Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs
| Autor: | David M. Jameson, Robert Wright, Sara E. Wingate, Randy M. Wadkins, Samantha M. Reilly, John J. Correia, Daniel F. Lyons |
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| Rok vydání: | 2014 |
| Předmět: |
Circular dichroism
Biophysics Cytidine 010402 general chemistry 01 natural sciences Proto-Oncogene Proteins c-myc 03 medical and health sciences chemistry.chemical_compound Humans A-DNA Nucleotide Motifs Promoter Regions Genetic Fluorescent Dyes 030304 developmental biology 0303 health sciences Temperature DNA Fluorescence Random coil 0104 chemical sciences Kinetics chemistry Biochemistry Hydrodynamics Proteins and Nucleic Acids Cytosine Fluorescence anisotropy |
| Zdroj: | Biophysical Journal. 107(7):1703-1711 |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2014.08.014 |
| Popis: | The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs. |
| Databáze: | OpenAIRE |
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