MECHANICAL STABILITY EVALUATION OF I-MOTIF AND G-QUADRUPLEX STRUCTURES UNDER DIVERSE CIRCUMSTANCES A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Soma Dhakal May 2013 i Dissertation written by Soma Dhakal B.S., Tribhuvan University, Nepal, 2001 M.S., Tribhuvan University, Nepal, 2003 Ph. D., Kent State University, USA, 2013 Approved by ___________________________________, Chair, Doctoral Dissertation Committee Hanbin Mao, Ph. D ___________________________________, Member, Doctoral Dissertation Committee Mietek Jaroniec, Ph. D ___________________________________, Member, Doctoral Dissertation Committee Soumitra Basu, Ph. D ___________________________________, Member, Doctoral Dissertation Committee Chanjoong Kim, Ph. D Accepted by ___________________________________, Chair, Dept. of Chemistry & Biochemistry Michael Tubergen, Ph. D __________________________________, Associate Dean, College of Arts and Sciences Raymond A. Craig, Ph. D ii TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................... vi LIST OF TABLES……………………………………………………………………...…x ACKNOWLEDGEMENTS………………………………………………………………xi Chapter 1 Introduction and background...…………………..………. ………..……….1 1.1 G-quadruplex ........................................................................................... 1 1.2 i-Motif ..................................................................................................... 3 1.3 G-quadruplex and i-motif in human ILPR .............................................. 5 1.4 G-quadruplex and i-motif in duplex DNA .............................................. 6 1.5 Biological roles of G-quadruplex and i-motif ......................................... 7 1.6 Material applications of G-quadruplex and i-motif ................................ 9 1.7 Laser Tweezers for single-molecule manipulation ............................... 11 2 Materials and methods.……………..…..…...…………………….…….….14 2.1 Materials and Methods .......................................................................... 14 2.1.1 DNA Samples .................................................................... …….14 2.1.2 DNA construct for ILPR i-motif and telomeric G-quadruplex in ssDNA ........................................................................................ .15 2.1.3 DNA construct for i-motif and G-quadruplex in ILPR duplex .. .18 2.1.4 Single-Molecule Experiment ..................................................... .19 iii 2.1.5 Calculation of change in contour length .................................... .21 2.1.6 Deconvolution of Populations.................................................... .21 2.1.7 Control Experiments for Single-Molecule Study…….………..22 2.1.8 Calculation of Percentage Formation............................. ……….22 2.1.9 Calculation of ∆Gunfold ............................................................... .22 2.1.10 CD Spectroscopy ..................................................................... .23 2.1.11 UV Spectroscopy …………… .…….……… . ….…….…..24 2.1.12 Preparation of 5' end radiolabeled DNA .................................. .24 2.1.13 Electrophoretic Mobility Shift Assay (EMSA)........................ .24 2.1.14 Bromine Footprinting of ssDNA ………………….….….…...25 2.1.15 DMS and Bromine footprinting of dsDNA ................. ………..26 3 Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single molecular level…...….......................................................................................……….29 3.1 Introduction .......................................................................................... 29 3.2 Results and Discussion ........................................................................ 33 3.3 Conclusions .......................................................................................... 48 4 Intramolecular folding in human ILPR fragment with three C-rich repeats………………………….……….......……………………….….….49 4.1 Introduction ........................................................................................... 49 4.2 Results ................................................................................................... 52 4.3 Discussion ............................................................................................. 69 4.4 Conclusions……………………………………………………………71 iv 5 G-quadruplex and i-motif are mutually exclusive in double-stranded ILPR DNA…………....……….......…………………………………….…….….73 5.1 Introduction ........................................................................................... 73 5.2 Results ................................................................................................... 