Conformational Transitions in Thymidine Bulge-containing Deoxytridecanucleotide Duplexes ROLE OF FLANKING SEQUENCE AND TEMPERATURE IN MODULATING THE EQUILIBRIUM BETWEEN LOOPED OUT AND STACKED THYMIDINE BULGE STATES*

Structural features at extra thymidine bulge sites in DNA duplexes have been elucidated from a two-dimen- sional NMR analysis of through-bond and through- space connectivities in the otherwise self-complemen- tary d(C-C-G-T-G-A-A-T-T-C-C-G-G) (GTG 13-mer) and d(C-C-G-G-A-A-T-T-C-T-C-G-G) (CTC 13-mer) duplexes in aqueous solution. These studies establish that the extra thymidine flanked by guanosines in the GTG 13-mer duplex is in a conformational equilibrium between looped out and stacked states. The looped-out state is favored at low temperature (0 “C), whereas the equilibrium shifts in favor of the stacked state at ele- vated temperatures (35 “C) prior to the onset of the duplex-strand transition. By contrast, the extra thy- midine flanked by cytidines in the CTC 13-mer duplex is looped out independent of temperature in the duplex state. Our results demonstrate that temperature and flanking sequence modulate the equilibrium between looped-out and stacked conformations of single base thymidine bulges in DNA oligomer duplexes.


Structural
features at extra thymidine bulge sites in DNA duplexes have been elucidated from a two-dimensional NMR analysis of through-bond and throughspace connectivities in the otherwise self-complementary d
These studies establish that the extra thymidine flanked by guanosines in the GTG 13-mer duplex is in a conformational equilibrium between looped out and stacked states. The looped-out state is favored at low temperature (0 "C), whereas the equilibrium shifts in favor of the stacked state at elevated temperatures (35 "C) prior to the onset of the duplex-strand transition. By contrast, the extra thymidine flanked by cytidines in the CTC 13-mer duplex is looped out independent of temperature in the duplex state. Our results demonstrate that temperature and flanking sequence modulate the equilibrium between looped-out and stacked conformations of single base thymidine bulges in DNA oligomer duplexes.
Early optical and photochemical research on synthetic RNAs established that extra pyrimidines loop out into solution (l-3). The application of high resolution NMR techniques to nucleic acid oligomer duplexes has provided structural information at the individual base pair level in solution (4,5) and is readily applicable to pyrimidine bulge containing duplexes. The earliest NMR study established that an extra cytidine in an A-C-A segment loops out of a DNA oligomer duplex (6). These studies were extended to hydrogen-exchange NMR measurements to monitor the migration of a cytidine bulge along a (G)n. (C)n DNA mutational hot spot tract (7). More recent NMR studies on an extra cytidine in a G-C-G segment detected a temperature dependent confor-ma%onal equilibrium between looped-out (predominant at low temperature) and stacked (predominant at elevated temperature) states at the cytidine bulge site in the duplex state (8). NMR studies on thymidine/uridine bulges show additional interesting and unexpected conformational features when comparing DNA and RNA oligomers of the same sequence.
For identical sequences, the extra thymidine stacks into the DNA helix (9), whereas the extra uridine loops out of the RNA helix (10) at room temperature. It appears that the nature of the helix (B form for DNA and A form for RNA) shifts the equilibrium between stacked and looped out conformations.
Systematic footprinting studies have also been undertaken on bulges in the stem of RNA hairpins (ll), demonstrating that structural perturbations are propagated away from the bulge site.
