Regulation of Nucleic Acid Structure and Function with Peptoid, Small Molecules and Bpna(+)

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Regulation of nucleic acid structure and function with peptoid, small molecules and

bPNA(+)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Jie Mao

Graduate Program in Chemistry

The Ohio State University

2017

Dissertation Committee:

Dr. Dennis Bong, Advisor

Dr. Venkat Gopalan

Dr. Psaras McGrier

Copyrighted by

Jie Mao

2017

Abstract

Nucleic acids are not only central to the storage and expression of genomic information, but also key to many biological activities. Much effort has been dedicated to the research of molecular recognition of nucleic acids, to understand their structure and function, as well as possibly developing methods to regulate them. We have previously demonstrated that triaminotriazine

(melamine, M*) moieties installed on peptide and polyacrylate backbones could trigger the formation of non-native secondary structures from unfolded T/U rich nucleic acids due to the

Janus-Wedge type hydrogen bonding pattern between thymine/uracil and melamine. In this dissertation, we developed novel melamine displaying peptoids, small molecules and a new type of peptide called bPNA(+), all of which could trigger folding of T/U rich nucleic acids into defined triplex structures.

In Chapter 2, we demonstrated the synthesis of melamine displaying peptoids, which are analogous to bPNAs. These peptoids could bind with oligo T DNAs, supported by CD spectra changes and corporative melting of the peptoid-DNA complexes. The backbone change from α-

PNA (bPNA) to peptoid decreased the thermal stability of the complex, possibly due to the lack of backbone hydrogen bonding and the elimination of chiral centers.

In Chapter 3, we reported that small molecule t3M, which was synthesized via one step arylation from tris(2-aminoethyl) amine (tren), could induce folding of oligo T DNAs. Further analysis ii showed that only two out the three melamines are needed to form hydrogen bonds with T rich

DNA, thus creating the minimal binding unit t2M. It binds to a tetrathymidine bulge in DNA duplex with micromolar affinity. Functionalization of the tren backbone with t2M creates dendritic small molecule t4M, which exhibits much increased affinity towards oligo T buldges

(50 nM) due to multivalency. These small molecules could act as allosteric switches for nucleic acid. Hammerhead ribozymes whose stem and/or loop regions are replaced with oligo U could refold in the presence of t4M, self-cleavage reactions are then initiated due to the restoration of both secondary and tertiary interactions. We also reported for the first time, the regulation of nucleic acid functions using small molecule as a molecular bridge, by mimicking crucial native tertiary interaction. This regulation pathway is similar with the chemically induced dimerization in protein regulation. The facile and general method describe here could be used for installation of t2M motif on any primary amine, thus stimulating the design of synthetic allosteric riboswitches and small molecule-nucleic acid complexes.

In Chapter 4, we descried the selective fluorescence turn on of Spinach aptamer mutants and native human telomer G quadruplex with synthetic small molecules. Covalent linking of the triplex triggering small molecules melamine and fluorogenic moieties DFHBI yielded ligands that selectively turn on fluorescence of the U rich mutants of Spinach aptamer due to potential formation of U-M*-U triplex. Due to the structural similarities of Spinach and Mango (turns on thiazole orange fluorescence) aptamers, namely, both are composed of G quadruplexes in the fluorogenic dye binding interface, we created new aptamers for thiazole orange (TO) and its derivatives with melamine and t2M by rational redesign of Spinach aptamer, suggesting a new way of obtaining aptamers, through de novo designed instead of in vitro selection. t2M

iii conjugates of TO was also found to selectively turn on human telomere core sequence

(GGGTTA)3GGG, due to the possible triplex formation with the TTA loop region. Further optimizations and applications are currently underway.

In Chapter 5, we synthesized a new class of peptide bPNA(+) by reductive alkylation on the amine side chains of native peptides, creating t2M motifs. The display of t2M motifs instead of single melamine in bPNA decreases the total chain length of the peptide, thus reducing the entropic cost when hybridizing with nucleic acids. We observed an increase affinity in binding with T rich DNAs and much higher thermal stability of the complex. Nucleic acid imaging and probing of structural change could be achieved thanks to this affinity enhancement.

In summary, we demonstrated in this dissertation novel synthetic small molecules and oligomers that could trigger the folding of oligo T/U rich nucleic acids. The structural change induced by these melamine displaying compounds turns on nucleic acid functions such as hammerhead ribozyme catalysis and aptamer fluorescence turn on. Further applications such as nucleic acid packaging, delivery and labeling with the synthetic molecules discussed in this dissertation are currently underway.

Acknowledgments

I would like to express my sincere gratitude to my advisor, Dr. Dennis Bong, for his intelligence, motivation and patience as a mentor. His guidance over several exciting projects has helped me not only in learning new knowledge, but also in developing problem solving abilities. His thorough and careful attitude, together with his enthusiasm and persistence in research has inspired me and will continue beyond graduate school.

I would also like to thank Drs. Venkat Gopalan and Psaras McGrier for serving on my committee, providing constructive and encouraging advice.

Many thanks to the Bong group members, past and current, for creating a great environment to work in, for the helpful discussions and late night hangouts.

Last by, I would like to thank my parents, father Huajun Mao and mother Shuqin Tao, for their endless support and unconditional love throughout the years that I was away from home.

Vita

2004 to 2007 ...... Wuhu No.1 High School, Wuhu, China

2007 to 2011 ...... B.S. Chemistry, Tsinghua University, China

2011 to 2017 ...... PhD. Chemistry, Department of Chemistry,

The Ohio State University

Publications

Mao, J. and Bong, D*. (2015) “Synthesis of DNA-Binding Peptoids.” Synlett., 26, 1581-

1585.

Piao, X.; Xia, X.; Mao, J. and Bong, D*. (2015) “Peptide ligation and RNA cleavage via an abiotic interface.” J. Am. Chem. Soc., 137, 3751-3754.

Mao, J.; DeSantis, C. and Bong, D*. (2017) “Small molecule recognition triggers secondary and tertiary interactions in DNA folding and hammerhead ribozyme catalysis.”

J. Am. Chem. Soc., DOI: 10.1021/jacs.7b05448.

Fields of Study

Major Field: Chemistry

Table of Content

Abstract ...... ii

Acknowledgments...... ii

Vita ...... ii

Table of Content ...... iii

List of Tables ...... viii

List of Figures ...... ix

Chapter 1 : Structure and recognition of nucleic acids ...... 1

1.1 Native nucleic acid structures ...... 2

1.1.1 DNA duplex ...... 3

1.1.2 G quadruplex ...... 3

1.1.3 RNA secondary structure ...... 4

1.2 Triple helical nucleic acids ...... 5

1.3 Peptide nucleic acid (PNA) for nucleic acid recognition ...... 6

1.4 Triplex forming artificial nucleobases ...... 8

1.5 PNA with native α peptide backbone ...... 10

iii

1.6 Melamine displaying bifacial peptide nucleic acid (bPNA) as allosteric switch 11

1.7 Small molecule nucleic acid binders ...... 13

1.7.1 B-form DNA minor groove binders ...... 13

1.7.2 G-quadruplex binders...... 14

1.7.3 Mismatch sites targeting small molecules ...... 17

1.7.4 Small molecule RNA binders, aptamers, riboswitches ...... 18

1.8 Small molecule induced dimerization ...... 24

1.9 References...... 26

Chapter 2 : DNA binding peptoids ...... 47

2.1 Triazines and DNA molecular recognition ...... 48

2.2 Synthesis of DNA-binding peptoids ...... 49

2.3 Peptoid-DNA binding studies ...... 52

2.4 Synthesis of monomers ...... 57

2.5 Peptoid synthesis and purification ...... 58

2.6 UV-melting...... 59

2.7 Circular Dichrosim (CD) spectroscopy ...... 59

2.8 Compound characterization...... 59

2.9 References...... 63

iii

Chapter 3 : Small molecule recognition triggers secondary and tertiary interactions in

