Abstract
DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence supporting G-quadruplex involvement in a number of important aspects of genome functions, including transcription, replication, and genomic stability, and that protein and enzyme recognition of G-quadruplexes may represent a key event to regulate physiological or pathological pathways. Two important techniques to study G-quadruplexes and their protein interactions are the electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay. EMSA, one of the most sensitive and robust methods for studying the DNA-protein interactions, can be used to determine the binding parameters and relative affinities of a protein for the G-quadruplex. DMS footprinting is a powerful assay for the initial characterization of G-quadruplexes, which can be used to deduce the guanine bases involved in the formation of G-tetrads under physiological salt conditions. DMS footprinting can also reveal important information in G-quadruplex-protein complexes on protein contacts and regional changes in DNA G-quadruplex upon protein binding. In this paper, we will provide a detailed protocol for the EMSA and DMS footprinting assays for characterization of G-quadruplexes and G-quadruplex-protein complexes. Expected outcomes and references to extensions of the method will be further discussed.
| Original language | English (US) |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press Inc. |
| Pages | 201-222 |
| Number of pages | 22 |
| DOIs | |
| State | Published - Jan 1 2019 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 2035 |
| ISSN (Print) | 1064-3745 |
| ISSN (Electronic) | 1940-6029 |
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Keywords
- Dimethyl sulfate (DMS) footprinting
- DNA
- Electrophoresis
- Electrophoretic mobility shift assay (EMSA)
- G-quadruplex
- Protein
ASJC Scopus subject areas
- Molecular Biology
- Genetics
Cite this
Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quadruplex-Protein Complexes. / Onel, Buket; Wu, Guanhui; Sun, Daekyu; Lin, Clement; Yang, Danzhou.
Methods in Molecular Biology. Humana Press Inc., 2019. p. 201-222 (Methods in Molecular Biology; Vol. 2035).Research output: Chapter in Book/Report/Conference proceeding › Chapter
}
TY - CHAP
T1 - Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quadruplex-Protein Complexes
AU - Onel, Buket
AU - Wu, Guanhui
AU - Sun, Daekyu
AU - Lin, Clement
AU - Yang, Danzhou
PY - 2019/1/1
Y1 - 2019/1/1
N2 - DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence supporting G-quadruplex involvement in a number of important aspects of genome functions, including transcription, replication, and genomic stability, and that protein and enzyme recognition of G-quadruplexes may represent a key event to regulate physiological or pathological pathways. Two important techniques to study G-quadruplexes and their protein interactions are the electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay. EMSA, one of the most sensitive and robust methods for studying the DNA-protein interactions, can be used to determine the binding parameters and relative affinities of a protein for the G-quadruplex. DMS footprinting is a powerful assay for the initial characterization of G-quadruplexes, which can be used to deduce the guanine bases involved in the formation of G-tetrads under physiological salt conditions. DMS footprinting can also reveal important information in G-quadruplex-protein complexes on protein contacts and regional changes in DNA G-quadruplex upon protein binding. In this paper, we will provide a detailed protocol for the EMSA and DMS footprinting assays for characterization of G-quadruplexes and G-quadruplex-protein complexes. Expected outcomes and references to extensions of the method will be further discussed.
AB - DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence supporting G-quadruplex involvement in a number of important aspects of genome functions, including transcription, replication, and genomic stability, and that protein and enzyme recognition of G-quadruplexes may represent a key event to regulate physiological or pathological pathways. Two important techniques to study G-quadruplexes and their protein interactions are the electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay. EMSA, one of the most sensitive and robust methods for studying the DNA-protein interactions, can be used to determine the binding parameters and relative affinities of a protein for the G-quadruplex. DMS footprinting is a powerful assay for the initial characterization of G-quadruplexes, which can be used to deduce the guanine bases involved in the formation of G-tetrads under physiological salt conditions. DMS footprinting can also reveal important information in G-quadruplex-protein complexes on protein contacts and regional changes in DNA G-quadruplex upon protein binding. In this paper, we will provide a detailed protocol for the EMSA and DMS footprinting assays for characterization of G-quadruplexes and G-quadruplex-protein complexes. Expected outcomes and references to extensions of the method will be further discussed.
KW - Dimethyl sulfate (DMS) footprinting
KW - DNA
KW - Electrophoresis
KW - Electrophoretic mobility shift assay (EMSA)
KW - G-quadruplex
KW - Protein
UR - http://www.scopus.com/inward/record.url?scp=85071493326&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85071493326&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-9666-7_11
DO - 10.1007/978-1-4939-9666-7_11
M3 - Chapter
C2 - 31444751
AN - SCOPUS:85071493326
T3 - Methods in Molecular Biology
SP - 201
EP - 222
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -