutionary pathways that random nucleotide libraries follow when generating aptamers to a small molecule under different stringencies. A novel bioinformatics pipeline for the analysis of high throughput (HT) SELEX data from multiple selection strategies has been developed and implemented. The conventional method of gas chromatography mass spectrometry (GC-MS) was used to analyse water samples from waterways in the North Island of New Zealand. Technical nonylphenol equivalents and oxybenzone were detected above the minimum detection limit (7.5 ng/L and 0.5 ng/L respectively) at all sampled sites. Concentrations of nonylphenol exceeding environmental guidelines were detected in the Waiwhetu Stream, a small stream within an industrial area in Lower Hutt. The pesticide terbuthylazine was detected at all sampled sites with particular high concentrations in the Waiwhetu and Porirua Stream. Carbendazim (a fungicide) and hexazinone (a herbicide) were also highly prevalent being detected at 87.5% and 75% of sites respectively. Glyphosate was detected at 800 ng/L in both the Waiwhetu Stream and the Porirua Stream. In general, contaminant load was much higher in urban areas than rural or forested areas. These results indicate that EOCs are present in NZ waterways and are likely to be having an impact on aquatic species. The selection of aptamers to three key EOC’s was completed using standard (glyphosate and nonylphenol) and high-throughput (HT) (oxybenzone) SELEX methodologies. DNA aptamers for glyphosate and oxybenzone were successfully generated and characterised. The GLY04 (glyphosate) and OXY-ED7-C1 (oxybenzone) aptamers were characterised using micro-scale thermophoresis and exhibited a Kd of 158 and 107.5 nM, respectively. This is the first report of a glyphosate-binding aptamer in the literature. Attempts to generate a DNA aptamer for nonylphenol were unsuccessful. Whilst five aptamer candidates were generated through 20 rounds of selection, they did not show any evidence of binding to the target molecule. A HT-SELEX approach was utilised to study the effect of different selection parameters on the same starting library during the generation of an oxybenzone aptamer. Six strategies, compared to a standard protocol, were assessed including mutation via error-prone PCR, increased washing volume, increased detergent concentration, higher incubation temperature and negative selection and counter selection. Within each strategy, the frequency and enrichment of candidates at each SELEX round was determined using a novel bioinformatics pipeline. On average, higher frequency candidates were present at the end of SELEX within strategies using higher stringency. Higher enrichment was also observed in the strategy using the most stringent conditions. In summary, this PhD study presents a number of novel findings. The wide-scale presence of key EOC’s in New Zealand waterways was determined. The generation of aptamers that bind to glyphosate and oxybenzone with a nanomolar affinity reveals that aptamers can be generated to such small molecules. This study also resulted in the development of a novel bioinformatics pipeline for HT-SELEX analysis that resulted in a number of recommendations on the design of such experiments. The findings presented herein highlight the possibilities and pitfalls of selecting future aptamers for EOC’s and for implementing HT-SELEX experiments.</p>

The generation and evolutionary study of novel aptamers for environmental contaminants