DNA folding and hammerhead catalysis ...... 69

3.1 Overview ...... 70

3.2 A minimal binding motif for T/U rich nucleic acids ...... 70

3.3 Extension of t2M motif: t4M ...... 74

3.4 Quaternary t3M : qt3M ...... 75

3.5 Ribozyme rescue by small molecule triggers ...... 75

3.6 Conclusion ...... 79

3.7 Materials and general experimental procedures ...... 79

3.7.1 Nucleic acid sequences used ...... 80

3.7.2 UV-melting ...... 83

3.7.3 Circular Dichroism(CD) spectroscopy...... 84

3.7.4 Isothermal Titration Calorimetry (ITC)...... 84

3.7.5 Differential Scanning Calorimetry (DSC)...... 84

3.7.6 Fluorescence quenching assay...... 85

3.7.7 Fluorescence anisotropy...... 85

3.7.8 Electrophoretic mobility assay (EMSA) ...... 86

3.7.9 Additional scaffolds tested for DNA binding...... 87

3.8 Additional data ...... 89

3.9 Synthesis...... 94

3.10 Ribozyme cleavage data ...... 107

3.10.1 U-(2,3) ribozyme...... 107

3.10.2 S-U4n ribozyme ...... 110

3.10.3 Binary ribozyme...... 113

3.11 Compound characterization...... 117

3.12 References...... 133

Chapter 4 : Small molecule G quadruplex sensor ...... 141

4.1 G quadruplex in aptamers ...... 142

4.2 Selective imaging of G quadruplexes...... 143

4.3 Engineering the Spinach aptamers ...... 144

4.4 DFHBI derivatives with modified Spinach aptamers ...... 145

4.5 Thiazole orange with modified Spinach aptamers ...... 147

4.6 De novo design of G quadruplex containing aptamer for selective targeting. . 152

4.7 Conclusions...... 153

4.8 Compound preparations...... 154

4.9 Fluorescence turn on experiment...... 160

4.10 Apparent Kd determination...... 160

4.11 References...... 162

Chapter 5 : bPNA(+) ...... 165

5.1 Minimal T-T/U-U recognition motif: t2M ...... 166

5.2 Integration of t2M motif on peptide backbone: bPNA(+)...... 166

5.3 Biophysical study of bPNA(+) ...... 167

5.4 Applications of bPNA(+) ...... 172

5.5 Synthesis and characterization of bPNA(+) ...... 174

5.5.1 Monomer synthesis...... 174

5.5.2 Typical procedure for synthesis of bPNA(+)...... 176

5.5.3 Synthesis and characterization of bPNA(+) ...... 177

5.6 General experimental procedures ...... 186

5.6.1 Materials and general experimental procedures...... 186

5.6.2 UV-melting ...... 186

5.6.3 Differential Scanning Calorimetry (DSC)...... 187

5.6.4 Fluorescence anisotropy...... 187

5.6.5 Electrophoretic mobility shift assay (EMSA)...... 188

5.6.6 Strand invasion...... 188

5.7 Additional UV-melting data ...... 189

5.8 Kd fitting from fluorescence anisotropy ...... 189

5.9 References...... 191

References ...... 195

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List of Tables

Table 2.1. Thermal Stability of peptoid-DNA complexesa ...... 55

Table 3.1 DNA complexation dataa...... 88

Table 3.2 Summary of tren derivatives DNA binding data from DSC...... 89

Table 3.3 Summary of cleavage rates of binary ribozymes ...... 116

Table 4.1. Summary of DFHBI derivatives tested...... 146

Table 4.2. Summary of TO derivatives tested...... 148

Table 5.1. DNAa hydridization data for bPNAb and bPNA (+) ...... 172

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List of Figures

Figure 1.1. Chemical structures of nucleic acids. Figure is adopted from reference1...... 2

Figure 1.2. Three forms of DNA duplexes. Figure is adapted from references3-4...... 3

Figure 1.3. (A) G-quartet structure, M+ indicates monovalent ions such as K+ and Na+.

(B) Intermolecular G-quadruplexes. (C). Different forms of intramolecular G- quadruplexes. Figure is reproduced from reference14...... 4

Figure 1.4. Common RNA secondary and tertiary structures. Figure is reproduced from reference20 ...... 5

Figure 1.5. Top: Watson-Crick base pairing; Bottom: Base triplets TAT and C+GC.

Reference is reproduced from reference28...... 6

Figure 1.6. Top: Chemical structure comparison between protein, PNA and DNA.

Bottom: PNA binding mode. Figure from reference32...... 7

Figure 1.7. Artificial nucleobases that can form triplexes. Adapted from reference43...... 8

Figure 1.8. Janus-Wedge type triplex formation45, 47...... 9

Figure 1.9. Acridine melamine conjugates binds to CUG repeat48...... 10

Figure 1.10. Bifacial melamine-thymine recognition...... 11

Figure 1.11. bPNA as allosteric switch in rescuing aptamer-protein binding, aptamer- small molecule binding and ribozyme cleavage54...... 12

Figure 1.12. Polyamide targets DNA duplex minor groove56...... 14

Figure 1.13. TMpyP4 photocleavage of the c-MYC NHE...... 15

Figure 1.14. Small molecule binders to G-quadruplex in the telomere (left) and gene promoters (right). Figure is reproduced from reference69 and reference70...... 17

Figure 1.15. A metallo-intercalator bound to matched and mismatched sites...... 18

Figure 1.16. Various aminoglycoside that target the 16S ribosomal RNA79...... 19

Figure 1.17. RNA Riboswitches, whose aptamer domains have been solved95...... 21

Figure 1.18. In vitro selection of various dyes109...... 23

Figure 1.19. General principle of chemically induced dimerization. Figure is reproduced from reference 125...... 24

Figure 1.20. Dimerization of FKBP by FK1012 and intracellular signaling initiated by the dimerization125...... 25

Figure 1.21. Principle of small molecule induced protein splicing125...... 26

Figure 2.1. Strucutres of conventional PNA, α-PNA (bPNA), and peptoid backbones. .. 49

Figure 2.2. Submonomer synthesis of polyamino peptoids on Rink amide resin...... 51

Figure 2.3. Side-chain installation of melamine bases. A representative reaction to produce (AbM)6 is shown...... 52

Figure 2.4. UV melting and CD of DNA-peptoids complexes...... 54

Figure 2.5. HPLC trace of (HbM)6, using the gradient described in the beginning of the section...... 60

Figure 2.6. MALDI-TOF MS of (HbM)6...... 60

Figure 2.7. HPLC trace of (AbM)6, using the gradient described in the beginning of the section...... 61

Figure 2.8. MALDI-TOF of (AbM)6 ...... 61

Figure 2.9. HPLC trace of (AbM)8, using the gradient described in the beginning of the section...... 62

Figure 2.10. MALDI-TOF of (AbM)8...... 62

Figure 3.1. Overview of small molecule and RNA structures used in this chapter...... 71

Figure 3.2. Folding of dT10C4T10 DNA with t3M...... 73

Figure 3.3. Binding ratio and affinity of t2M and t4M to T rich DNAs...... 74

Figure 3.4. U23 cleavage restored by t4M...... 76

Figure 3.5. S-U4n and S-U4-2bp cleavage restored by t4M...... 77

Figure 3.6. Binary ribozyme cleavage restored by t4M...... 78

Figure 3.7. Secondary structure representation of the binary HHRs...... 83

Me Figure 3.8. UV-melting of t3M and t3M with dT10C4T10...... 89

Figure 3.9. UV-melting of Tren derivatives with dT10C4T10...... 89

Me Figure 3.10. UV-melting t2M and t2M with dT10C4T10...... 90

Me Figure 3.11. UV melting of t4M and t4M with dT10C4T10...... 90

Figure 3.12. Job plots of molecular beacon fluorescence quenching with t3M and t2M. 91

Figure 3.13. ITC raw heat of t3M (600µM, 50µl, 1XPBS) injected into dT10C4T10 (10µM,

300µl, 1XPBS)...... 91

Figure 3.14. ITC raw heat of t3M (200µM, 50µl, 1XPBS) injected into 12-T2-12 (20µM,

300µl, 1XPBS)...... 92

Figure 3.15. DSC curves of 25 µM dT10C4T10 with Boc t2M or t3M...... 92

Figure 3.16. DSC trace of qt3M alkyne with dT10C4T10...... 93

Figure 3.17.Gel shift of Cy5-t2M with 12-T2-12 (left) and 12-T0-4 (right)...... 93

Figure 3.18. UV melting of 50 µM t3M with vaious concentrations of dT10C4T10...... 94

Figure 3.19. Synthetic scheme for t3M...... 94

Figure 3.20. Synthetic scheme for t2M and t2MMe...... 95

Figure 3.21. Synthetic scheme for t2M alkyne...... 97

Figure 3.22. Synthetic scheme for FITC-t2M...... 99

Figure 3.23. Synthetic scheme for Cy5-t2M...... 100

Figure 3.24. Synthetic scheme for N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6-triamine.