We report below on an NMR study of thymidine bulges as a function of flanking sequence and temperature in selfcomplementary tridecanucleotide duplexes. This paper compares the structural features of thymidine bulges flanked by guanosines in the G-T-G segment of the otherwise self-complementary d(C-C-G-E-G-A-A-T-T-C-C-G-G) duplex (designated GTG 13-mer, Scheme 1) and of thymidine bulges flanked by cytidines in the C-T-C segment of the otherwise self-complementary d(C-C-G-G-A-A-T-T-C-E-C-G-G) duplex (designated CTC 13-mer, Scheme 2). The extra thymidine at the bulge site is designated TX in Schemes 1 and 2. Our results establish that the flanking residues play an important role in defining the conformation of pyrimidine bulge sites which contrasts with our earlier demonstration that the conformation of purine bulges are independent of flanking sequence in solution (12).  L  >   TX  /  '  C,-c2-G,  G,-AA-AA(-T,-T8-Co-C  IO-GI~-G,Z   G12-~,,-~,0-~O-~I-~7-~6-~6-~,,   TX' G,-C,-C,  The assignments of cross-peaks A to D in A are as follows: NOES are detected between the imino protons of adjacent T7 and T8 (peak A), adjacent G4 and T8 (peak B), adjacent G3 and G4 (peak C), and adjacent G3 and Gil (peak D). The chemical shift of the imino proton TX is indicated by a box on the diagonal. The assignments of cross-peaks A to Y in B are as follows: The 14.01-ppm imino proton of T8 exhibits NOE cross-peaks to the H2 proton of A5 (peak C) and A6 (peak A) and to the hydrogen-bonded amino proton of A5 (peak B). The 13.86-ppm imino proton of T7 exhibits NOE cross-peaks to the H2 protons of A6 (peak F) and A5 (peak C) and to the exposed amino protons of A5/A6 (peaks H/I). The 13.26-ppm imino proton of Gil exhibits NOE cross-peaks to the hydrogen-bonded (peak K) and exposed (peak L) amino protons and the H5 proton (peak M) of C2. The 12.97-ppm imino proton of G3 exhibits NOE cross-peaks to the hydrogen-bonded (peak N) and exposed (peak 0) amino protons and the H5 proton (peak P) of ClO. The 12.61-ppm imino proton of G4 exhibits NOE cross-peaks to the hydrogen-bonded (peak Q) and exposed (peak S) amino protons and the H5 proton (peak U) of C9 and to the H2 proton of A5 (peak R). Strong exchange cross peaks with the solvent HZ0 are detected for the imino protons of G12 (peak V) and TX (peak Y), whereas weak exchange cross-peaks are detected for the imino protons of G3 (peak W) and G4 (peak X).
observed within each base pair coupled with those NOES observed to protons of adjacent base pairs provides the specific resonance assignments (Table I) and cross-peak assignments (caption to Fig. 2). These exchangeable proton assignments were obtained following analysis of the NOESY spectrum of the GTG 13-mer duplex acquired in Hz0 solution.
An NOE cross-peak is detected between the imino protons of G3 and G4 (peak C, Fig. 2A) which flank the extra thymidine, TX, in the d(G3-TX-G4).d(CS-0) segment of the GTG 13-mer duplex at 0 "C. The imino proton of TX exchanges quite rapidly with the solvent during the 120-ms a 0.1 M NaCl, 10 InM phosphate, H20, pH 6.0. b Hydrogen-bonded amino protons. ' Exposed amino protons. d 0.1 M NaCl, 10 mM phosphate, D20, pH 6.5. mixing time as evidenced by the strong exchange cross-peak with H20 (peaiz Y, Fig. 2B) and the absence of a diagonal resonance (see box, Fig. 2A). The absence of an NOE between the imino proton of TX and the imino protons of G3 and G4 has been confirmed by one-dimensional NOE experiments, as is the presence of an NOE between the imino protons of G3 and G4 in the GTG 13-mer duplex. Both one-and twodimensional NOE experiments also establish that the CH, group of TX, like its imino proton, does not exhibit NOES to the imino proton of flanking G3.ClO and G4.C9 base pairs in the GTG 13-mer duplex at 5 "C. The exchangeable proton resonance assignments in the GTG 13-mer duplex in Hz0 buffer, pH 6.0 at 0 "C are listed in Table I.