<p>The contamination of waterways by environmental pollutants is of growing global concern. The bio-accumulative properties of these contaminants suggest long-term impacts on many species, even those not directly exposed. There is ample evidence of the presence of environmental contaminants within biological fluids of humans, but their effects on health are largely unknown. Understanding the extent of this problem is hampered by labour-intensive extraction techniques that require expensive instrumentation and highly specialised technical expertise. Due to the prohibitive nature of routine analysis, the occurrence of many of these compounds in New Zealand waterways is unknown. Thus, a robust, portable and sensitive biosensor is urgently needed to guide regulatory agencies worldwide. Aptamers are single-stranded nucleic acid molecules that can bind to a specific target molecule with high affinity. Whilst the use of aptamers presents a novel technology to monitor small molecule environmental contaminants, the generation of high affinity aptamers has been limited. The objectives of this PhD study were to: (1) measure key emerging organic contaminants (EOC’s) in a selection of New Zealand waterways covering different land-use types; (2) generate and characterise aptamers that bind three key EOC’s, namely glyphosate, nonylphenol and oxybenzone, and; (3) explore the evolutionary pathways that random nucleotide libraries follow when generating aptamers to a small molecule under different stringencies. A novel bioinformatics pipeline for the analysis of high throughput (HT) SELEX data from multiple selection strategies has been developed and implemented. The conventional method of gas chromatography mass spectrometry (GC-MS) was used to analyse water samples from waterways in the North Island of New Zealand. Technical nonylphenol equivalents and oxybenzone were detected above the minimum detection limit (7.5 ng/L and 0.5 ng/L respectively) at all sampled sites. Concentrations of nonylphenol exceeding environmental guidelines were detected in the Waiwhetu Stream, a small stream within an industrial area in Lower Hutt. The pesticide terbuthylazine was detected at all sampled sites with particular high concentrations in the Waiwhetu and Porirua Stream. Carbendazim (a fungicide) and hexazinone (a herbicide) were also highly prevalent being detected at 87.5% and 75% of sites respectively. Glyphosate was detected at 800 ng/L in both the Waiwhetu Stream and the Porirua Stream. In general, contaminant load was much higher in urban areas than rural or forested areas. These results indicate that EOCs are present in NZ waterways and are likely to be having an impact on aquatic species. The selection of aptamers to three key EOC’s was completed using standard (glyphosate and nonylphenol) and high-throughput (HT) (oxybenzone) SELEX methodologies. DNA aptamers for glyphosate and oxybenzone were successfully generated and characterised. The GLY04 (glyphosate) and OXY-ED7-C1 (oxybenzone) aptamers were characterised using micro-scale thermophoresis and exhibited a Kd of 158 and 107.5 nM, respectively. This is the first report of a glyphosate-binding aptamer in the literature. Attempts to generate a DNA aptamer for nonylphenol were unsuccessful. Whilst five aptamer candidates were generated through 20 rounds of selection, they did not show any evidence of binding to the target molecule. A HT-SELEX approach was utilised to study the effect of different selection parameters on the same starting library during the generation of an oxybenzone aptamer. Six strategies, compared to a standard protocol, were assessed including mutation via error-prone PCR, increased washing volume, increased detergent concentration, higher incubation temperature and negative selection and counter selection. Within each strategy, the frequency and enrichment of candidates at each SELEX round was determined using a novel bioinformatics pipeline. On average, higher frequency candidates were present at the end of SELEX within strategies using higher stringency. Higher enrichment was also observed in the strategy using the most stringent conditions. In summary, this PhD study presents a number of novel findings. The wide-scale presence of key EOC’s in New Zealand waterways was determined. The generation of aptamers that bind to glyphosate and oxybenzone with a nanomolar affinity reveals that aptamers can be generated to such small molecules. This study also resulted in the development of a novel bioinformatics pipeline for HT-SELEX analysis that resulted in a number of recommendations on the design of such experiments. The findings presented herein highlight the possibilities and pitfalls of selecting future aptamers for EOC’s and for implementing HT-SELEX experiments.</p>

Development and identification of candidate aptamers for growth differentiation factor 9

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted growth factors that are essential for fertility and ovarian development in many mammalian species. It is hypothesised that the ratios of these growth factors within ovarian follicles are unique to each species and determine litter size in mammals. The current detection method for these proteins is by Western blotting with monoclonal antibodies, however this methodology generates semi-quantitative results at best. Systematic evolution via exponential enrichment (SELEX) is a method involving iterative rounds of affinity maturation. It has been used for generating synthetic DNA- and RNA-based sequences (aptamers) that are capable of recognising a target molecule with high sensitivity. To understand more about the biological functions of BMP15 and GDF9, reliable detection methods need to be developed so these proteins can be measured in biological samples. The generation of aptamers that recognise BMP15 and GDF9 of multiple species could enable a novel detection system to be developed to further explore the role of BMP15 and GDF9 in determining litter size in mammals. In this study, recombinant BMP15 and GDF9 proteins from species that vary in litter size were produced and purified by IMAC and HPLC for aptamer validation. Membrane-SELEX was used to generate candidate aptamers against recombinant human GDF9. After ten rounds of SELEX, seven candidate aptamers were identified and sequenced. Alignment of the sequences revealed a conserved region of 29bp in four of the seven candidate aptamers. These sequences also showed a high (85-90%) guanine/thymine content consistent with G-quadruplex forming aptamers, a tertiary structure that increases specificity to the target molecule. These four sequences also showed homology with that of a previously published aptamer for BMP15. This is interesting as BMP15 and GDF9 are both members of the transforming growth factor beta superfamily and as such, share a high peptide homology suggesting convergent evolution occurred in two unrelated SELEX experiments against related proteins. Whilst time constraints did not allow for characterisation of the resultant GDF9 aptamer candidates, nor their validation using recombinant ovine and mouse GDF9 produced herein, this study showed that GDF9 aptamer candidates were generated by SELEX that exhibited sequence similarities to an aptamer generated against a structurally-related protein.</p>

Development and identification of candidate aptamers for growth differentiation factor 9

