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Techniques for the Initiation and Study of the Self-Assembly of Biopolymers.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Techniques for the Initiation and Study of the Self-Assembly of Biopolymers./
Author:	Aghvami, Seyedmohammadali .
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2020,
Description:	121 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-09, Section: B.
Contained By:	Dissertations Abstracts International81-09B.
Subject:	Physics. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=27735747
ISBN:	9781392779392

Techniques for the Initiation and Study of the Self-Assembly of Biopolymers.
Aghvami, Seyedmohammadali .

Techniques for the Initiation and Study of the Self-Assembly of Biopolymers. - Ann Arbor : ProQuest Dissertations & Theses, 2020 - 121 p.

Source: Dissertations Abstracts International, Volume: 81-09, Section: B.

Thesis (Ph.D.)--Brandeis University, 2020.

This item must not be sold to any third party vendors.

The growing interest in bottom-up approaches for fabrication of functional materials has led to development of novel methods for the study of the hierarchical self-assembly of nanoscale building blocks. Several examples of complex self-assembled biomolecular structures formed through multiple levels of hierarchical organization can be found in nature, i.e. the hierarchical assembly of polypeptides (protein) and polynucleotides (DNA/RNA). These two types of biopolymers dominate life, forming a myriad of structural and functional biomaterials in living systems and providing inspiration to material scientists who look to nature for design principles for materials with desirable properties. The hierarchical structure of proteins can be studied at multiple length scales. While a protein is a polymer consisting of a linear sequence of amino acids, its function depends on its three-dimensional structure. X-ray crystallography was developed to resolve the 3D structure of the protein in its crystalline phase. However, crystallization is an activated process that requires a supersaturated protein solution to cross a nucleation barrier. Exposing a supersaturated protein solution to an intense laser has been shown to induce crystal nucleation and promote crystal growth, however, the mechanism of its action is not well understood. The first chapter, describes how two optical techniques, fluorescence lifetime microscopy (FLIM) and second-harmonic generation are used to study laser induced crystallization in solutions of xylanase, a globular protein. We stimulate fluorescence within a xylanase molecule using a femtosecond pulsed laser and quantify the emission lifetime by Time Correlated Single Photon Counting (TCSPC). We observe two major fluorescence decay rates that are consistent with tryptophan, of which there are 6 per xylanase. The fast decay rate has two components. One is the intrinsic decay rate of an individual tryptophan, the other varies with denaturation of the protein in a fashion that that indicates quenching between two nearby tryptophans, or between one tryptophan and a tyrosine, of which there are 17 per xylanase. We measured the spectral emission in the range of 400nm – 600nm and observed a peak around 500 nm that is consistent with the emission from oxidized tryptophan. Second-harmonic generation (SHG) is measured simultaneously with FLIM. SHG is a coherent scattering phenomenon in which two photons “combine” into one with twice the energy. This can happen only for proteins in a crystal that lacks an inversion symmetry and cannot occur in a disordered aggregate. Hence SHG discriminates between crystalline and amorphous sub-resolution protein condensates. We characterize the relation between the decay rates of tryptophan emission and the increase in its intensity after high power laser pulses, correlate the decay rates with the SHG signal and hypothesize a mechanism explaining laser induced protein crystallization.The DNA double helix is another example of the hierarchical assembly of biomaterials. Bionanotechnology has made a step forward in using DNAs as building blocks of nanomaterials by developing several techniques based on base-pairing and base-stacking interactions of DNA strands. DNA origami is the technique with many advantages over other bionanotechnological platforms, such as the ease of design and robustness of the resulting structure. In chapter two I will talk about using the DNA origami platform to encapsulate organic and inorganic cargo in virus-like DNA origami structures. We demonstrate a robust method for self-assembly of virus like capsids DNA nanostructure of various sizes by designing specific capsomers with programmable shape-complementary interactions. These DNA nanostructures are constructed from highly modular building blocks, allowing us to successfully functionalize them for encapsulation of organic and inorganic cargo particles. DNA origami structures are biocompatible and have highly controllable size and shape, making them potential candidates for biomaterial engineering purposes such as drug delivery and gene therapy. We also develop a monitoring system based on in-situ TEM of DNA-origami capsids to attempt to visualize the assembly of the capsids in real time at the resolution of individual monomer subunits to elucidate the assembly kinetics. Advancements in soft matter experiments have only been possible because of development of new experimental methods. In chapter three, I will explain our novel method developed for inexpensive and rapid prototyping of microfluidic devices constructed using the thermoplastic Cyclic Olefin Copolymer (COC) which is a biocompatible, cost effective material and suitable for studying biomaterials. The method has attracted the interest from different biotechnology companies interested in fabricating devices for a variety of medical applications.We demonstrate fabrication of microfluidic devices comprised of two molded pieces joined together to create a sealed device. The first piece contains the microfluidic features and the second contains the inlet and outlet manifold, a frame for rigidity and a viewing window. We demonstrated the application of our method by making x-ray transparent microfluidic devices for protein crystallization and in situ structure determination. We also introduce the XScreen chip, which we propose as a microfluidic liquid handling device for screening protein crystallization conditions using counter-diffusion.