Once the imino proton assignments have been determined, the effects of temperature and pH at the individual base pair level can be monitored through the imino proton chemical shifts and line widths. Specifically, the imino protons of TX and the terminal Cl. G12 base pair broaden out above 20 "C, followed by the onset of broadening of the imino protons of the C2.Gll and G3.ClO base pairs by 40 "C in the GTG 13mer duplex at pH 6.34 (Fig. LA). The temperature dependence of the imino proton chemical shifts of TX, G3, and G4 centered about the bulge site in the GTG 13-mer duplex are plotted in Fig. SlA (see Miniprint Section).3 The imino proton of TX broadens significantly on raising the pH from 5.74 to 7.82, whereas the imino protons of the base pairs are unaffected for the GTG 13-mer duplex at -5 "C (Fig. 1B).
Two-dimensional NOESY spectra of the GTG 13-mer in Hz0 were also recorded at ambient temperature.
The rapid imino proton exchange with Hz0 successfully competes with 3 Portions of this paper (including Figs. SlLS4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
the NOE at this temperature thereby reducing or eliminating the intensity of many of the cross-peaks preventing any fruitful analysis.
Nonexchangeable Protons-The nonexchangeable protons in the GTG 13-mer duplex have been monitored in NOESY (250 ms mixing time) spectra recorded in D20 buffer, pH 6.5 between 5 and 33 "C. An expanded region of the NOESY spectrum establishing distance connectivities between the base protons (7.0-8.4 ppm) and the sugar Hl' and cytidine H5 protons (5.2-6.4 ppm) at 5 "C! is contour-plotted in Fig.   3A. Cross-peaks are detected between the base (purine H8 or pyrimidine H6) protons and their own and 5'-sugar Hl' protons, and the observed directionality (21) permits the chain to be traced from Cl to Gl2. Several breaks in the connectivities occur where NOES are absent between the H6 of TX and the Hl' of G3, between the H8 of G4 and the Hl' of TX, and between the H8 of Gil and the Hl' of Cl0 in the 5 "C data set (Fig. 3A). These breaks are detected primarily at the G3-TX-G4 segment centered about the thymidine bulge site. The same expanded NOESY contour plot for GTG 13mer data at 33 "C is shown in Fig. 3B. The breaks detected in the connectivities at 5 "C are retained at 33 "C with additional disruptions corresponding to the absence of NOES between the H8 of G3 and the Hl' of C2 and also between the H6 of C2 and the Hl' of Cl at the higher temperature ( Fig. 3B).
Severe spectral overlap prevents the monitoring of the potential NOE between the H6 of C9 and the H5 of Cl0 within the C9-Cl0 step opposite the bulge site in the GTG 13-mer duplex recorded at 5 "C ( Fig. 3A) and 33 "C ( Fig. 3B) DzO, pH 6.5, at 5 "C (A) and 33 "C (B). The cross-peaks between the H6 and H5 protons of cytidine are designated by asterisks. The labeled NOE cross-peaks are listed below. A, Cl(H6) to C2(H5); B, T8(H6) to C9(H5); C, A6(H2) to T7(Hl'); D, A6(H2) to T8(Hl'); E, A5(H2) to C9(Hl'); The regions of the NOESY spectrum (250 ms mixing time) establishing the distance connectivities between the base protons and the sugar H2', 2" protons similarly lack NOES between the H6 proton of TX and the H2',2" protons of G3 and between the H8 protons of G4 and the H2',2" protons of TX in the G3-TX-G4 segment at 5 "C ( Fig. 4A) and 33 "C ( Fig. 4B). NOES are also absent between the H6 proton of TX and the H8 protons of either G3 or G4 at both 5 and 33 "C.
An important difference is detected between the NOESY spectra of the GTG 13-mer duplex recorded at 5 and 33 "C. An NOE is observed between the H8 of G4 and the Hl' of G3 in the G3-TX-G4 segment at 5 "C (peak G, Fig. 3A) which is absent in the 33 "C spectrum (Fig. 3B).