...... 101

Figure 3.25. Synthetic scheme for N2-(2,2-dimethoxyethyl)-N4,N4,N6,N6-tetramethyl-

1,3,5-triazine-2,4,6-triamine...... 101

Figure 3.26. Synthetic scheme for t4M ...... 102

Figure 3.27. Synthetic scheme for t4MMe...... 103

Figure 3.28. Synthesis scheme for qt3M alkyane ...... 105

Figure 3.29. U-(2,3) ribozyme activity rescued by 10 µM tren derivative...... 108

Figure 3.30. U-(2,3) ribozyme activity rescued by 10µM tren derivative...... 108

Figure 3.31. U-(2,3) ribozyme activity rescue by 100µM tren derivative...... 109

Figure 3.32. U-(2,3) ribozyme cleavage quantification...... 109

Figure 3.33. S-U4n ribozyme rescued by t4M...... 110

Figure 3.34. S-U4n ribozyme rescue by t4M...... 111

Figure 3.35. S-U4n ribozyme rescue control...... 111

Figure 3.36. S-U4n-2bp ribozyme rescue by t4M...... 112

Figure 3.37. S-U4n ribozyme cleavage data quantification...... 112

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Figure 3.38. S-U4n and S-U4n-2bp ribozyme cleavage data quantification...... 113

Figure 3.39. Binary ribozyme cleavage data quantification...... 114

Figure 3.40. Binary ribozyme cleavage data quantification...... 114

Figure 3.41. Binary ribozyme cleavage data quantification...... 115

Figure 3.42. Representative gel image for binary ribozyme cleavage...... 115

Figure 3.43. Binary ribozyme cleavage data quantification...... 116

1 Figure 3.44. H NMR (400 MHz, D2O, pH=3) of t3M. TFA from HPLC...... 117

13 Figure 3.45. C NMR (100 MHz, D2O) of t3M...... 117

1 Figure 3.46. H NMR (400 MHz, D2O, pH=6) of t3M. TFA from HPLC...... 118

13 Figure 3.47. C NMR (100 MHz, D2O+NaOD,pH=6) of t3M. TFA from HPLC...... 118

1 Figure 3.48. H NMR (400 MHz, D2O) of Boc t2M...... 119

13 Figure 3.49. C NMR (100 MHz, D2O) of Boc t2M...... 119

1 Figure 3.50. H NMR (400 MHz, D2O, pH=3) of t2M. TFA from HPLC...... 120

13 Figure 3.51. C NMR (125 MHz, D2O) of t2M. TFA from HPLC...... 120

1 Figure 3.52. H NMR (400 MHz, D2O, pH=3) of t2M. TFA from HPLC...... 121

13 Figure 3.53. C NMR (125 MHz, D2O+NaOD, pH=6) of t2M. TFA from HPLC...... 121

1 Me Figure 3.54. H NMR (400 MHz, CDCl3) of Boc t2M ...... 122

13 Me Figure 3.55. C NMR (100 MHz, CDCl3) of Boc t2M ...... 122

1 Me Figure 3.56. H NMR (400 MHz, CD3CN:D2O=1:1) of t2M ...... 123

13 Me Figure 3.57. C NMR (125 MHz, CD3CN:D2O=1:1) of t2M ...... 123

1 2 Figure 3.58. H NMR (400 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)-1,3,5-triazine-

2,4,6-triamine...... 124

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13 2 Figure 3.59. C NMR (100 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)-1,3,5-triazine-

2,4,6-triamine...... 124

1 2 4 4 6 6 Figure 3.60. H NMR (400 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)-N ,N ,N ,N - tetramethyl-1,3,5-triazine-2,4,6-triamine...... 125

13 2 4 4 6 6 Figure 3.61. C NMR (100 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)-N ,N ,N ,N - tetramethyl-1,3,5-triazine-2,4,6-triamine...... 125

1 Figure 3.62. H NMR (400 MHz, D2O+NaOD, pH=6, water suppression) of t4M...... 126

13 Figure 3.63. C NMR (125 MHz, D2O+NaOD, pH=6) of t4M...... 126

1 Me Figure 3.64. H NMR (400 MHz, CD3CN:D2O=1:1) of t4M . TFA from deprotection of

Boc...... 127

13 Me Figure 3.65. C NMR (125 MHz, CD3CN:D2O=1:1) of t4M . TFA from deprotection of Boc...... 127

1 Figure 3.66. H NMR (400 MHz, D2O, pH=3) of t2M alkyne...... 128

13 Figure 3.67. C NMR (125 MHz, D2O, pH=3) of t2M alkyne...... 128

1 Figure 3.68. H NMR (400 MHz, CDCl3) of tri Boc quat tren alkyne...... 129

13 Figure 3.69. C NMR (100 MHz, CDCl3) of tri Boc quat tren alkyne...... 129

1 Figure 3.70. H NMR (400 MHz, D2O, water suppression) of qt3M alkyne...... 130

1 Figure 3.71. H NMR (400 MHz, MeOD) of FITC-N3...... 130

Figure 3.72. HPLC and ESI spectra of t3M...... 131

Figure 3.73. HPLC and ESI spectra of t2M...... 132

Figure 3.74. HPLC and ESI spectra of t4M...... 132

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Figure 4.1. G quadruplexes in Spinach (left) and Mango (right) aptamers. Figure is adapted from references 7 and 9...... 142

Figure 4.2. Spinach aptamer-DFHBI interaction...... 144

Figure 4.3. Spinach U mutant sequences. Mutations are highlighted in red...... 145

Figure 4.4. Selective fluorescence turn on by DFHBI-4C-M*...... 147

Figure 4.5. Metal ion dependence of 5bp S U2 aptamer with TO-t2M...... 149

Figure 4.6. Selective turn on of Spinach aptamers by TO-t2M...... 150

Figure 4.7. Selective turn on of Spinach aptamers by TO-4C-M*...... 151

Figure 4.8. Two relevant structures of human telomere G quadruplex core sequence

(GGGTTA)3GGG, TT mimatches are highlighted in red...... 152

Figure 4.9. Fluorescence turn on of (GGGTTA)3GGG with TO-t2M...... 153

Figure 4.10. Synthetic scheme for DFHBI-4C-M* and DFHBI-4C-M*4M...... 155

Figure 4.11. Synthetic scheme for TO derivatives...... 157

Figure 4.12. HPLC spectra of TO-t2M...... 158

Figure 4.13. HPLC spectra of TO-t2MMe...... 158

Figure 4.14. HPLC spectra of TO-4C-M*...... 159

Figure 4.15. HPLC spectra of TO-4C-M*4M...... 159

Figure 4.16. Apparent Kd determination of DFHBI-4C-M* with Spinach RNAs...... 161

Figure 4.17. Apparent Kd determination of TO derivatives with Spinach RNAs...... 161

Figure 5.1. Structure and synthesis scheme for bPNA(+) ...... 167

Figure 5.2. Circular dichroism spectra showing 1:1 bPNA(+)-DNA complexation (10µM

+ each) of dT6C4T6 alone (---) and dT6C4T6 complex with (EK )3 (─) ...... 168

Figure 5.3. UV melting traces (left) and first derivatives (right) of hexamer bPNA(+) with dT6C4T6 and pentamer bPNA(+) with dT10C4T10...... 169

Figure 5.4. Hexapeptide bPNA(+) hybridization with dT6C4T6 DNA...... 170

Figure 5.5. Strand invasion of DNA dupelx by bPNA(+)...... 173

Figure 5.6. Synthesis of N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6-triamine ...... 174

1 2 Figure 5.7. H NMR(400 MHz, d6-DMSO) of N -(2,2-dimethoxyethyl)-1,3,5-triazine-

2,4,6-triamine...... 175

13 2 Figure 5.8. C NMR(100 MHz, d6-DMSO) of N -(2,2-dimethoxyethyl)-1,3,5-triazine-

2,4,6-triamine...... 175

+ Figure 5.9. HPLC traces for reductive alkylation to bPNA(+) (SK )3 ...... 177

+ Figure 5.10. HPLC trace of ABA- Ala-(EK )3-G, using the gradient described at the beginning of the section...... 178

+ Figure 5.11. MALDI spectrum of ABA- Ala-(EK )3-G...... 178

+ Figure 5.12. HPLC trace of ABA- Ala-(EO )3-G, using the gradient described at the beginning of the section...... 179

+ Figure 5.13. MALDI spectrum of ABA- Ala-(EO )3-G...... 179

+ Figure 5.14. HPLC trace of ABA- Ala-(EB )3-G, using the gradient described at the beginning of the section...... 180

+ Figure 5.15. MALDI spectrum of ABA- Ala-(EB )3-G...... 180

Figure 5.16. HPLC trace of ABA- Ala-(SK+)3-G, using the gradient described at the beginning of the section...... 181

+ Figure 5.17. MALDI spectrum of ABA- Ala-(SK )3-G...... 181

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+ Figure 5.18. HPLC trace of ABA- Ala-(SO )3-G, using the gradient described at the beginning of the section...... 182

+ Figure 5.19. MALDI spectrum of ABA- Ala-(SO )3-G...... 182

+ Figure 5.20. HPLC trace of ABA- Ala-(SB )3-G, using the gradient described at the beginning of the section...... 183

+ Figure 5.21. MALDI spectrum of ABA- Ala-(SB )3-G...... 183

+ Figure 5.22. HPLC trace of ABA- Ala-(EK )5-G, using the gradient described at the beginning of the section...... 184

+ Figure 5.23. MALDI spectrum of ABA- Ala-(EK )5-G...... 184

+ Figure 5.24. HPLC trace of ABA- Ala-(SK )5-G, using the gradient described at the beginning of the section...... 185

+ Figure 5.25. MALDI spectrum of ABA- Ala-(SK )5-G...... 185

Figure 5.26. UV-melting of bPNA(+) (SK+)3 and control peptides...... 189

+ Figure 5.27. Fluorescence anisotropy assay of T6C4T6 titrating into FITC-βAla-(EK )3-G.

