title: Exploring the role of binding surface in enzymatic ligation of modified and synthetic substrates

authors: Dutta, Aakashdeep

subjects: XNA

abstract: Deoxyribonucleic acid (DNA) ligases are essential enzymes that catalyze the formation of phosphodiester bonds to repair nicks in DNA during replication and repair processes. Their ability to ligate synthetic non-canonical nucleic acids (NCNAs), such as unnatural base pairs (UBPs) and backbone-modified DNA/RNA, has significant implications for synthetic biology, biotechnology, and therapeutic applications. This study aimed to evaluate the activity of various DNA ligases on UBP and backbone-modified DNA/RNA substrates, optimize ligation conditions, and explore the relationship between ligase structure and substrate preference.

The study focused on three main objectives: (1) establishing baseline ligation activity for UBP and backbone-modified substrates across multiple ligases, (2) investigating the impact of ligase binding domain size on substrate preference, and (3) optimizing ligation conditions to enhance enzyme activity. A panel of ligases, including Human DNA ligase I, Escherichia coli ligase A, Neisseria gonorrhoeae ligase A, and several ATP-dependent ligases (Lig 3, Lig 4, Lig 5, Lig 12, Lig 15), were tested on substrates containing UBPs (P-Z and S-B systems) and backbone modifications (phosphorothioate (PPT) and 2’-Methoxyethoxy (MOE) RNA).

Results revealed that ligases exhibited varying levels of activity depending on the substrate type and modification position. H-Lig I and Lig 4 demonstrated the highest ligation activity on UBP substrates, particularly those with modifications at the 5’ end of the nick. In contrast, substrates with UBPs on both ends of the nick were poorly tolerated. Gel shift assays confirmed that low ligation activity was not due to binding failure but rather steric hindrance during the adenyl group transfer step. For backbone-modified substrates, Lig 3 and Lig 5 emerged as the most effective, with PPT modifications being better tolerated than MOE modifications. The addition of 10% polyethylene glycol (PEG) 3350 as a crowding agent significantly improved ligation efficiency, particularly for MOE substrates, but inhibited the activity of larger ligases like Lig 12 and H-Lig I.

In conclusion, this study provides valuable insights into the activity of DNA ligases on non-canonical substrates, highlighting the importance of ligase structure and reaction conditions in determining ligation efficiency. The findings contribute to the growing toolbox of synthetic biology and pave the way for further exploration of ligase-substrate interactions, enzyme engineering, and the development of novel biotechnological applications.

text: Exploring the role of binding surface in enzymatic ligation of modified and synthetic substrates Deoxyribonucleic acid (DNA) ligases are essential enzymes that catalyze the formation of phosphodiester bonds to repair nicks in DNA during replication and repair processes. Their ability to ligate synthetic non-canonical nucleic acids (NCNAs), such as unnatural base pairs (UBPs) and backbone-modified DNA/RNA, has significant implications for synthetic biology, biotechnology, and therapeutic applications. This study aimed to evaluate the activity of various DNA ligases on UBP and backbone-modified DNA/RNA substrates, optimize ligation conditions, and explore the relationship between ligase structure and substrate preference.

The study focused on three main objectives: (1) establishing baseline ligation activity for UBP and backbone-modified substrates across multiple ligases, (2) investigating the impact of ligase binding domain size on substrate preference, and (3) optimizing ligation conditions to enhance enzyme activity. A panel of ligases, including Human DNA ligase I, Escherichia coli ligase A, Neisseria gonorrhoeae ligase A, and several ATP-dependent ligases (Lig 3, Lig 4, Lig 5, Lig 12, Lig 15), were tested on substrates containing UBPs (P-Z and S-B systems) and backbone modifications (phosphorothioate (PPT) and 2’-Methoxyethoxy (MOE) RNA).

Results revealed that ligases exhibited varying levels of activity depending on the substrate type and modification position. H-Lig I and Lig 4 demonstrated the highest ligation activity on UBP substrates, particularly those with modifications at the 5’ end of the nick. In contrast, substrates with UBPs on both ends of the nick were poorly tolerated. Gel shift assays confirmed that low ligation activity was not due to binding failure but rather steric hindrance during the adenyl group transfer step. For backbone-modified substrates, Lig 3 and Lig 5 emerged as the most effective, with PPT modifications being better tolerated than MOE modifications. The addition of 10% polyethylene glycol (PEG) 3350 as a crowding agent significantly improved ligation efficiency, particularly for MOE substrates, but inhibited the activity of larger ligases like Lig 12 and H-Lig I.

