An FTIR investigation of flanking sequence effects on the structure and flexibility of DNA binding sites.
Fourier transform infrared (FTIR) spectroscopy and a library of FTIR marker bands have been used to examine the structure and relative flexibilities conferred by different flanking sequences on the EcoRI binding site. This approach allowed us to examine unique peaks and subtle changes in the spectra of d(AAAGAATTCTTT)(2), d(TTCGAATTCGAA)(2), and d(CGCGAATTCGCG)(2) and thereby identify local changes in base pairing, base stacking, backbone conformation, glycosidic bond rotation, and sugar puckering in the studied sequences. The changes in flanking sequences induce differences in the sugar puckers, glycosidic bond rotation, and backbone conformations. Varying levels of local flexibility are observed within the sequences in agreement with previous biological activity assays. The results also provide supporting evidence for the presence of a splay in the G(4)-C(9) base pair of the EcoRI binding site and a potential pocket of flexibility at the G(4) cleavage site that have been proposed in the literature. In sum, we have demonstrated that FTIR is a powerful methodology for studying the effect of flanking sequences on DNA structure and flexibility, for it can provide information about the local structure of the nucleic acid and the overall relative flexibilities conferred by different flanking sequences.
Kahn
Talia R
TR
Joint Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, California 91711, USA.
Fong
Kimberly K
KK
Jordan
Brian
B
Lek
Janista C
JC
Levitan
Rachel
R
Mitchell
Patrick S
PS
Wood
Corrina
C
Hatcher
Mary E
ME
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D003855:Deoxyribose / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry; D018583:Pliability; D017550:Spectroscopy, Fourier Transform Infrared; D014732:Vibration
D010710:Phosphates; D003855:Deoxyribose; D004247:DNA