...... 189

+ Figure 5.28. Fluorescence anisotropy assay of T6C4T6 titrating into FITC-βAla-(EO )3-G.

...... 190

+ Figure 5.29. Fluorescence anisotropy assay of T6C4T6 titrating into FITC-βAla-(EB )3-G.

...... 190

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Chapter 1 : Structure and recognition of nucleic acids

1.1 Native nucleic acid structures

Native nucleic acids consist of deoxyribonucleic acid (DNA) and ribonucleic acid

(RNA). They play a central role in the storage and readout of genetic information, as well as in the catalysis of many crucial reactions. Nucleic acids are made up of nucleotide monomers and linked by phosphodiester bonds. Each nucleotide is composed of phosphate groups, a five-carbon sugar and a nucleobase. Adenine (A), thymine (T), cytosine (C) and guanine (G) nucleobases are observed in DNA, while A,C,G and uracil

(U) are observed in RNA. Hydrogen bonding and shape complementarity of nucleobases, combined with the structural and electrostatic properties of sugar-phosphate backbone lead to multiple types of nucleic acid structure.

Figure 1.1. Chemical structures of nucleic acids. Figure is adopted from reference1.

1.1.1 DNA duplex

The elucidation of DNA duplexes by Watson and Crick2 is arguably the most iconic moment in the 20th century scientific discoveries. Generally, DNA duplexes adopt the double helix structure with three forms (A, B and Z). B-form DNA is most common, which is right-handed with 10.5 base pairs (bps) per turn, 20 Å diameter and C2'-endo sugar pucker. The helical nature of DNA duplexes creates major grooves which are 22 Å wide and minor grooves, which are 12 Å wide. A-form DNA is also right-handed, but adopts C3’-endo sugar pucker, which increases the diameter to 22 Å. Z-form DNA is left- handed and elongated along the axis, with a diameter of 18 Å.

Figure 1.2. Three forms of DNA duplexes. Figure is adapted from references3-4.

1.1.2 G quadruplex

G quadruplex, one of the most stable nucleic acid structures , is extremely common throughout the human genome, such as in the telomere and promoter region5-6 as well as in in vitro selected aptamers (Spinach7, Mango8 etc.), recently RNA G quadruplexes are 3 also found to exist in vivo9-10 and are responsible for regulation of translation9, 11.

Through coordination of guanine O6 to monovalent cations such as K+ and Na+, G quadruplexes are kinetically and thermodynamically12-13 stable. They can be formed both intermolecularly and intramolecularly, with various type of arrangement of guanine residues (parallel, antiparallel, hybrid), such versatility and importance makes G quadruplexes one of the most popular targets in nucleic acid research.

Figure 1.3. (A) G-quartet structure, M+ indicates monovalent ions such as K+ and Na+. (B) Intermolecular G-quadruplexes. (C). Different forms of intramolecular G- quadruplexes. Figure is reproduced from reference14.

1.1.3 RNA secondary structure

Although the only chemical difference of RNA compared to DNA is the additional 2'- hydroxyl group in the ribose backbone, RNA has much more structural features. A single-stranded RNA mostly likely will form secondary and even tertiary structures by folding back onto itself to form Watson-Crick base pairings, Hoogsteen interactions15 or

4 even wobble parings16-17, leading to structures like hairpins, bulges or pseudoknots18. The structural complexity of RNA makes it functionally versatile, besides its place in the central dogma (mRNA), RNAs also appear in the form of ribozymes, riboswitches, regulatory non-coding RNAs and cell machinery19.

Figure 1.4. Common RNA secondary and tertiary structures. Figure is reproduced from reference20

1.2 Triple helical nucleic acids

Triple helix DNA is found to form between two oligopyrimidine strands and one oligopurine strand21-24 in the presence of Mg2+ and in slightly acidic media. The third strand interacts within the major groove of the duplex at the Hoogsteen edge of the purines. Triplex forming oligonucleotide (TFO) is considered a major groove ligand that sequence specifically binds to DNA duplexes with high affinity, and can be used to direct

DNA modifying agents to selected sequences25. RNA triplexes are found in vivo26-27, and are responsible for regulation of RNA function and lifetime.

Figure 1.5. Top: Watson-Crick base pairing; Bottom: Base triplets TAT and C+GC. Reference is reproduced from reference28.

1.3 Peptide nucleic acid (PNA) for nucleic acid recognition

The term peptide nucleic acid (PNA) was first coined by Peter Nielsen, during their effort to find a DNA mimic that can form triplex structure. Nielsen and coworkers synthesis an oligomer that displayed nucleobases on a pseudopeptide (polyamide) backbone, with N- substituted (2-aminoethyl) glycine as monomer units. Due to the lack of anionic charge in the backbone, PNA-DNA hybrid is thermally more stable than DNA duplexes. In the

6 original attempt to form DNA2-PNA triplex, PNA invaded into the duplex region that

29 sequence complementary to the PNA, and created a PNA2-DNA triplex . Using a homopurine decamer PNA, duplex invasion mode was observed30, while using sterically compromised base pair, double duplex invasion mode was observed31.

Figure 1.6. Top: Chemical structure comparison between protein, PNA and DNA. Bottom: PNA binding mode. Figure from reference32.

The high thermal stability and resistance to enzymatic degradation33, makes PNAs widely used in biological and medicinal applications, such as interference of transcription

7 process via antigene strategy34-36 and inhibition of the translation process through complexation with mRNA37-38(antisense strategy).

1.4 Triplex forming artificial nucleobases

Traditionally, TFO and PNA form triplexes by targeting the Hoogsteen face of the purine bases, if the hydrogen bonding face is expanded across the purine-pyrimidine pair, one would imagine higher stability and selectivity. Many artificial nucleobases have been designed for this purpose. In 1995, Sasaki and coworkers synthesized benzaminoimidazole-glycyl (BIG) nucleobase that targets CG base pair39, similarly, phthalimide based nucleobases developed by Lengeler and Weisz forms the same kind of hydrogen bonding pattern with CG pair40. An aminophenyl-thiazole nucleobase was incorporated into TFO and could target TA pair specifically41-42.

Figure 1.7. Artificial nucleobases that can form triplexes. Adapted from reference43.

Different from traditional triplex formation where the third strand binds on the Hoogsteen face, McLaughlin44-45 and Tor46 reported Janus-Wedge47 type of triplex where the third strand can base pair at the Watson-Crick face of the two target strands. 8

Figure 1.8. Janus-Wedge type triplex formation45, 47.

(Left) Nucleobase that poses D-A-A and A-D-D faces (D is hydrogen bond donor, A is hydrogen bond acceptor) could insert into a G-C base pair, while nucleobase that poses A-D and D-A-D faces could insert into a A-T base pair. (Right) Examples of Janus- Wedge type diazine and triazine nucleobases.

Nucleobases based on diazine or triazine heterocycles were readily modified by

McLaughlin to insert into AT or GC pairs. 1,3,5-Triazine-2,4,6-triamine (Melamine) was known to have the hydrogen bonding face to form Janus type recognition with T-T or U-

U mismatches47, Zimmerman group utilized this interaction, coupled with an acridine intercalator, to design a ligand that binds CUG repeat specifically at low micromolar affinity.

Figure 1.9. Acridine melamine conjugates binds to CUG repeat48.

1.5 PNA with native α peptide backbone

Traditional PNA developed by Nielsen et al. is based on pseudopeptide backbone, which requires non-trivial monomer synthesis. Eschenmoser49-50 and Ghadiri51 have reported peptide nucleic acid based on native α peptide backbone which require little modification of the commercially available α amino acids for monomer preparation. These peptide nucleic acids have alternate nucleobase displaying residue and solubilizing residue, leading to minimal non-specific electrostatic interaction with the phosphate backbone of nucleic acids. In the thorough study by Eschenmoser and coworkers49-50, substituted triazine and pyrimidine nucleobases were displayed alternatively on native peptide and peptoid backbone. These oligomers which could form under primordial conditions, could recognize native oligonucleotides. Ghadiri and coworkers develop thioester peptide nucleic acid (tPNA)51, which could reversibly dock nucleobases and selectively recognized the complementary nucleic acid oligomers.

1.6 Melamine displaying bifacial peptide nucleic acid (bPNA) as allosteric switch

1,3,5-Triazine-2,4,6-triamine, also known as melamine, can form bifacial hydrogen bonding with cyanuric acid52, thymine or uracil nucleobases have a have hydrogen bonding face identical to that of cyanuric acid, thus melamine could form Janus-type recognition pattern with T-T or U-U mismatches47. Using this interaction, Bong and coworkers developed a peptide with alternating glutamic acid and lysine, with melamine moiety at the end of each lysine residue, they name this peptide bifacial peptide nucleic acid (bPNA)53. Oligothymine could interact with bPNA to form triplex structure in an associative fashion, unstructured T rich DNA T10C10T10 could found into defined hairpin structure when titrate in bPNA. The binding affinity of bPNA to T rich DNA is 4000 nM for the triplex and 2.7 nM for the hairpin.

