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determination of the conformation of biomolecules and also provides insights about the binding modes of small molecules with DNA using pattern recognition [28]_{. Binding of ligands}

to DNA can be easily identified by the changes exhibited in the CD pattern by monitoring the compound and DNA wavelengths. When an achiral ligand, which has no CD by itself in solution, binds to a chiral macromolecule, an induced CD (ICD) signal is observed in the wavelength region corresponding to the bound achiral ligand [29]_{. This ICD signal can be a}

weak positive or negative, as in the case of duplex DNA intercalators, or can be a large positive signal, as in the case of duplex DNA groove binders [26b, 30]_{. Compounds that are known to}

interact with quadruplex structures have demonstrated preferential stacking at the terminal G- tetrads. Quadruplex end stackers, similar to duplex DNA intercalators, also exhibit a weak positive or negative ICD signal upon complex formation. In some cases, compounds are shown to effectively stack at the ends even without exhibiting any significant ICD signals [31]_{. Groove}

binding with quadruplexes has been a rarely observed phenomenon. As a result, a clear CD pattern that can used to identify possible groove binding agents has not been established until now. Shafer et al., using docking method, identified a carbocyanine dye, DODC (Figure 2.1-B), as a potential groove binder, and studied its interaction with an intermolecular dimeric hairpin quadruplex using CD [22]_{. This was the first ever reported binding study of a quadruplex}

interacting agent using CD. DODC binding to the quadruplex resulted in an induced CD signal in the wavelength region corresponding to the absorbance of the bound DODC. The large positive and negative ICD signal was hypothesized to be a consequence of the dye molecules stacking in the grooves of the quadruplex (Figure 2.3-A, adopted from the above reference).

CD experiments were carried out with DODC with other very similar quadruplex systems to confirm that a similar ICD pattern is exhibited for groove binding. Figure 2.3-B shows the CD spectra of DODC also with a dimeric hairpin quadruplex (Materials and Methods, Section 2.2.1), recently reported to exist as a mixture of parallel and antiparallel conformers. DODC exhibits an exciton-type splitting upon complex formation suggesting that compounds that can form stacked species possibly in the grooves of the quadruplex have an exciton-type splitting in the region corresponding to the absorbance of the bound ligand. DODC also exhibited a very similar ICD pattern upon complex formation with the wild-type human telomeric sequence, Tel22. Therefore, the large ICD pattern exhibited by small molecules upon complex formation is characteristic of the ligands stacking in the grooves of quadruplex systems. RHPS4 (Figure 2.1-A), a well-known small molecule shown to stack at the ends of human telomeric quadruplex DNA by NMR, was evaluated by CD to see if a ICD pattern exists for this class of compounds. As expected, RHPS4 did not exhibit any ICD upon complex formation (Figure 2.3-C) further suggesting that end-stacking compounds have very weak to almost no ICD with quadruplexes.

DB832 was recently reported to bind to the human telomeric quadruplex DNA, Tel22 d[AG3(T2AG3)3], possibly in the grooves of the quadruplex conformation as exemplified by a

large positive and negative induced CD signals (Figure 2.5-A) [25]_{. The large magnitude of the}

induced CD was hypothesized due to the stacking of DB832 in different grooves of the telomeric quadruplex structure. Using the induced CD pattern exhibited by DB832 as a model for quadruplex groove recognition, CD studies of DB832 were carried out with Tel24, d[TTG3(T2AG3)3A], to investigate if a pattern similar exists (Figure 2.5-B). NMR studies have

shown that Tel24 sequence forms a mixed parallel/antiparallel hybrid type structure in K+

(Figure 2.2-A) [32]_{. When DB832 is titrated into Tel24, a large exciton-type ICD signal, very}

bound DB832. This suggests that the DB832 is also binding as a stacked species possibly in the grooves of this quadruplex system. Moreover, the DNA wavelength region does not exhibit much change upon compound binding suggesting that DB832 is preferentially binding to the pre-formed hybrid conformations.