The NOESY data sets have also been recorded at short mixing time (50 ms) on the GTG 13-mer duplex at 5 and 33 "C. The quality of the short mixing time NOESY data sets are marginal at 33 "C compared with their counterparts at 5 "C. Expanded regions establishing distance connectivities from the base protons to the sugar Hl' protons and to the sugar H2',2" protons are plotted in Figs. S2A and S3A, respectively (Miniprint Section) for the 5 "C data set. The sugar Hl', H2',2", H3', and H4' protons within each nucleotide in the GTG 13-mer duplex can be linked via bond connectivities in correlated two-dimensional data sets. The expanded region of the COSY spectrum establishing the coupling connectivities between sugar Hl' (5.2-6.4 ppm) and sugar H2',2" (1.6-3.2 ppm) protons in the GTG 13-mer duplex in D20 at 25 "C is plotted in Fig. 5A. The Hl'-H2' cross-peaks are upfield of the Hl'-H2" cross-peaks except for an inversion at G3 and terminal G12 residues An expanded region of the magnitude RELAY COSY spectrum of the GTG 13-mer duplex in DzO buffer at 25 "C is plotted in Fig. 5B. The cross-peaks grouped on the left side correspond to the four-bond relay connectivities between the sugar Hl' and H3' protons, whereas the cross-peaks grouped on the right side corresponding to the three-bond connectivities between the sugar H3' and H4' protons (Fig. 5B). We do not detect a four bond relay cross-peak between the Hl' and H4' of C9 nor do we detect a three-bond cross-peak between the H3' and H4' of the same residue in the GTG 13-mer duplex at 25 "C. The above analysis of the NOESY, COSY, and RELAY COSY data sets of the GTG 13-mer duplex in DzO at 25 "C yield the nonexchangeable base and sugar proton chemical shifts listed in Table I.

Phosphorus
Spectra-The phosphorus spectrum of the GTG 13-mer in D20 buffer at 25 "C is plotted in Fig. 6A. The majority of the phosphorus resonances are dispersed between -3.9 and -4.4 ppm with one phosphorus resonating downfield at -3.77 ppm. The phosphorus spectrum has been assigned by correlating the phosphorus resonances to the known sugar H3' and H2',H2" proton resonances in a phosphorus (observed)-proton COSY experiment. The contour plot (positive levels only) is shown in Fig. 6B and permits a complete assignment of the phosphorus spectrum of the GTG 13-mer duplex (Table II). A slice taken through the -3.77 ppm phosphorus resonance exhibits through bond correlations to the H2', H3', and H4' protons of G3 and the H4' proton of TX (Fig. 6C) permitting assignment to the GB-TX step.

CTC 13-mer Duplex
Exchangeable Protons-The imino proton spectra of the CTC 13-mer duplex in Hz0 buffer as a function of temperature at pH 6.42 is plotted in Fig. 7A and as a function of pH at -5 "C is plotted in Fig. 7B. The base-paired imino protons have been assigned following an analysis of the NOESY (120 ms mixing time) spectrum of the CTC 13-mer duplex in Hz0 buffer, pH 6.0, at 0 "C (  imino proton of G4.C9 resonates upfield at 12.53 ppm. An NOE is detected between the superpositioned imino protons at 13.15 ppm and the 12.53-ppm imino proton of G4 (peak C, Fig. 8B The exchangeable proton chemical shifts in the CTC 13mer duplex in Hz0 buffer, pH 6.0, at 0 "C are listed in Table  III. The imino proton of the extra thymidine in the CTC 13mer duplex is sensitive to both increase in temperature (Fig.  7A) and to increase in pH (Fig. 7B). The temperature dependence of the imino proton chemical shifts of TX, G3, and G4 centered about the bulge site in the CTC 13-mer duplex are plotted in Fig. SlB (

see Miniprint Section). Nonexchangeable
Protons-Two-dimensional NOESY (250 ms mixing time) spectra have been recorded on the CTC 13mer duplex in D20 buffer, pH 6.2, between 5 and 25 "C. Expanded regions of the NOESY spectrum plot establishing distance connectivities between base protons and sugar Hl' and cytidine H5 protons at 5 "C is plotted in Fig. 9A and at 25 "C! is plotted in Fig. 9B. The chain can be traced from Cl to G12 by monitoring the NOES between the base protons and their own and 5'-flanking sugar Hl' protons in the CTC 13-mer duplex at 5 "C ( Fig. 9A) and at 25 "C (Fig. 9B). Disruptions occur in the tracing at the CS-TX-Cl0 segment, since NOES are absent between the H6 of TX and the Hl' of C9 and between the H6 of Cl0 and the Hl' of TX at both 5 "C ( Fig. 9A) and 25 "C (Fig. 9B). An NOE is detected between the H5 proton of Cl0 and the H2',2" protons of C9 in the CS-TX-Cl0 segment of the CTC 13-mer duplex at 25 "C, whereas no conclusion can be made due to cross-peak overlaps at 5 "C.