Figure 1.10. Bifacial melamine-thymine recognition. Melamine displaying peptide (bPNA) could fold T rich DNA into defined triplex or stem- loop structure. Figure is from reference53.

The high selectivity combined with the robust binding affinity of bPNA makes it a perfect candidate as an allosteric switch. Through restructuring the mutated U stretch into a triplex structure, bPNA could rescue aptamer-protein binding (IgE with its aptamer), aptamer small molecule binding (Spinach aptamer with DFHBI) and hammerhead ribozyme self-cleavage reaction. This allosteric control of non-coding nucleic acid function provides a generally strategy to regulate nucleic acid function54.

Figure 1.11. bPNA as allosteric switch in rescuing aptamer-protein binding, aptamer-small molecule binding and ribozyme cleavage54.

1.7 Small molecule nucleic acid binders

Previous sections discussed mostly nucleic acids recognized by macromolecules, while small molecule nucleic acid binders have also been widely studied, as they are relatively ease to synthesize and modify. Small molecule nucleic acid binders came from both natural sources and de novo design. In either case, the major interactions involved in the binding of small molecules and nucleic acids are electrostatic interaction, hydrogen binding, base stacking and covalent linking.

1.7.1 B-form DNA minor groove binders

The minor grooves in B-form DNAs are shallower than its major groove, making it an ideal target for synthetic small molecules. Typical examples are natural molecule distamycin A and synthetic diarylamidines such as DAPI, Berenil and pentamidine as well as bis-benzimidazoles such as Hoechest 33528. These reagents rely on electrostatic interaction and intercalation as the major driving force in binding with DNAs. Dervan and coworkers elegantly designed a type of polyamide containing pyrrole (Py), hydroxypyrrole (Hp) and imidazole (Im) amino acids that targets DNA minor grooves55.

Using hydrogen bonding from the heterocycles as well the amide backbone, an Im/Py pair recognizes a G-C base pair, whereas a Py/Im combination targets a C-G base pair. An

Hp/Py pair distinguishes T-A from A-T base pairs. Using a simple molecular shape and a three-letter amino-acid code, eight-ring pyrrole-imidazole polyamides achieve affinities and specificities comparable to DNA-binding proteins.

Figure 1.12. Polyamide targets DNA duplex minor groove56.

1.7.2 G-quadruplex binders

The interest in developing small molecule binders to stabilize G-quadruplex structures arise largely from the common occurrence of G-quadruplexes throughout the genomes of many organisms. They could potentially form in the telomere and the regulatory regions of a number of genes. Telomeres appear at the ends of eukaryotic chromosomes, and are responsible in protecting against degradation and genome instability. In a majority of human cancers, telomerases, an enzyme that catalyzes telomere extension, is overexpressed. Compounds that can stabilize G-quadruplexes in the telomere could inhibit telomerase activity57, thus these small molecules could be used as a potential anticancer drugs. Promoter region of oncogenes is also a hot target for G quadruplex binding ligands. In 2002, Hurley and coworkers published their work that a small molecule

(TMpyP4) could repress transcription of the MYC proto-oncogene (c-MYC), which is over-expressed in 80% of all solid tumors58. The putative mechanism is that the small molecule could target the potential G quadruplex in the nuclease hypersensitive element

(NHE). Apart from the oncogene promoter region, regulation of chicken β-globin gene59-

60, c-kit oncogene61, human BCL-262, VEGF gene63 and hypoxia inducible factor 1α64 are also linked to the G quadruplex structures.

Figure 1.13. TMpyP4 photocleavage of the c-MYC NHE. Blue arrow shows possible binding position of TMpyP4 to the DNA58.

Due to the planarity of the G quadruplex, many ligands that possess planar aromatic moieties could intercalate; cationic side chains, if present, are also very beneficial due to the anionic nature of nucleic acids. Overall, the majority of the ligands is designed to have the common features of extended electron-deficient planar aromatic chromophores, together with side chains having terminal cationic groups, as shown in Figure 1.13, regardless of their desired target region. Telomestatin, a natural product isolated from

Streptomyces anulatus, is found to be a very potent telomerase inhibitor65. It has a 70-fold 15 selectivity for intramolecular G quadruplex over DNA duplex, as well as the ability to induce and stabilized G quadruplex structure in the absence of added monovalent cations66. Structurally telomestatin is a macrocycle made of oxazole and thiazole heterocycles connected by sp2-sp2 C-C bond, making it a huge planar structure. Synthetic analogue of telomestatin have been made, in particular, by introducing a bisamide linker into a heptaoxazole macrocycle, resulting in a potent binder of the intramolecular antiparallel G quadruplex structure67. TmPyP4, on the hand, preferentially facilitate the formation of intermolecular G quadruplexes66, it is a cationic porphyrin structure, with side chains being methylated pyridines. Simply by changing the position of the N-methyl group from para to ortho, telomerase inhibition IC50 is dropped by 10-fold, according to an extensive structure-activity relationship study was carried out by Hurley group68.

Apart from DNA G-quadruplex binders, recently a small molecule pyridostatin is found to bind RNA G-quadruplexes preferably over DNA quadruplexes10 and can be used to visualized RNA G-quadruplex structures in the cytoplasm of human cells.

Figure 1.14. Small molecule binders to the G-quadruplex in the telomere (left) and promoters (right). Figure is reproduced from reference69 and reference70.

1.7.3 Mismatch sites targeting small molecules

Nucleobase mismatches are very common in the genome due to gene mutations, many leading to diseases. For example, single nucleotide polymorphisms (SNP) in APOE gene is associated with a higher risk of Alzheimer’s disease71, while CTG repeat in the myotonic protein kinase (DMPK) gene may lead to myotonic dystrophy72. Design of small molecules that target nucleotide mismatch usually involves hydrogen bonding and base stacking. Nakatani and coworkers found that naphthyridine derivative could be used to target G-G73 mismatch while 8-azaquinolone derivatives could be used to target A-A mismatch73. The Zimmerman group developed a small molecule conjugate of melamine 17 and acridine that binds to T-T/U-U mismatches, which could be used to inhibit the binding of MBNL protein to the CUG repeats48. Metallo-intercalators are another powerful tool to detect mismatched DNA base pairs, developed by Jacqueline Barton group, these positively charged polycyclic aromatic compound selectively insert into the mismatch site, using their metal center, radicals could be generated when photoactivated, causing cleavage into the mismatched DNA74-76.

Figure 1.15. A metallo-intercalator bound to matched and mismatched sites. C shows intercalation into the major groove of the matched oligonucleotide while D shows insertion into the AC mismatched site while ejecting adenosine (green) and cytosine (blue). Figure from reference74.

1.7.4 Small molecule RNA binders, aptamers, riboswitches

As discussed previously, RNAs possess much more structural features than their DNA counterpart, so in terms of small molecule binders, one can image them being as colorful as the kaleidoscope, here we will briefly review their role in medicinal and biological applications.

Antibiotics

Aminoglycoside antibiotic is well known class of RNA binders, more specifically, they tend to bind to the internal loop or bulged region of RNA. A typical example is the binding of paromoycin to the 16S ribosomal RNA in E. Coli, which causes misreading of the genetic code and inhibit translocation77. However, they are notoriously non-specific, will bind to many RNA sequences with low micromolar affinity78.

Figure 1.16. Various aminoglycoside that target the 16S ribosomal RNA79.

Natural occurring riboswitches

Riboswitches are noncoding RNAs that often appear in the 5’-UTR of mRNAs. The binding of small molecules, often metabolites, to the riboswitch is coupled with the structural change of the RNA, leading to gene expression control in bacteria as well as in some plants and fungi80-82. Riboswitches are highly selective and exhibit high affinity toward their target, usually ranging from nM to low µM. They are composed of (i) the aptamer domain, which binds to the ligand, and (ii) the expression platform, which modulate gene expression. Thus, ligand binding has an allosteric control on gene expression. Sequence and secondary-structure analysis indicates that more than 2% of all genes in some species are regulated at least in part by a riboswitch mechanism80, 83-86.

Well known example of riboswitches found in vivo could be regulated by small molecules such as guanine80, adenine87, thiamine pyrophosphate83, 85, lysine88-89, glycine90, S-adenosylmethionine91-93.

Adenine and guanine riboswitches are similar in structure, with a single nucleotide mutation (U74C) changing the affinity of an adenine riboswitch to guanine binding87.

These riboswitches are important in regulation of translation in certain species. Adenine riboswitch from Vibrio vulnificus could switch on translation by releasing the Shine-

Dalgarno sequence (SD sequence) and start codon94, while the guanine riboswitch from

B. subtilis could act as an off switch. Thiamine pyrophosphate (TPP) and S- adenosylmethionine (SAM) riboswitches are also involved in the regulation of transcription initiation and termination, while functions such as regulation of mRNA stability was also found to involve these riboswitches88. It is not surprising that a single

20 riboswitch could regulate multiple events since the aptamer domain and expression platform are separate parts, the allosteric regulation arise from the structural change upon ligand binding could be utilized in different ways depending on the surrounding environment.