74 5.3 Discussion ............................................................................................. 90 5.4 Conclusion…..……………………………….………………………..92 6 CONCLUDING REMARKS……...…..……...........….....………………….94 PERMISSION TO REUSE COPYRIGHTED MATERIAL…………...………….…….97 REFERENCES……………….……………………..……................................……….100 v LIST OF FIGURES Figure 1.1 Formation of G-quadruplex and its general types based on the strand orientation ……………………………………...………………………..…. 2 Figure 1.2 Formation of an i-motif structure by intercalation of C:CH + pairs.…….....….4 Figure 2.1 Schematic of the laser tweezers instrument…. …...…………………………. 15 Figure 2.2 Flow chart of making DNA constructs for single-molecule experiment…….17 Figure 2.3 Schematic representation of sample preparation and mechanical unfolding of DNA structures in single-molecule experiment …. …………………………. 20 Figure 2.4 A typical force-extension ( F-X) curve for the unfolding of i-motif structure at pH 5.5………………………………………………………………………...21 Figure 3.1 Schematic of i-motif structure and a typical force extension (F-X) curve……………………………………..….…...………………………….34 Figure 3.2 CD experiments and Br 2 footprinting of the ILPR C-rich sequence, 5'- (TGTCCCCACACCCC) 2TGT ……………………………………………..35 Figure 3.3 UV (295 nm) melting for the i-motif forming sequence 5'-(TGTCCCCACA CCCC) 2TGT…….………………………….……………………..………...36 Figure 3.4 All of the stable structures in the sequence 5'-(TGTCCCCACACCCC) 2TGT at 25 °C ………………………………..….…...…………………………...37 Figure 3.5 Calculation of number of nucleotides contained in a secondary structure in the sequence 5'-(TGTCCCCACACCCC) 2TGT ..…...………………………….39 vi Figure 3.6 A representative pulling curve with two ~5 nm change in contour length ( ∆L) unfolding events in the sequence 5'-(TGTCCCCACACCCC) 2TGT ……………………………………………. .…...…………………………...40 Figure 3.7 Histogram of change in contour length ( ∆L) of the DNA secondary structure formed in 5′-TGTC 4ACAC 4TGTC 4ACA .….…...………………………….41 Figure 3.8 ∆L histograms at different pH (23 oC) .…..….…...………………………….43 Figure 3.9 The percentage formation of the i-motif and the partially folded structure at different pH under 23 ºC..………………………….……………………….44 Figure 3.10 Rupture force histograms of partially folded structure and i-motif structures different pH under 23 oC.………………..….…...………………………….46 Figure 4.1 CD experiments of ILPR-I3 at different pH and temperature in a 10 mM sodium phosphate buffer with 100 mM KCl and 5 µM DNA concentration……………………………………………………………….54 Figure 4.2 Electrophoretic mobility shift assays (EMSA) and thermal melting/reannealing of the ILPR-I3…...………………………………….55 Figure 4.3 CD spectra of sequences described in Table 4.1………………..………….56 Figure 4.4 A typical force-extension ( F-X) curve obtained from the mechanical unfolding of the secondary structure in the ILPR-I3 at pH 5.5………….…………….58 Figure 4.5 Single-molecule study of the ILPR-I3 at different pH using laser tweezers ………………………………………………………………..…………….59 vii Figure 4.6 Four possible structures that employ C:CH + pair stacking in the ILPR-I3 sequence ……………………………………………….……..…………….60 Figure 4.7 Mutation analysis in a 10 mM sodium phosphate buffer (pH 5.5) with 100 mM KCl …………………………………………………………………….64 Figure 4.8 Formation of an intermolecular i-motif…………………………………….66 Figure 4.9 Intensity scan for ILPR-I3 bands and the fold protection for ILPR-I4 and ILPR-I3 for Br 2 footprinting in a 10 mM sodium phosphate buffer at pH 5.5 with 100 mM KCl………………………………………………………….67 Figure 4.10 Calculation of the unfolding rate constant ( kunfold ) at 0 pN for the intramolecular i-motif and the 45 pN population in the ILPR-I3/ILPR-I1 mixture…………………………………………………………….……….68 Figure 5.1 Chemical footprinting of a double-stranded 87-bp ILPR DNA at different pH and salt conditions at 23 °C. ……………………………..………………….75 Figure 5.2 Single-molecule investigation of G-quadruplex/i-moif in the double-stranded ILPR DNA ………………………………………………………….……….79 Figure 5.3 Calculation of the number of nucleotides ( N) involved in the tetraplex structures. …………………………………………………..…….………….81 Figure 5.4 Histogram of change in contour length (∆L) in a pH 7.4 Tris buffer with 100 mM Li + at 23 °C…………………………………………………..………….84 viii Figure 5.5 Kinetic analyses of G-quadruplex and i-motif in the double-stranded ILPR DNA at pH 5.5.………………………………………………..….………….87 Figure 5.6 Major species under different pH and ionic conditions…………………….90 ix LIST OF TABLES Table 3.1 Summary of change in contour length (∆L) , rupture force ( F), free energy change of unfolding ( ∆G ), and 286 nm CD melting temperature ( Tm) at pH 5.5-7.0…………………………………………………….………………….37 Table 4.1 Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study