Based on the CD studies of Tel22 and Tel24, however, it is rather difficult to definitively establish the groove binding aspect of DB832. Therefore, if such a system exists, where the grooves of the quadruplex systems are rendered sterically inaccessible, small molecules that selectively bind in the grooves can be completely prevented from interacting in the grooves. Structural features of G-quartets reveal that the aromatic proton, H8, of the guanine bases is directly pointed into the grooves of the quadruplex. If those protons could be replaced by a bulky substituent, small molecules can be directly inhibited from interacting in the grooves. Bromo-guanines turn out to be the best possible candidate to test such a hypothesis. Sugiyama and coworkers have successfully employed these modified guanine bases and shown that stable quadruplex architectures could be maintained with this modification [12a, 33]_{. The bromine}

substitution on the C8 position of guanine further locks the syn glycosidic conformation of guanine nucleotides further adding to the stability of the fold [34]_{. Sugiyama and coworkers}

recently showed that ODN9, d(AG2GGTTAG8GGTTAGGGTTAG20GG), folds in a mixed

parallel/antiparallel hybrid conformation, which is likely the structure the unmodified human telomere adopts in solution in the presence of K+ [34]_{. The three bromoguanine substitutions}

correspond to the guanines in the terminal tetrad at the 5‟-end of the hybrid fold. These bulky bromine substituents selectively inhibit binding in all the three accessible grooves of the quadruplex (Figure 2.2-C), thereby, effectively hindering the interaction of any groove binding molecules. The fourth groove in the hybrid quadruplex conformation is inaccessible due to a diagonally running loop connecting two adjacent strands.

CD studies of DB832 were conducted with the ODN9 sequence to further test the groove binding mode. CD spectra of DB832 titrated into ODN9 shown in figure 2.5-C. Most interestingly, DB832 does not exhibit the characteristic exciton-type splitting, that is observed with unmodified telomeric sequences, with the ODN9 sequence. A weak ICD signal at significantly high ratios of DB832 is observed, suggesting that the interaction of DB832 with ODN9 is primarily at the ends. Since DB832 does not exhibit any exciton-type splitting with this hybrid quadruplex structure when the grooves are rendered inaccessible by bromo- guanine substitution, it provides evidence that the mode of binding of this compound to the human telomere is groove binding as a stacked species.

Another modified human telomeric sequence with bromine atoms at different positions

was also tested to confirm the groove binding of DB832. ODN4,

d(AGGGTTAGGGTTAG14G15G16TTAGGG), was also shown to fold into a mixed

parallel/antiparallel hybrid conformation, similar to the unmodified human telomeric sequence [34]_{. However, in this case, the three bromine substitutions were located in a single}

quadruplex groove (Figure 2.2-D). Therefore, small molecules that interact in the grooves should still be able to bind in the remainder of the two accessible grooves. CD studies of DB832 were conducted with ODN4 sequence (Figure 2.5-D). Interestingly, exciton-type ICD signal is still observed with this sequence upon titration of DB832. However, the magnitude of the ICD is comparatively smaller than what is observed with the unmodified telomeric sequences. The ICD signal generated upon complex formation is due to the formation of stacked species of DB832 in the remaining two grooves of the hybrid fold, resulting in decreased intensity.

The 27-nt G-rich sequence in the NHE-III promoter element of the c-myc oncogene has been shown to fold into a parallel quadruplex architecture [35]_{. The connecting loop bases}

parallel scaffold in different variants of c-myc. Moreover, the connecting bases form a double- chain reversal loop and completely block all the grooves of the quadruplex. This parallel quadruplex fold is also an excellent system to test for small molecules that interact primarily in the grooves of the quadruplex. The loop bases should act as a natural steric blockage for groove interacting ligands. To test this idea, CD titrations of DB832 were carried out with a dual mutant c-myc variant, d[T(AGGGTGGGG)2AA], that was shown by NMR to form a stable

parallel conformation [35b]_{. Figure 2.6-C shows the CD spectra of DB832 with c-myc sequence.}

The absence of any exciton-type ICD signal indicates that the compound is not able to form stacked species due to the steric blockage of the grooves. This also suggests the preferential affinity of DB832 for the hybrid-type quadruplex scaffolds and the exciton-type splitting observed with the telomeric sequences is as a result of stacking molecules in the grooves.

The combined CD results of Tel22, Tel24, ODN9, ODN4 and c-myc strongly suggest the groove binding mode of DB832 with the human telomeric quadruplex sequence.

2.3.2 DB832 is the First Reported Quadruplex Groove Binder for Biologically Relevant