The regions of the NOESY spectrum (250 ms mixing time) establishing distance connectivities between the base protons and the sugar H2',2" protons in the CTC 13-mer duplex at 5 and 25 "C are plotted in Fig. 10, A and B, respectively. Cross-peak overlaps have prevented us from monitoring NOES between base protons and their 5'-flanking sugar H2',2" protons in the (G3-G4). (CS-TX-ClO) segment centered about the bulge site at both 5 and 25 "C.
The NOESY data sets have also been recorded at short mixing times (50 ms) on the CTC 13-mer duplex at 5 and 25 "C. The short mixing time experimental data recorded at low temperature was of better quality than the corresponding NOES are detected between the imino protons of adjacent T7 and T8 (peak A), adjacent G4 and T8 (peak B), and adjacent G3 and G4 (peak C). The chemical shift of the imino proton of TX is indicated by a box on the diagonal.
The assignments of cross-peaks A-Y in B are as follows: the 13.93-ppm imino proton of T8 exhibits NOE cross-peaks to the H2 protons of A5 (peak C) and A6 (peak A) and to the hydrogenbonded amino protons of A5 (peak C) and A6 (peak 23) and exposed amino protons of A5 (peak E) and A6 (peak D). The 13.67-ppm imino proton of T7 exhibits NOE cross-peaks to the H2 proton of A6 (peak F) and A5 (peak G) and to the hydrogen-bonded amino protons of A5 (peak G) and A6 (peak H) and exposed amino protons of A5 (peak J) and A6 (peak I). The 13.15-ppm imino proton of Gil exhibits NOE cross-peaks to the hydrogen-bonded (peak K) and exposed (peak L) amino protons and the H5 proton (peak M) of C2.
The 13.11-ppm imino proton of G3 exhibits NOE cross-peaks to the hydrogen-bonded (peak N) and exposed (peak 0) amino protons and the H5 proton (peak P) of ClO. The 12.53-ppm imino proton of G4 exhibits NOE cross-peaks to the hydrogen-bonded (peak Q) and exposed (peak S) amino protons of C9 and to the H2 proton of A5 (peak R). The cross-peak T may correspond to an NOE between imino and amino protons of G4. Strong exchange cross-peaks with the solvent Hz0 are detected for the imino protons of G12 (peak V) and TX (peak Y), whereas a weak exchange cross-peak is detected for the imino protons of G4 (peak X).
data at ambient temperature. Expanded regions establishing distance connectivities from the base protons to the sugar Hl' protons and to the sugar H2', 2" protons are plotted in Figs. S2B and S3B, respectively (Miniprint Section) for the 5 "C data set.
The sugar proton connectivities in the CTC 13-mer duplex in DzO buffer, pH 6.2, at 25 "C have been identified following an analysis of the magnitude COSY (Fig. 1lA) and the mag- nitude RELAY COSY ( Figure 11B) spectra. The coupling connectivities between the sugar Hl' and sugar H2',2" protons in the COSY spectrum of the CTC 13-mer duplex are plotted in Fig. 11A. The H2',2" protons are superpositioned for C9, whereas the H2',2" protons are separated by 0.77 ppm and shifted to high field for Cl0 in the CTC 13-mer duplex. We do not detect four bond Hl'-H3' connectivities for G3, G4, and Gil in the Relay COSY spectrum of the CTC 13-mer duplex at 25 "C (Fig. 11B).
