An FTIR Investigation of Flanking Sequence Effects on the Structure and Flexibility of DNA Binding Sites

Abstract

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. KeywordsFTIR; DNA; backbone conformations; flexibility; indirect readout During both replication and transcription, amino acids of the protein factor involved recognize and interact electrostatically with the base pairs of the specific DNA sequence containing the recognition site (direct readout) with remarkable specificity. Upon binding of a protein in this region, a bending of the DNA occurs which enhances the stability of the complex by increasing the number of favorable contacts made between the DNA and the protein 1; 2 . The four classifications of these interactions are: (i) van der Waals interactions between aliphatic amino acids and the C5-methyl groups of thymines or the C5-hydrogen groups of cytosines, (ii) hydrogen bonds between the nucleic acids and the amide and carbonyl groups of the peptide backbone, (iii) hydrogen bonding bridges constructed with water molecules between amino acids and base-pairs 3; 4; 5 , and (iv) interlacing of amino acid side-chains between two bases 6 . Although these are the major interactions, direct contacts at the recognition site alone do not account for the conformation of the DNA-protein complex; base pairs on either side of the *Author to whom correspondence should be addressed: Mary E. recognition site, referred to as flanking sequences, are also thought to play a role in the conformation of the DNA-protein complex (indirect readout) 1; 7; 8 .In most cases, it is necessary for the protein to distort the DNA in order to achieve the transition state. To compensate for the energetic costs of deformation, the DNA-protein complex may make any modification that improves the binding free energy such as releasing water from non-polar surfaces and/or increasing the number of d...

Role of Minor Groove Width and Hydration Pattern on Amsacrine Interaction with DNA

“…For instance, spectra of duplex (AGAGCAACAGAG)2 and (AGAGACCAGAGA)2 exhibit subtle N-type sugar conformation features (∼860 cm −1 and ∼808 cm −1 ). Presence of these features is consistent with the given fact that increasing content of G/C base pairs favor N type sugar conformation in DNA [33], [34]. Spectral presence of these minute features signifies the single nucleotide level sensitivity of ATR-FTIR approach that is also supported by some earlier observations [28], [34].…”

“…This band splits into two bands at 836 cm −1 and 824 cm −1 in the complexes (Figure 3, 4 panel 1). Formation of the new band at 824 cm −1 is indicative of some N type (C3′-endo/anti) sugar conformation (A-DNA conformation) features in amsacrine-ctDNA complexes [31], [33]. Occurrence of N type sugar conformation is further confirmed by the presence of a shoulder around 861 cm −1 in the complexes (Figure 3, 4 panel 1) [31], [34].…”

“…S (C2′-endo/anti) type sugar pucker corresponds to global B-DNA conformation. Split into this band (∼842 and ∼824 cm −1 ) and presence of new shoulders around ∼865and ∼808 cm −1 in the complexes (Figure 3, 4) are indicative of some N type (C3′-endo/anti) sugar features in complexes [31], [33], [34]. N (C3′-endo/anti) type sugar pucker represents A-DNA conformation.…”

Fourier Transform Infrared Spectroscopy of the DNA of the Chuxiong Population ofCamellia reticulataLindl. of China

“…[1,2] So, it has been widely applied to qualitative and quantitative analyses in chemistry, [3] biology, [4] medicinal chemistry, [5] disease diagnosis, [6,7] and environmental science, and so forth. [8] In recent years, with the rapid development of chemo metrics, computer science, and molecular biology, the study on DNA molecules by using FTIR is particularly eye-catching.…”

Nucleotides and nucleic acids; oligo- and polynucleotides

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