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted growth factors that are essential for fertility and ovarian development in many mammalian species. It is hypothesised that the ratios of these growth factors within ovarian follicles are unique to each species and determine litter size in mammals. The current detection method for these proteins is by Western blotting with monoclonal antibodies, however this methodology generates semi-quantitative results at best. Systematic evolution via exponential enrichment (SELEX) is a method involving iterative rounds of affinity maturation. It has been used for generating synthetic DNA- and RNA-based sequences (aptamers) that are capable of recognising a target molecule with high sensitivity. To understand more about the biological functions of BMP15 and GDF9, reliable detection methods need to be developed so these proteins can be measured in biological samples. The generation of aptamers that recognise BMP15 and GDF9 of multiple species could enable a novel detection system to be developed to further explore the role of BMP15 and GDF9 in determining litter size in mammals. In this study, recombinant BMP15 and GDF9 proteins from species that vary in litter size were produced and purified by IMAC and HPLC for aptamer validation. Membrane-SELEX was used to generate candidate aptamers against recombinant human GDF9. After ten rounds of SELEX, seven candidate aptamers were identified and sequenced. Alignment of the sequences revealed a conserved region of 29bp in four of the seven candidate aptamers. These sequences also showed a high (85-90%) guanine/thymine content consistent with G-quadruplex forming aptamers, a tertiary structure that increases specificity to the target molecule. These four sequences also showed homology with that of a previously published aptamer for BMP15. This is interesting as BMP15 and GDF9 are both members of the transforming growth factor beta superfamily and as such, share a high peptide homology suggesting convergent evolution occurred in two unrelated SELEX experiments against related proteins. Whilst time constraints did not allow for characterisation of the resultant GDF9 aptamer candidates, nor their validation using recombinant ovine and mouse GDF9 produced herein, this study showed that GDF9 aptamer candidates were generated by SELEX that exhibited sequence similarities to an aptamer generated against a structurally-related protein.</p>

Split Aptamers Immobilized on Polymer Brushes Integrated in a Lab-on-Chip System Based on an Array of Amorphous Silicon Photosensors: A Novel Sensor Assay

Innovative materials for the integration of aptamers in Lab-on-Chip systems are important for the development of miniaturized portable devices in the field of health-care and diagnostics. Herein we highlight a general method to tailor an aptamer sequence in two subunits that are randomly immobilized into a layer of polymer brushes grown on the internal surface of microfluidic channels, optically aligned with an array of amorphous silicon photosensors for the detection of fluorescence. Our approach relies on the use of split aptamer sequences maintaining their binding affinity to the target molecule. After binding the target molecule, the fragments, separately immobilized to the brush layer, form an assembled structure that in presence of a “light switching” complex [Ru(phen)2(dppz)]2+, emit a fluorescent signal detected by the photosensors positioned underneath. The fluorescent intensity is proportional to the concentration of the target molecule. As proof of principle, we selected fragments derived from an aptamer sequence with binding affinity towards ATP. Using this assay, a limit of detection down to 0.9 µM ATP has been achieved. The sensitivity is compared with an assay where the original aptamer sequence is used. The possibility to re-use both the aptamer assays for several times is demonstrated.

Recognition of Symmetry as a Powerful Tool in Natural Product Synthesis

The design of concise and efficient synthetic strategies to access naturally occurring, pharmaceutically active complex molecules is of utmost importance in current chemistry. It not only enables rapid access to these molecules and their analogues but also provides sufficient quantities for their biological evaluation. Identification of any symmetric or pseudosymmetric synthetic intermediates upon retrosynthetic bond disconnection of the target molecule holds the promise to significantly streamline the route towards the compound of interest. This review will highlight recent examples of successful natural product syntheses reported within the past five years that benefited from the recognition of symmetry elements during the retrosynthetic design.

Intestinal Epithelial Cell Derived Exosomes Package MicroRNA-23a-3p Alleviate Gut Damage After Ischemia/Reperfusion via Targeting MAP4K4

Backgroud: Intestinal epithelial cells (IEC) contribute to regulate gut injury after intestinal isochemia/reperfusion (II/R). Exosomes are well documented to deliver bioactive molecules to recipient cells to modulate cell function. However, the role of IEC-derived exosomes in gut damage after II/R and the underlying mechanisms remains unclear. Methods IEC-derived exosomes were intravenously injected into the rats after the superior mesenteric artery occlusion for 45 min following reperfusion for 6h. The scores of gut injury, ZO-1 and IL-17A expression, TNF-α and endotoxin concentration were determined at 3 days after II/R assault. Exosomal microRNA-23a-3p and its downstream target molecule-MAP4K4 were also detected. Results Our results showed that the IEC-derived exosomes attenuated the damage of IECs after oxygen-glucose deprivation in vitro (p<0.05) and the degree of gut injury at 3 days after II/R assault in vivo (p<0.05). Injection of miR-23a-3p knockdown exosomes derived from IECs aggravated the II/R injury whereas PF-6260933, a small-molecule inhibitor of MAP4K4, was partly reversed the injuried degree by intraperitoneal injection. Underlying mechanism studies revealed that exosomal miR-23a-3p attenuated gut damage by regulating its downstream target MAP4K4. Conclusions We demonstrated that IEC-derived exosomes alleviated the II/R injury and promoted gut damage recovery via exosomal miR-23a-3p and its downstream target molecule-MAP4K4.