Figure 1.17. RNA Riboswitches, whose aptamer domains have been solved95. a) adenine riboswitch from V. vulnificus, b) guanine riboswitch from B. subtilis, c) SAM riboswitch from T. tengcongensis, d) TPP riboswitch from E. coli, and e) glmS ribozyme from T. tengcongensis.

Glms Ribozyme is an intriguing RNA that acts both as a ribozyme and a riboswitch, it is found in the 5’-UTR of mRNA encoding GlcN6P synthase in numerous Gram-positive bacteria96. Glms ribozyme can catalyze the production of glucosamine-6-phosphate

(GlcN6P), while also relies on the binding of GlcN6P to catalyze self-cleavage97. Since 21

GlcN6P is a key metabolic precursor of the bacteria cell wall96, glms ribozyme is crucial to the survival of these bacteria.

Overall, the discovery of riboswitch gave us a glimpse into the simple yet profound cellular regulation machinery, some could even say it supports the idea of RNA world83,

98-99 since it does not contain any protein components. Since riboswitches are an effective in controlling gene expression in vivo, interest is high in using engineered artificial riboswitches as potential method for gene therapy100-102.

Aptamer via in vitro selection

Much like the riboswitch, aptamers obtained from in vitro selection (or SELEX) binds certain ligands selectively. The range of ligands with which artificial aptamers bind to, however, are much wider than metabolites. Small molecules, proteins103-104, even cells105-

108 have been examined as targets for aptamer selection. In terms of small molecule ligands, interest arise from fluorogenic ligands as they could be used as sensors and reporter in vivo. One of the first in vitro selections was performed using different dyes as targets109, in which a random pool of 155-nucleotide long DNAs were synthesized with a mixture of equimolar of four bases, achieving approximately 1015 individual sequences.

The DNA pool was then selected against a number of dyes for six rounds using the procedure shown in Figure 1.18, aptamers selected against each dye was then shown to have selectivity over binding with other dyes.

Figure 1.18. In vitro selection of various dyes109.

Since then, several dozen of small molecules aptamer have been developed, ligands range from ATP110, vitamin B12111, dopamine112 to reaction intermediates113 and dye molecule that induces site-specific cleavage upon laser irradiation114.

This method of obtaining nucleic acid aptamer that binds selectively to any molecule of choice became a very powerful tool in biotechnology. However the problem for aptamers obtained from SELEX method is the discrepancy between the in vitro function and in vivo activity. There is only a handful of in vivo active aptamers while several dozen have been selected in vitro. The problem maybe from the fact that big conformation change upon ligand binding is needed for in vivo function, not only high binding affinity115.

Efforts have been made to address such issue, as the Batey group used partially structured libraries based on native riboswitch scaffolds as the starting point for in vitro selection, and obtained aptamers for new ligands that are active in vivo116.

Among the handful of in vivo active small molecule aptamers, Spinach developed by

Jaffrey and coworkers is one of the most widely used. The RNA aptamer could bind a 23 fluorogenic small molecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and emit green light, making it a RNA mimic of GFP7. Native riboswitch sequence could be inserted into the Spinach aptamer, thus generating a real-time imaging probe for metabolites in live cells117-118. Similarly, Mango, an aptamer that binds the thiazole orange dye and has about 1000 times fluorescence turn on was discovered by Unrau and coworkers through in vitro selection8.

1.8 Small molecule induced dimerization

Chemically induced dimerization (CID) is the controlled dimerization of two dispersed proteins (same of different), via a dimerizer. The increased local concentration of the proteins could be used to regulated a biological event. The dimerizer could be proteins119-

120, nucleic acids121-123 or small molecules. Here we focus on the small molecule induced dimerization.

Figure 1.19. General principle of chemically induced dimerization. Figure is reproduced from reference 125.

In 1993, Schreiber, Crabtree, and coworker published a paper demonstrating that the bivalent derivative of small molecule FK506 (FK1012) can induce the dimerization of

FK506 binding protein (FKBP), more importantly, the dimerization of FKBP-TCR fusion induced by FK1012 lead to the activation of endogenous signal transduction cascade124.

Figure 1.20. Dimerization of FKBP by FK1012 and intracellular signaling initiated by the dimerization125.

Since the initial report of small molecules CID, much effort has been invested into the understanding the mechanism and discovery of new ligands. For example, homodimer formation of dihydrofolate reductase (DHFR)126-127 and bacterial DNA gyrase B subunit

(GyrB)128-129 have been induced by small molecules methotrexate derivative and natural product coumermycin. 25

With the emergence of more dimerizers, heterodimer formation became an attractive possibility, the idea of using small molecules to bring two nonequivalent proteins together precisely would provide new ways for modulation of biological event in vivo.

For example, post translational control could be achieved by dimerization of two engineered intein halves130, as show in Figure 1.21 . The inteins are covalently attached with FKBP and FRB, respectively, no splicing was observed in the naïve state, while with the addition of rapamycin derivative, two components were brought together to initiate protein splicing. Applications such as transcriptional control131, proximity sensing132 and bio screening133-135 have also been exploited.

Figure 1.21. Principle of small molecule induced protein splicing125.

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Chapter 2 : DNA binding peptoids

2.1 Triazines and DNA molecular recognition

Complexation of 1,3,5-triazines based on melamine and cyanuric acid have been well- studied in the solid state1-3, in organic solvents4-5, and in water1, 6-13. Like DNA base stacking14, aqueous-phase triazine assembly is strongly exothermic6. Indeed, melamine derivatives can dock thymine15 or uracil16 bases via their Watson–Crick faces. The bifacial symmetry of monosubstituted melamine allows recognition of a T–T or U–U site in DNA or RNA17-18. Melamine-displaying α-peptides (bifacial PNA or bPNA) bind to unstructured oligothymidine and oligouridylate tracts to form obligate triplex hybrid structures19-20 by virtue of base-triple recognition and stacking (Figure 2.1). This triplex interface can effectively compete with enzymatic processing of nucleic acids21, but can also serve as a structural trigger for noncoding DNA and RNA function22 as well as peptide bond formation23. The structure of conventional PNA24, α-PNA25-26, and peptoids27 are similar in their polyamide backbones as well as the spacing of nucleic acid interacting residues, which are separated by seven backbone atoms in each macromolecule. The absence of intervening side chains in conventional PNA leads to a neutral backbone, while alternate residues in α-PNA and peptoids afford an opportunity to tune electrostatic interactions and water solubility through installation of polar side chains. As potential biomedical reagents, the peptoid scaffold holds considerable appeal in increased protease stability28-29. Furthermore, as a result of the tertiary amide backbone, peptoids are much less polar than peptides composed of secondary amide linkages leading to increased transmembrane passage and intracellular delivery30. Herein

48 we describe the synthesis of triazine-displaying peptoid macromolecules and their DNA- binding properties. We find peptoids to be less efficient in binding DNA relative to their peptide analogues.

Figure 2.1. Strucutres of conventional PNA, α-PNA (bPNA), and peptoid backbones. With identical spacing of DNA-binding side chains. Thymine-melamine-thymine base triple interface is shown (right).

2.2 Synthesis of DNA-binding peptoids

Bifacial PNA is prepared from solid-phase peptide synthesis using Fmoc-lysine derivatized on the ε-nitrogen with melamine. In contrast, peptoids can be prepared with much simpler and cheaper starting materials in the submonomer31 approach, which avoids the use of densely functionalized and chiral reactants such as α-amino acids. In the submonomer approach, the amino acid peptoid unit is constructed in two steps through terminal nitrogen bromoacetylation, followed by bromide displacement with a primary

49 amine bearing the desired side-chain functionality (Figure 2.2). The resulting resin-bound secondary amine is bromoacetylated again to grow the chain. Triazine-displaying peptoids were designed to mimic bPNA structures, which display melamine at alternate residues, with solubilizing residues in the remaining positions. Carboxylic acid and hydroxy groups were chosen for solubilization, with the expectation that the negatively charged carboxylate group would minimize nonspecific binding, but also decrease affinity through electrostatic repulsion with the polyanionic DNA backbone. Thus, peptoid–DNA binding should be responsive to overall peptoid charge. Peptoid side-chain length was varied to probe the steric requirements of complexation; this is more easily achieved using the submonomer approach to peptoids relative to preparation of amino acid derivatives for bPNA synthesis. Thus, peptoid cognates of bPNA were constructed using alkyl chains of two, three, and four carbons to link melamine to the peptoid backbone at alternate positions and carboxylic acid or hydroxy substituents in the remaining positions. The carboxylic acid side chains were installed using tert-butyl esters of glycine (G) and β-alanine (A) while unprotected ethanolamine was used to install the hydroxyl (H) side chain (Table 2.1). The synthetic base melamine (M) was incorporated in a solution-phase step following after solid-phase peptoid synthesis from the polyamino peptoid. Amine side chains were derived from mono-Boc-protected ethylene, propylene, and butylene diamines. Each peptoid was capped with glycine and 4-cetamidobenzamide

(ABA) as a chromophore to determine concentration. Following capping, the peptoids were globally deprotected, cleaved from resin, and purified by HPLC. Melamine bases were coupled to the amino side chains via nucleophilic aromatic substitution on 2-chloro-

4,6-diamino-1,3,5-triazine (Figure 2.3) and purified to homogeneity by HPLC. The chlorotriazine was readily prepared from double aminolysis of cyanuric chloride with ammonium hydroxide.