The above analysis of the through-space and through-bond connectivities in the CTC 13-mer duplex in D20 at 25 "C yield the nonexchangeable proton chemical shift assignments listed in Table III. Phosphorus Spectra-The phosphorus resonances in the CTC 13-mer duplex in D20 buffer at 25 "C ( Fig. 12A) have been assigned by monitoring the coupling connectivities in the phosphorus (observed)-proton COSY contour plot recorded in Fig. 12B (Table IV). The phosphorus resonance to lowest field is assigned to the CS-TX step, and a slice through this -3.73 ppm resonance exhibits coupling connectivities to the H2',2", H3', and H4' protons of C9 and the H4' protons of TX in the CTC 13-mer duplex (Fig. 12C).

Sequence-dependent Parameters at Thymidine Bulge Sites
Melting Transition-The helix-to-strand transition of the GTG 13-mer and CTC 13-mer duplexes in 0.1 M NaCl, 10 mM phosphate, D20 can be monitored by measuring the chemical shift of the CH, resonance of T8 between 5 and 80 "C (Fig. S4,  The cross-peaks between the H6 and CH, protons of thymidine are designated by asterisk. The labeled crosspeaks correspond to NOES between base and their own sugar H2',2" protons in the (G3-G4).
T8 in the GTG 13-mer and CTC 13-mer duplexes in 0.1 M NaCl, 10 mM phosphate, DzO between 5 and 70 "C are plotted in Fig. 13, A and B, respectively. The CH3 protons of T7 and T8 in the GTG 13-mer and CTC 13-mer undergo upfield shifts upon lowering the temperature from 70 to 45 "C asso- ciated with the strand to duplex transition and then exhibit temperature-independent chemical shifts between 40 and 5 "C in the duplex state (Fig. 13).
The CHB protons of TX in the GTG 13-mer shift upfield on lowering the temperature from 70 to 40 "C but then shift dramatically downfield on lowering the temperature from 40 to 5 "C (Fig. 13A). By contrast, the CHs protons of TX in the CTC 13-mer exhibit a temperature-independent chemical shift between 70 and 5 "C! (Fig. 13B). DISCUSSION Our previous NMR study focused on single adenosine bulges in the GAG 13-mer and the CAC 13-mer duplexes which concluded that the extra adenosine stacks into the duplex independent of temperature and flanking sequence (12). The current study focuses on the role of flanking sequence and temperature on the conformation of thymidine bulge sites in the GTG 13-mer and CTC 13-mer duplexes. The bases flanking the thymidine bulge were chosen to be either both purines or both pyrimidines to cover the two extremes.
Sequence-dependent Stability of the Duplex-The CH3 proton of T8 located towards the center of the GTG 13-mer and CTC 13-mer duplexes provides a convenient monitor of the relative stabilities of the two self-complementary duplexes through the helix-coil transition.
The GTG 13-mer duplex (T, = 47.5 "C) is more stable than the CTC 13-mer duplex (!I',,, = 42.5 "C) in the 0.1 M NaCl solution (Fig. S4)   is observed in the temperature-dependent line width behavior of the imino protons, where the imino protons of the CTC 13mer duplex, pH 6.42 (Fig. 7A), are broadened to a greater extent at a given elevated temperature than the same imino protons of the GTG 13-mer duplex, pH 6.34 (Fig. IA).
The greater stability of the GTG 13-mer relative to the CTC 13-mer duplex parallels the earlier result of greater stability of the GAG 13-mer relative to the CAC 13-mer duplex (12). Thus, purines flanking an extra base are more stabilizing than pyrimidines flanking an extra base in the interior of DNA oligomer duplexes.