In conclusion, this study provides valuable insights into the activity of DNA ligases on non-canonical substrates, highlighting the importance of ligase structure and reaction conditions in determining ligation efficiency. The findings contribute to the growing toolbox of synthetic biology and pave the way for further exploration of ligase-substrate interactions, enzyme engineering, and the development of novel biotechnological applications.

year: 2025

title: Boat electrofishing survey of the Awaiti and Bancrofts canals (Tee Head Canal), Piako River system

authors: Hicks, Brendan J.

abstract: This purpose of this survey of Awaiti and Bancrofts canals was to establish the presence of fish in the canals and to determine their relative density and biomass by boat electrofishing. These canals drain the Kopuatai Peat Dome and adjacent farmland into Tee Head Canal, which is part of the Piako River system in the Waikato Region. Awaiti Canal had patches of the emergent macrophyte Eurasian reed sweet grass (Glyceria maxima) and floating common duckweed (Lemna minor). Bancrofts Canal had dense beds of reed sweet grass and water pepper (Persicaria hydropiper, syn. Polygonum hydropiper) that extended into the water along most of its margin to the extent that the bank was not visible. The water of both canals was tannin-stained, but Bancrofts Canal had heavier staining than Awaiti Canal.
We boat electrofished five sites in Awaiti Canal and five sites in Bancrofts Canal for 10 mins each on 21 February 2018. Water temperature was 24.3°C in Awaiti Canal and 23.1°C in Bancrofts Canal. In Awaiti Canal, ambient electrical conductivity was 313.5 µS cm⁻¹, specific conductivity was 317.6 µS cm⁻¹, and black disc distance was 0.18 m. In Bancrofts Canal, ambient conductivity was 174.3 µS cm⁻¹, and specific conductivity was 180.8 µS cm⁻¹.
We caught 31 fish in total, 28 in Awaiti Canal and 3 in Bancrofts Canal. Species included shortfin eels (Anguilla australis), inanga (Galaxias maculatus), goldfish (Carassius auratus), brown bullhead catfish (Ameiurus nebulosus) and gambusia (Gambusia affinis). Two inanga at site 4 (Awaiti Canal) were retrieved dead on capture, as was the one eel retrieved in Bancrofts Canal.
Fish densities were very low, up to a maximum of 1.02 fish 100 m-2 for all species combined at site 3 in Awaiti Canal. Only gambusia were found alive in Bancrofts Canal. Biomass per site and areal biomass were similarly very low, and this was partly attributable to the small size of individuals, which suggested that, with the exception of gambusia, they were young, newly recruited fish.
Awaiti Canal had a low diversity of small fish species that are generally tolerant of poor water quality. This fish diversity is particularly low considering the close proximity of the sites to the coast (about 30 km from the Firth of Thames). The most diverse fish communities in New Zealand are found close to the coast. Typical mean eel densities in the Waikato River assessed by boat electrofishing were 0.7-1.3 eels 100 m⁻² compared to the mean of 0.12 fish 100 m⁻² (range of 0-0.29 eels 100 m⁻² ) at the five sites Awaiti Canal. Bancroft Canal had only gambusia, and the one shortfin eel found dead could have drifted in or died in the canal, probably as result of low DO concentrations.