Figure 2.2. Submonomer synthesis of polyamino peptoids on Rink amide resin. Reagents and conditions: a) 2 M bromoacetic acid, DIC, HOBt in DMF; b) 2 M amine, DMF; c) 1) 2 M Fmoc-Gly, DIC, HOBt, DMF, 2) Fmoc deprotection and wash, 3) 2 M ABA, DIC, HOBt in DMF, 4) TFA cleavage from resin. Amines with R1 and R2 groups used are shown below.

Using these general procedures, a small family of triazine-displaying peptoids was constructed (Table 2.1). Though this peptoid family could in theory also be accessed using a preformed amino-alkyl melamine derivative rather than through reaction of a polyamino peptoid (Figure 2.3), alkylation with melamine submonomers was inefficient, as judged by the chloroanil test for secondary amines. This was likely due to the poor

51 solubility of the hydrochloride salt in DMF/NMP solvents, with low yields when in situ salt neutralization with a non-nucleophilic acid scavenger was attempted32. Thus, synthesis was simplified by installation of the triazines on the side chains after complete peptoid construction. Even with the mono-Boc-protected diamines, alkylation reactions were sluggish and not detectably accelerated through heating. Fortunately, microwave treatment33 in a conventional oven led to significantly improved yield sand was applied in both alkylation and acylation steps.

Figure 2.3. Side-chain installation of melamine bases. A representative reaction to produce (AbM)6 is shown.

2.3 Peptoid-DNA binding studies

The triazine peptoids were assessed for DNA binding by incubation with DNA of the general form dTnC4Tn. It was anticipated that the melamine bases would each coordinate with two thymine bases on the terminal tracts, folding the DNA into a triplex hairpin, as observed with bPNA. Peptoids with repeat units of n = 6 and 8 were prepared and

52 incubated with dT6C4T6 or dT8C4T8 DNA strands. Indeed, these peptoids were generally found to elicit a decrease in DNA (thymine) UV absorption; heating triggered a cooperative melting transition of the peptoid–DNA complex, indicating recognition of the thymine-rich tracts. The melts revealed a single cooperative binding interaction with

DNA, as indicated by a single peak in the first derivative analysis of the transition.

Additionally, incubation of peptoid with single stranded DNA resulted in a clear transition to a folded DNA structure by circular dichroism (Figure 2.4). Upon complexation, the positive Cotton peak observed in unstructured dT6C4T6 at λ = ca. 277 nm was diminished and the negative peak at λ = ca. 250 nm was red-shifted and increased in magnitude. Additionally, a large positive Cotton effect developed at lower wavelengths; all these spectral changes are consistent with triplex folding as observed with bPNA19-20.

A family of peptoids was prepared with variation in the linker length connecting melamine to the backbone as well as charge (Table 2.1). When glycine was used to incorporate an ethanoic acid side chain with an ethyl linker between the backbone and melamine to produce peptoid (GeM)6, there was no detectable binding with dT6C4T6

DNA. Similarly, (GpM)6, with a propyl linker between the backbone and melamine, also did not bind DNA. Interestingly, extension of the linker to butyl in a 5mer peptoid

(GbM)5 elicited decrease in thymine extinction, signifying base stacking, and a cooperative melting transition.

Figure 2.4. UV melting and CD of DNA-peptoids complexes. Top: Thermal transitions of 1:1 DNA-peptoids complexes monitored by UV absorbance at λ=260nm, with peptoids as indicated and dT6C4T6 DNA at 2µM concentration. (AbM)8 was complexed with dT8C4T8. Bottom: Representative circular dichroism spectra showing 1:1 peptoid-DNA complexation (20µM each) of dT6C4T6 alone (---) and dT6C4T6 complex with (HbM)6 (─)

This trend is consistent with relief of sterics that are prohibitive of macromolecular interaction upon lengthening the triazine linker to four carbons from three. This was further supported by the clear complexation signatures of (AbM)6, which features a β- alanine side chain and a butyl linker between backbone and

54 melamine. As expected, increasing the length of the interface increased the thermal stability of the complex, as observed in the complexation of (AbM)8 with dT8C4T8, which has a Tm of 25 °C as compared to 19 °C for the 6mer. The same thermal stability could be observed in a 6mer with neutral hydroxyethyl side chains, (HbM)6, instead of anionic carboxylates. This was expected based on decreased electrostatic repulsion between the neutral peptoid and the negatively charged DNA strand.

Table 2.1. Thermal Stability of peptoid-DNA complexesa

aThermal transitions were monitored at 260nm at 2µM peptoid and DNA in PBS buffer (pH=7.4). For comparison, 6mer and 8mer peptide analogues (bPNA) exhibit Tm values of 38℃ and 49℃, respectively.

Notably, the peptoids bound DNA much more weakly than their α-peptide bPNA cognates; a 6mer bPNA binds to dT6C4T6 DNA with a complex transition temperature of

20 38 °C . The origin of this difference is not clear. The 6mer peptoid (AbM)6 is isomeric with 6mer bPNA (Figure 2.1). Thus, the side chains were identical in both peptide and peptoid, but simply placed on the amide nitrogen in the peptoid rather than the α-carbon of the peptide. This peptoid–DNA complex is 19 °C lower in Tm than the isomeric bPNA–DNA complex. While disappointing, this is also a curious finding. Given the identical length of the side chains, the origin of the difference in DNA-binding affinity between peptide and peptoid isomers must derive from the backbone itself. The peptoid backbone is less polar than the peptide as a result of the tertiary amide linkages; the peptoid thus lacks amide NH hydrogen bond donors and only has carbonyl–hydrogen bond acceptors. Additionally, the peptide is composed of all L-amino acids, while the peptoid lacks chiral centers. These physical characteristics lead to conformational differences that could lead to decreased peptoid–DNA affinity27, 34-35. Indeed, backbone tertiary amides in conventional PNA backbones does not lead to DNA binding27, and peptoid backbones have been previously shown to display weaker DNA binding than peptides36. Thus, despite the identical spacing of the triazine bases in the peptoid and bPNA backbones, complexation is markedly more efficient with bPNA. It is possible that alternative side-chain designs could elevate the affinity of peptoids for DNA. These, and further structural studies, are currently under way.

2.4 Synthesis of monomers

General procedure for mono-Boc protection of diamines37

Diamine 1 (100mmol) was dissolved in 50mL DCM and cooled in ice bath. Boc anhydride (12.5mmol) was dissolved in 100mL DCM added to the diamine solution over

2 hours. The reaction was slowly warmed up to room temperature and stirred overnight.

The solvent was evaporated under vacuum and re-dissolved in 100mL saturated NaHCO3 solution, the water layer was extracted with DCM 50mL×3. The organic layer was dried with Na2SO4, then solvent was evaporated to get 2 as an oil.

6-chloro-1,3,5-triazine-2,4-diamine (3)38

Cyanuric chloride (100mmol, 18.4g) was dissolved in 150mL acetone, then poured into

150mL ice-cold water. Ammonium hydroxide 28% solution in H2O (53.7mL, 400mmol) was added into the reaction solution drop-wise under ice bath. The reaction was slowly warmed up to room temperature, then heat up to 50 ℃ in a sand bath. After reacting for another hour, the solution was cooled down to room temperature and filtered. The solid was washed with H2O three times and dried over vacuum overnight to obtain the desired product.

2.5 Peptoid synthesis and purification

Rink Amide resin (0.71mmol/g) was swelled in N,N-dimethylformamide (DMF anhydrous) at room temperature for 0.5h. The beads were then deprotected with 20%

(v/v) piperidine in DMF for 5min. The acylation step was carried in a microwave oven for 15s×2 with 2M bromoacetic acid in DMF and DIC/HOBt. After completion of this step (monitored by Kaiser test or chloranil test), 2M amine DMF solution was then subjected to the beads and react in the microwave oven for 15s×2. The above acylation and substitution steps were repeated until the desired length was achieved. The beads were then washed three times with DMF and three times with DCM, finally shrunk with

MeOH and dried over vacuum. 2mL of 95% trifluoroacetic acid (TFA) in H2O was added to the beads and incubated at room temperature for 2 hours. The slurry was filtered to remove the resin beads, the cleavage solution obtained was then evaporated under N2.

