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: Using online citizen science to develop students' science capabilities

authors: Anderson, Dayle

subjects: Social Sciences

abstract: In 2018, a research project funded by New Zealand’s teaching and learning research initiative (TLRI) explored the impact of online citizen science (OCS) projects on the science educa primary school children in New Zealand. This article provides an intion of depth case study from this project in order to share ways in which one teacher used OCS projects specifically to develop students’ science capability, ‘critique evidence’. Comparin g data from students’ conversations and work with the progress indicators for the science capabilities released by the Ministry of Education in 2019 suggests that many of the students were achieving at or above the expected level. We suggest that teacher p rofessional learning and development in relation to the intent of science in the NZC, and the purpose of the science capabilities, was a significant enabler.

text: Using online citizen science to develop students' science capabilities In 2018, a research project funded by New Zealand’s teaching and learning research initiative (TLRI) explored the impact of online citizen science (OCS) projects on the science educa primary school children in New Zealand. This article provides an intion of depth case study from this project in order to share ways in which one teacher used OCS projects specifically to develop students’ science capability, ‘critique evidence’. Comparin g data from students’ conversations and work with the progress indicators for the science capabilities released by the Ministry of Education in 2019 suggests that many of the students were achieving at or above the expected level. We suggest that teacher p rofessional learning and development in relation to the intent of science in the NZC, and the purpose of the science capabilities, was a significant enabler.

year: 2020-01-01
This is an author’s accepted version of an article published in the Curriculum Matters. © 2020 NZCER

title: Te Tarata

authors: Maxwell, Te Kahautu

abstract: This composition placed second in the Haka Composition Section. Te Tarata this composition commemorates the battle of Te Tarata in Te Whakatōhea 15 October 1865. This was the aftermath of the killing of the missionary Rev Carl Sylvius Volkner on the 2 March 1865. At the battle of Te Tarata it is recorded as the only calvary charge in the New Zealand Land Wars. Te Whakatōhea ingeniously wove around the palisade walls tī kouka (Cordyline australis) or cabbage tree. When the canon ball hit the palisades the tī kouka absorbed the force of the canon ball and rebounded the ball. 20 Te Whakatohea were killed and a number of others were wounded. This composition was performed by Ōpōtiki Mai Tawhiti at the Te Matatini World Championship Kapa Haka Competition 2015.

text: Te Tarata This composition placed second in the Haka Composition Section. Te Tarata this composition commemorates the battle of Te Tarata in Te Whakatōhea 15 October 1865. This was the aftermath of the killing of the missionary Rev Carl Sylvius Volkner on the 2 March 1865. At the battle of Te Tarata it is recorded as the only calvary charge in the New Zealand Land Wars. Te Whakatōhea ingeniously wove around the palisade walls tī kouka (Cordyline australis) or cabbage tree. When the canon ball hit the palisades the tī kouka absorbed the force of the canon ball and rebounded the ball. 20 Te Whakatohea were killed and a number of others were wounded. This composition was performed by Ōpōtiki Mai Tawhiti at the Te Matatini World Championship Kapa Haka Competition 2015.

year: 2014-10-10