Conformational Equilibria
in Duplex State-The temperature dependence of the chemical shift of the CH, protons of TX in the temperature range (5-40 "C) prior to the onset of the melting transition provides an excellent marker for monitoring the potential changes in the conformational equilibria at the bulge site in the duplex state. The CH, proton of TX in the GTG 13-mer duplex exhibits a chemical shift at 5 "C which is very close to its strand value (Fig. 13A) indicating that the extra thymidine is looped out of the helix. By contrast, this CH, resonance shifts upfield by 0.2 ppm on raising the temperature to 35 "C ( Fig. 13A), indicating that the extra thymidine is stacked into the GTG 13-mer duplex and experiencing the ring current contributions to the chemical shift from the flanking GC pairs. The H6 proton of TX similarly shifts upfield on raising the temperature from 5 "C ( Fig. 3A) to 33 "C ( Fig. 3B) in the NOESY spectra of the GTG 13-mer duplex. These results indicate that the extra thymidine equilibrates between a looped-out conformation predominant at low temperature (5 "C) and a stacked conformation predominant at elevated temperatures (40 "C) in the GTG 13-mer duplex prior to the onset of the melting transition.
In striking contrast, the CH, protons of TX in the CTC 13mer duplex exhibit an essentially temperature-independent chemical shift between 5 and 65 "C (Fig. 13B). Thus, the similarity in the chemical shift of the CH3 protons of TX in the strand state (65 "C) and the duplex state (5-35 "C) indicates that TX is looped-out both at low (5 "C) and elevated (35 "C) temperatures prior to the onset of the melting transition. This conclusion is further supported by the identical chemical shift of the H6 proton of TX at 5 "C ( Fig. 9A) and at 25 "C ( Fig. 9B) in the expanded NOESY spectra of the CTC I3-mer duplex.
The results of these studies on the GTG 13-mer and CTC 13-mer duplexes establish the importance of flanking sequence and temperature in defining the conformation at thymidine bulge sites. A previous NMR study on an extra thymidine located in a G-T-G segment established that this thymidine stacked into the DNA oligomer duplex at ambient temperature (9). Our research predicts that this thymidine will loop out of the duplex if the studies (9) are extended to low temperature.

Right-handed
Helices with Watson-Crick Pairing-The local and global features of the GTG 13-mer and CTC 13-mer duplexes are reflected in the observed NOE and chemical shift parameters. The NOES detected between thymidine imino and adenosine H2 protons establish Watson-Crick A. T pairing, whereas the observed NOES between guanosine imino and cytidine amino protons establish Watson-Crick G. C pairing in the extra thymidine 13-mer duplexes (Figs. 2B and 8B). Furthermore, intact G3.ClO and G4.C9 base pairs are detected flanking the extra thymidine sites for the GTG 13-mer and CTC 13-mer duplexes (Figs. 1 and 6).
These results establish anti-glycosidic torsion angles with Watson-Crick pairing for all base pairs along the entire length of right-handed GTG 13-mer and CTC 13-mer duplexes.
Thymidine Imino Proton Exchange-We observe faster exchange rates at the imino proton of the extra thymidine compared with the thymidines paired with adenosines in A. T base pairs in the GTG 13-mer and the CTC 13-mer duplexes at low temperature. This is reflected in the observed increase in line width of the extra thymidine imino proton with increasing pH in both the GTG 13-mer duplex (Fig. lA) and the CTC 13-mer duplex (Fig. 7B) at -5 "C. Further support for this conclusion follows from the absence of a diagonal peak (boxed region) and the presence of an exchange crosspeak with solvent HZ0 (peak Y) for the extra thymidine imino proton in NOESY plots of the GTG 13-mer duplex (Fig. 2) and the CTC 13-mer duplex (Fig. 8) at 0 "C. The absence of the diagonal peak reflects complete chemical exchange of the thymidine imino proton with solvent HZ0 during the 120-ms mixing time period of the NOESY experiment. Hydrogen exchange studies on thymidine imino protons have established previously that exchange lifetimes for internal A. T pairs are on the order of 250 ms at pH 6.5 and 0 "C (22,23). Thus, the thymidine imino protons in A. T base pairs are observable along the diagonal in the 120-ms mixing time NOESY spectra of the two duplexes, whereas the extra thymidine imino proton whose exchange lifetime is much shorter than 120 ms is not detected.

