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Modeling an Enzyme Active Site using Molecular Visualization Freeware

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3D visualization of macromolecule synthesis.

Timothy J Duerr¹, Ester Comellas^2,3, Eun Kyung Jeon¹

¹Department of Biology, Northeastern University, Boston, United States.

|October 14, 2020

Summary

This summary is machine-generated.

This study introduces a novel method for in vivo macromolecule synthesis measurement using click chemistry and light sheet microscopy. This technique allows for detailed analysis of DNA, RNA, protein, and glycosylation in developing and regenerating tissues.

Keywords:

Axolotl click-chemistry developmental biology light sheet fluorescence microscopy macromolecules regenerative medicine stem cells whole mount

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Area of Science:

Developmental Biology
Molecular Biology
Biotechnology

Background:

Understanding cellular and tissue development requires accurate measurement of macromolecule synthesis.
Existing methods may lack the resolution or scope to capture dynamic synthesis processes in vivo.
Investigating tissue regeneration and response to stimuli necessitates quantitative analysis of molecular synthesis.

Purpose of the Study:

To develop and validate a novel in vivo method for quantifying nascent macromolecule synthesis.
To enable high-resolution imaging and analysis of DNA, RNA, protein, and glycosylation synthesis.
To apply this method to study tissue development and regeneration, such as in axolotl limb regeneration.

Main Methods:

In vivo injection of alkyne- or azide-modified nucleoside and amino acid analogs.
Whole-mount click chemistry-based fluorescent staining for labeling synthesized macromolecules.
Light sheet fluorescent microscopy for three-dimensional volumetric imaging.
Development of an image processing pipeline for cell and tissue segmentation.

Main Results:

Successfully labeled and imaged nascent DNA, RNA, protein, and glycosylation synthesis in axolotl tissues.
Demonstrated the method's sensitivity by detecting changes in DNA synthesis after limb denervation.
Quantitatively analyzed macromolecule synthesis at organ, cellular, and molecular levels.

Conclusions:

The developed method provides a powerful tool for interrogating in vivo macromolecule synthesis.
This approach allows for quantitative analysis in heterogeneous tissues and complex biological processes.
Enables deeper understanding of cellular and tissue dynamics during development and regeneration.