text: Boat electrofishing survey of the Awaiti and Bancrofts canals (Tee Head Canal), Piako River system This purpose of this survey of Awaiti and Bancrofts canals was to establish the presence of fish in the canals and to determine their relative density and biomass by boat electrofishing. These canals drain the Kopuatai Peat Dome and adjacent farmland into Tee Head Canal, which is part of the Piako River system in the Waikato Region. Awaiti Canal had patches of the emergent macrophyte Eurasian reed sweet grass (Glyceria maxima) and floating common duckweed (Lemna minor). Bancrofts Canal had dense beds of reed sweet grass and water pepper (Persicaria hydropiper, syn. Polygonum hydropiper) that extended into the water along most of its margin to the extent that the bank was not visible. The water of both canals was tannin-stained, but Bancrofts Canal had heavier staining than Awaiti Canal.
We boat electrofished five sites in Awaiti Canal and five sites in Bancrofts Canal for 10 mins each on 21 February 2018. Water temperature was 24.3°C in Awaiti Canal and 23.1°C in Bancrofts Canal. In Awaiti Canal, ambient electrical conductivity was 313.5 µS cm⁻¹, specific conductivity was 317.6 µS cm⁻¹, and black disc distance was 0.18 m. In Bancrofts Canal, ambient conductivity was 174.3 µS cm⁻¹, and specific conductivity was 180.8 µS cm⁻¹.
We caught 31 fish in total, 28 in Awaiti Canal and 3 in Bancrofts Canal. Species included shortfin eels (Anguilla australis), inanga (Galaxias maculatus), goldfish (Carassius auratus), brown bullhead catfish (Ameiurus nebulosus) and gambusia (Gambusia affinis). Two inanga at site 4 (Awaiti Canal) were retrieved dead on capture, as was the one eel retrieved in Bancrofts Canal.
Fish densities were very low, up to a maximum of 1.02 fish 100 m-2 for all species combined at site 3 in Awaiti Canal. Only gambusia were found alive in Bancrofts Canal. Biomass per site and areal biomass were similarly very low, and this was partly attributable to the small size of individuals, which suggested that, with the exception of gambusia, they were young, newly recruited fish.
Awaiti Canal had a low diversity of small fish species that are generally tolerant of poor water quality. This fish diversity is particularly low considering the close proximity of the sites to the coast (about 30 km from the Firth of Thames). The most diverse fish communities in New Zealand are found close to the coast. Typical mean eel densities in the Waikato River assessed by boat electrofishing were 0.7-1.3 eels 100 m⁻² compared to the mean of 0.12 fish 100 m⁻² (range of 0-0.29 eels 100 m⁻² ) at the five sites Awaiti Canal. Bancroft Canal had only gambusia, and the one shortfin eel found dead could have drifted in or died in the canal, probably as result of low DO concentrations.

year: 2018

title: Spot sampling of nutrient concentrations in the Puarenga catchment, Rotorua

authors: Abell, Jonathan Michael

subjects: New Zealand

abstract: The Centre for Biodiversity and Ecology Research was approached by Tūhourangi Tribal Authority for assistance with measuring water quality in streams in the Puarenga Stream catchment. Water sampling was subsequently undertaken on 18 July 2011 and samples were analysed to determine concentrations of total and dissolved fractions of nitrogen and phosphorus. 
Nitrogen and phosphorus are both essential plant nutrients which, when present in excess, can cause eutrophication and associated water quality decline of freshwaters. High concentrations of dissolved forms of nitrogen can also be toxic to aquatic organisms. Excessive nitrogen and phosphorus concentrations are typically the result of pollution due to human activities, although groundwater in the Central Volcanic Plateau region can have elevated concentrations of phosphorus arising from natural geological sources.
This report summarises the methods used, presents the results and places measured concentrations in context by drawing comparisons with both guideline and regional mean values.

text: Spot sampling of nutrient concentrations in the Puarenga catchment, Rotorua The Centre for Biodiversity and Ecology Research was approached by Tūhourangi Tribal Authority for assistance with measuring water quality in streams in the Puarenga Stream catchment. Water sampling was subsequently undertaken on 18 July 2011 and samples were analysed to determine concentrations of total and dissolved fractions of nitrogen and phosphorus. 
Nitrogen and phosphorus are both essential plant nutrients which, when present in excess, can cause eutrophication and associated water quality decline of freshwaters. High concentrations of dissolved forms of nitrogen can also be toxic to aquatic organisms. Excessive nitrogen and phosphorus concentrations are typically the result of pollution due to human activities, although groundwater in the Central Volcanic Plateau region can have elevated concentrations of phosphorus arising from natural geological sources.
This report summarises the methods used, presents the results and places measured concentrations in context by drawing comparisons with both guideline and regional mean values.

year: 2011-09