Thymidine
Bulge Site in the GTG 13-mer Duplex-The temperature-dependent chemical shift data of the CH3 protons of TX in the GTG 13-mer indicate a predominant loopedout conformation for TX at 5 "C and a predominant stacked conformation for TX at 35 "C! with rapid equilibrium between these forms at intermediate temperature values. The thymidine CH3 of TX shifts as a narrow average resonance over the 0.2-ppm chemical shift separation so that the exchange rate of the extra thymidine between the looped-out and stacked states is much greater than 600 s-i [k >>2z (As)].
Both conformations exhibit interruptions in the NOE connectivities between base protons and their 5'-flanking sugar Hl' protons in the G3-TX and TX-G4 steps in the GTG 13mer duplex (Fig. 3, A and B). Furthermore, both conformations show no NOE between the H8 proton of G3 and the H5 proton of TX in the G3-TX step. The absence of these NOES characteristic of regular right-handed duplexes must reflect the disruption of the helix by the TX bulge site and are independent of whether TX is looped out at low temperature or stacked in at elevated temperature.
The NOES between imino protons can only be followed at low temperature, conditions under which the NOE competes effectively against hydrogen exchange. Specifically, the observed NOE between the G3 and G4 imino protons (peak C, Fig. 2A) and the absence of NOES between the thymidine imino/CHB protons and the flanking G3 and G4 imino protons establish that the extra thymidine TX is looped out and the G3 .ClO and G4.C9 base pairs stack on each other in the GTG 13-mer duplex at low temperature.
The observed NOE between the H8 of G4 and the Hl' of G3 at 5 "C (peak G, Fig. 3A) supports further the stacking of the G3. Cl0 and G4. C9 base pairs in the TX looped-out conformation of the GTG 13-mer duplex. By contrast, the absence of this NOE at 33 "C ( Fig. 3B) reflects the conformational change from a looped out TX at 5 "C to a stacked TX at 33 "C in the GTG 13-mer duplex.
The NOE cross-peak between the H6 and Hl' protons of TX is much stronger than all other base to their own Hl' NOE cross-peaks in the expanded 50-ms mixing time NOESY plot of the GTG 13-mer duplex at 5 "C! (Fig. S2A). Measurements of volume integrals establishes that the intensity under the H6-Hl' crosspeak of TX is 0.35 that under the H6-H5 of cytidine (2.45 A fixed distance) which translates into an interproton distance between H6 and Hl' of TX of 2.9 A. This calculation is based on the inverse sixth power interproton dependence of the NOE under conditions of the two-spin approximation and the assumption that the correlation times of the measured and standard interproton vectors are similar.
Thymidine Bulge-containing Deoxytridecanucleotide Duplexes Summary-The present NMR studies on the role of flanking sequence and temperature on the conformation of thymidine bulge sites contrasts dramatically with related studies on the conformation of adenosine bulge sites at the DNA oligomer level in aqueous solution. The earlier studies established that an extra adenosine stacks into the duplex independent of flanking sequence and temperature in aqueous solution (12). By contrasts a greater conformational variability is detected for pyrimidine bulge sites. The present NMR study on DNA oligomers demonstrates that for extra thymidines flanked by purines, the pyrimidine base is looped out at low temperature but stacks into the duplex at elevated temperature prior to the onset of the melting transition. By contrast, extra thymidines flanked by pyrimidines are looped out independent of temperature in the duplex state. These observations and the demonstration of a subtle balance between a stacked extra thymidine in B-DNA (9) and a looped out extra uridine in A-RNA (10) at room temperature establish that a range of parameters modulate the equilibrium between stacked and looped states at pyrimidine bulge sites in solution.