Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis

Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis

Bibliographic Details
Main Author: Yangyuoru, Philip
Language:English
Published: Kent State University / OhioLINK 2014
Subjects:
Analytical Chemistry
Biochemistry
Molecular Biology
Biophysics
DNA aptamers
G-quadruplexes
mechanical stability
force-based assays
single molecules
laser tweezers
capillary electrophoresis
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=kent1406541346
id ndltd-OhioLink-oai-etd.ohiolink.edu-kent1406541346
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Analytical Chemistry
Biochemistry
Molecular Biology
Biophysics
DNA aptamers
G-quadruplexes
mechanical stability
force-based assays
single molecules
laser tweezers
capillary electrophoresis
spellingShingle Analytical Chemistry
Biochemistry
Molecular Biology
Biophysics
DNA aptamers
G-quadruplexes
mechanical stability
force-based assays
single molecules
laser tweezers
capillary electrophoresis
Yangyuoru, Philip
Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis
author Yangyuoru, Philip
author_facet Yangyuoru, Philip
author_sort Yangyuoru, Philip
title Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis
title_short Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis
title_full Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis
title_fullStr Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis
title_full_unstemmed Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis
title_sort investigation of the formation of some biologically relevant small molecules using laser tweezers and capillary electrophoresis
publisher Kent State University / OhioLINK
publishDate 2014
url http://rave.ohiolink.edu/etdc/view?acc_num=kent1406541346
work_keys_str_mv AT yangyuoruphilip investigationoftheformationofsomebiologicallyrelevantsmallmoleculesusinglasertweezersandcapillaryelectrophoresis
_version_ 1719436718958968832
spelling ndltd-OhioLink-oai-etd.ohiolink.edu-kent14065413462021-08-03T06:26:00Z Investigation of the Formation of some Biologically Relevant Small Molecules Using Laser Tweezers and Capillary Electrophoresis Yangyuoru, Philip Analytical Chemistry Biochemistry Molecular Biology Biophysics DNA aptamers G-quadruplexes mechanical stability force-based assays single molecules laser tweezers capillary electrophoresis The interaction between nucleic acids and small molecule ligands is continuously generating significant interest due to their widespread biological and bioanalytical applications. We investigated the mechanical property of the binding between aptamers and small molecules. Using an ATP binding aptamer as an example, we observed that the mechanical stability of the aptamers that are bound with ATP is higher than those without a ligand. Therefore, a force-based sensor can be developed to detect small molecules using aptamers as a platform. We determined the dissociation constant, Kd, for aptamer-ligand interactions at the single-molecule level by applying a Hess-like cycle. Our experiments allow the Kd determination from only one ligand concentration which was further validated by our capillary electrophoresis (CE) method. By using only one ligand concentration, such a method not only saves time and material, but also is less susceptible to reduced reproducibility due to run-to-run fluctuations. G-quadruplex forming sequences which are wide spread in the genome particularly in telomeres and promoter regions have been shown to be therapeutic targets. G-quadruplex structures have been extensively studied using mostly conventional methods. However, the mechanical stability, thermodynamics, kinetics properties of these interactions at the single-molecule level, remains to be fully understood. While the formation of these G-quadruplex structures is highly dynamic, they are mechanically stabilized upon ligand binding which may affect their biological functions. Small molecules which bind to nucleic acid structures may interfere with vital cellular processes such as transcription and protein translation during cell division by acting as energy barriers or mechanical blockage. Therefore, understanding stability of nucleic structures from a mechanical stability stand point is critical to fully explore their therapeutic potentials. We have investigated the human telomeric repeat containing RNA (TERRA) G-quadruplex structures. By using a TERRA sequence that hosts only one G-quadruplex at the single-molecule level in a laser-tweezers instrument, we are able to investigate intramolecular G-quadruplex without implications from high order structures due to the tendency of the TERRA sequence to self-associate. Using a mechanical unfolding and refolding method, we revealed multiple TERRA G-quadruplexes in a solution mixture. One of the structures is consistent with the parallel conformation determined by NMR/X-ray techniques. In addition, we identified a partially folded structure that serves as an intermediate to the unfolding and refolding of the TERRA G-quadruplex. By applying a force-jump approach, the refolding kinetics of the TERRA G-quadruplex and the intermediate were measured and compared with those of human telomeric DNA (hTelo) G-quadruplex. We have found that formation of the TERRA G-quadruplex is slower than that of hTelo G-quadruplex. Together with the higher thermodynamic and mechanical stability of the TERRA with respect to the hTelo G-quadruplex, these results suggest different regulatory capacities of RNA and DNA G-quadruplexes for processes associated with human telomeres.Lastly, a quantitative assay was developed to investigate sphingosine kinase 2 (SphK2) activity both in vitro and with cell lysates, using capillary electrophoresis with laser-induced fluorescence (CE-LIF) and sphingosine fluorescein as the substrate. Sphingosine and sphingosine-1-phosphate are found in very minute quantities in cells and therefore require highly sensitive techniques for quantitative analysis. 2014-07-31 English text Kent State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=kent1406541346 http://rave.ohiolink.edu/etdc/view?acc_num=kent1406541346 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Similar Items