ISBN: 9781392779392Subjects--Topical Terms:

564049
Physics.
Subjects--Index Terms:

Cargo encapsulation

Techniques for the Initiation and Study of the Self-Assembly of Biopolymers.
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300 $a 121 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-09, Section: B.
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520 $a The growing interest in bottom-up approaches for fabrication of functional materials has led to development of novel methods for the study of the hierarchical self-assembly of nanoscale building blocks. Several examples of complex self-assembled biomolecular structures formed through multiple levels of hierarchical organization can be found in nature, i.e. the hierarchical assembly of polypeptides (protein) and polynucleotides (DNA/RNA). These two types of biopolymers dominate life, forming a myriad of structural and functional biomaterials in living systems and providing inspiration to material scientists who look to nature for design principles for materials with desirable properties. The hierarchical structure of proteins can be studied at multiple length scales. While a protein is a polymer consisting of a linear sequence of amino acids, its function depends on its three-dimensional structure. X-ray crystallography was developed to resolve the 3D structure of the protein in its crystalline phase. However, crystallization is an activated process that requires a supersaturated protein solution to cross a nucleation barrier. Exposing a supersaturated protein solution to an intense laser has been shown to induce crystal nucleation and promote crystal growth, however, the mechanism of its action is not well understood. The first chapter, describes how two optical techniques, fluorescence lifetime microscopy (FLIM) and second-harmonic generation are used to study laser induced crystallization in solutions of xylanase, a globular protein. We stimulate fluorescence within a xylanase molecule using a femtosecond pulsed laser and quantify the emission lifetime by Time Correlated Single Photon Counting (TCSPC). We observe two major fluorescence decay rates that are consistent with tryptophan, of which there are 6 per xylanase. The fast decay rate has two components. One is the intrinsic decay rate of an individual tryptophan, the other varies with denaturation of the protein in a fashion that that indicates quenching between two nearby tryptophans, or between one tryptophan and a tyrosine, of which there are 17 per xylanase. We measured the spectral emission in the range of 400nm – 600nm and observed a peak around 500 nm that is consistent with the emission from oxidized tryptophan. Second-harmonic generation (SHG) is measured simultaneously with FLIM. SHG is a coherent scattering phenomenon in which two photons “combine” into one with twice the energy. This can happen only for proteins in a crystal that lacks an inversion symmetry and cannot occur in a disordered aggregate. Hence SHG discriminates between crystalline and amorphous sub-resolution protein condensates. We characterize the relation between the decay rates of tryptophan emission and the increase in its intensity after high power laser pulses, correlate the decay rates with the SHG signal and hypothesize a mechanism explaining laser induced protein crystallization.The DNA double helix is another example of the hierarchical assembly of biomaterials. Bionanotechnology has made a step forward in using DNAs as building blocks of nanomaterials by developing several techniques based on base-pairing and base-stacking interactions of DNA strands. DNA origami is the technique with many advantages over other bionanotechnological platforms, such as the ease of design and robustness of the resulting structure. In chapter two I will talk about using the DNA origami platform to encapsulate organic and inorganic cargo in virus-like DNA origami structures. We demonstrate a robust method for self-assembly of virus like capsids DNA nanostructure of various sizes by designing specific capsomers with programmable shape-complementary interactions. These DNA nanostructures are constructed from highly modular building blocks, allowing us to successfully functionalize them for encapsulation of organic and inorganic cargo particles. DNA origami structures are biocompatible and have highly controllable size and shape, making them potential candidates for biomaterial engineering purposes such as drug delivery and gene therapy. We also develop a monitoring system based on in-situ TEM of DNA-origami capsids to attempt to visualize the assembly of the capsids in real time at the resolution of individual monomer subunits to elucidate the assembly kinetics. Advancements in soft matter experiments have only been possible because of development of new experimental methods. In chapter three, I will explain our novel method developed for inexpensive and rapid prototyping of microfluidic devices constructed using the thermoplastic Cyclic Olefin Copolymer (COC) which is a biocompatible, cost effective material and suitable for studying biomaterials. The method has attracted the interest from different biotechnology companies interested in fabricating devices for a variety of medical applications.We demonstrate fabrication of microfluidic devices comprised of two molded pieces joined together to create a sealed device. The first piece contains the microfluidic features and the second contains the inlet and outlet manifold, a frame for rigidity and a viewing window. We demonstrated the application of our method by making x-ray transparent microfluidic devices for protein crystallization and in situ structure determination. We also introduce the XScreen chip, which we propose as a microfluidic liquid handling device for screening protein crystallization conditions using counter-diffusion.
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653 $a SHG microscopy
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