Cold diethyl ether (Et2O) was added to precipitate the peptoid, the crude pellet was washed with cold Et2O two times and dried over vacuum. Peptoids were purified by

HPLC on a reverse phase C18 column using a gradient from 0-20% solvent B in 50min

(solvent A=0.1%TFA in water, solvent B=0.1% TFA in 80% acetonitrile, 20% water).

The UV detector was set at 238 nm. The purified peptoids were lyophilized to dryness.

The identity of peptoid was checked by MALDI-TOF MS and purity checked by analytical HPLC on a C18 column.

The purified amino peptoid was then dissolved in 0.1M carbonate-bicarbonate buffer

(pH=10.0) to get a final concentration of 200 μM, 500 equivalence of 6-chloro-1,3,5- triazine-2,4-diamine was subjected to the solution and reacted at 85 ℃ overnight. The

58 reaction was then cooled down to room temperature and quenched with 1N HCl, passed through 0.22 μm filter and purified with HPLC to get the melamine containing peptoid.

2.6 UV-melting

UV-melting curves were measured on Carry-100 UV-vis spectrophotometer equipped with an air-circulating temperature controller. All measurements were carried out with temperature change rate of 1℃/min and monitored at 260nm. All samples are freshly annealed in 1X PBS buffer (pH=7.4) before measurements, concentration of DNAs and peptoids were both 2 μM.

2.7 Circular Dichroism (CD) spectroscopy

CD spectrums were obtained from Jasco J815 Circular Dichroism Spectrometer equipped with Peltier device and water circulator. All measurements were taken at 4 ℃ in a Hellma quartz cell (1mm path length) from 300-200 nm, data interval 0.5nm, band width 1nm and D.I.T. 2s. For each sample three scans were collected, averaged and corrected for blanks. All samples were freshly annealed in 1X PBS buffer (pH=7.4) before measurements.

2.8 Compound characterization

HPLC traces were obtain on a reverse phase C18 column using a gradient of (0-5 min 0% solvent B; 5-20 min 0-20% solvent B; 20-25 min 20% solvent B; 25-27 min 20-100% solvent B; 27-32 min 100% solvent B; 32-35 min 100-0% solvent B). Solvent A=0.1%TFA in water, solvent B=0.1% TFA in 80% acetonitrile, 20% water. The UV detector was set at 238 nm.

Figure 2.5. HPLC trace of (HbM)6, using the gradient described in the beginning of the section.

4000 Intens. [a.u.] Intens.

3000

2266.247

2000

1000

0 1000 1500 2000 2500 3000 3500 m/z Figure 2.6. MALDI-TOF MS of (HbM)6. Mass calculated: [M+H]+=2266.482, mass found: [M+H]+=2266.247, [M+Na]+=2288.207, [M+K]+=2304.199.

Figure 2.7. HPLC trace of (AbM)6, using the gradient described in the beginning of the section.

Figure 2.8. MALDI-TOF MS of (AbM)6 . Mass calculated: [M+H]+=2434.542, mass found: [M+H]+=2433.070.

Figure 2.9. HPLC trace of (AbM)8, using the gradient described in the beginning of the section.

Figure 2.10. MALDI-TOF MS of (AbM)8. Mass calculated: [M+H]+=3167.299, mass found: [M+H]+=3166.871.

2.9 References.

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1753.

2. Zerkowski, J. A.; MacDonald, J. C.; Seto, C. T.; Wierda, D. A.; Whitesides, G.

M., Design of organic structures in the solid state: molecular tapes based on the network of hydrogen bonds present in the cyanuric acid. cntdot. melamine complex. Journal of the

American Chemical Society 1994, 116 (6), 2382-2391.

3. Zerkowski, J. A.; Seto, C. T.; Whitesides, G. M., Solid-state structures of rosette and crinkled tape motifs derived from the cyanuric acid melamine lattice. Journal of the

American Chemical Society 1992, 114 (13), 5473-5475.

4. Whitesides, G. M.; Simanek, E. E.; Mathias, J. P.; Seto, C. T.; Chin, D.;

Mammen, M.; Gordon, D. M., Noncovalent Synthesis: Using Physical-Organic

Chemistry To Make Aggregates. Accounts of Chemical Research 1995, 28 (1), 37-44.

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Thermodynamic Stability of Hydrogen‐ Bonded Nanostructures: A Calorimetric Study.

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6. Ma, M.; Bong, D., Determinants of cyanuric acid and melamine assembly in water. Langmuir 2011, 27 (14), 8841-8853.

7. Ma, M.; Bong, D., Protein assembly directed by synthetic molecular recognition motifs. Organic & biomolecular chemistry 2011, 9 (21), 7296-7299. 63

8. Ma, M.; Bong, D., Directed peptide assembly at the lipid− water interface cooperatively enhances membrane binding and activity. Langmuir 2010, 27 (4), 1480-

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9. Ma, M.; Paredes, A.; Bong, D., Intra-and intermembrane pairwise molecular recognition between synthetic hydrogen-bonding phospholipids. Journal of the American

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10. Ma, M.; Gong, Y.; Bong, D., Lipid membrane adhesion and fusion driven by designed, minimally multivalent hydrogen-bonding lipids. J. Am. Chem. Soc 2009, 131,

16919-16926.

11. Kimizuka, N.; Kawasaki, T.; Hirata, K.; Kunitake, T., Supramolecular membranes. Spontaneous assembly of aqueous bilayer membrane via formation of hydrogen bonded pairs of melamine and cyanuric acid derivatives. Journal of the

American Chemical Society 1998, 120 (17), 4094-4104.

12. Kawasaki, T.; Tokuhiro, M.; Kimizuka, N.; Kunitake, T., Hierarchical self- assembly of chiral complementary hydrogen-bond networks in water: reconstitution of supramolecular membranes. Journal of the American Chemical Society 2001, 123 (28),

6792-6800.

13. Ariga, K.; Kunitake, T., Molecular recognition at air− water and related interfaces: complementary hydrogen bonding and multisite interaction. Accounts of chemical research 1998, 31 (6), 371-378.

14. SantaLucia Jr, J.; Hicks, D., The thermodynamics of DNA structural motifs.

Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 415-440.

15. Lange, R. F. M.; Beijer, F. H.; Sijbesma, R. P.; Hooft, R. W. W.; Kooijman, H.;

Spek, A. L.; Kroon, J.; Meijer, E. W., Crystal Engineering of Melamine–Imide

Complexes; Tuning the Stoichiometry by Steric Hindrance of the Imide Carbonyl

Groups. Angewandte Chemie International Edition in English 1997, 36 (9), 969-971.

16. Zhou, Z.; Bong, D., Small-molecule/polymer recognition triggers aqueous-phase assembly and encapsulation. Langmuir 2012, 29 (1), 144-150.

17. Arambula, J. F.; Ramisetty, S. R.; Baranger, A. M.; Zimmerman, S. C., A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding. Proceedings of the National Academy of Sciences 2009, 106 (38), 16068-16073.

18. Jahromi, A. H.; Nguyen, L.; Fu, Y.; Miller, K. A.; Baranger, A. M.; Zimmerman,

S. C., A Novel CUGexp·MBNL1 Inhibitor with Therapeutic Potential for Myotonic

Dystrophy Type 1. ACS Chemical Biology 2013, 8 (5), 1037-1043.

19. Zeng, Y.; Pratumyot, Y.; Piao, X.; Bong, D., Discrete Assembly of Synthetic

Peptide–DNA Triplex Structures from Polyvalent Melamine–Thymine Bifacial

Recognition. Journal of the American Chemical Society 2012, 134 (2), 832-835.

20. Piao, X.; Xia, X.; Bong, D., Bifacial peptide nucleic acid directs cooperative folding and assembly of binary, ternary, and quaternary DNA complexes. Biochemistry

2013, 52 (37), 6313-6323.

21. Xia, X.; Piao, X.; Fredrick, K.; Bong, D., Bifacial PNA complexation inhibits enzymatic access to DNA and RNA. ChemBioChem 2014, 15 (1), 31-36.

22. Xia, X.; Piao, X.; Bong, D., Bifacial Peptide Nucleic Acid as an Allosteric Switch for Aptamer and Ribozyme Function. Journal of the American Chemical Society 2014,

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Abiotic Template Interface. Journal of the American Chemical Society 2015, 137 (11),

3751-3754.

24. Nielsen, P. E., Peptide Nucleic Acid. A Molecule with Two Identities. Accounts of Chemical Research 1999, 32 (7), 624-630.

25. Huang, Y.; Dey, S.; Zhang, X.; Sönnichsen, F.; Garner, P., The α-helical peptide nucleic acid concept: merger of peptide secondary structure and codified nucleic acid recognition. Journal of the American Chemical Society 2004, 126 (14), 4626-4640.

26. Garner, P.; Dey, S.; Huang, Y., α-Helical Peptide Nucleic Acids (αPNAs): A New

Paradigm for DNA-Binding Molecules. Journal of the American Chemical Society 2000,

122 (10), 2405-2406.

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28. Zuckermann, R. N.; Kodadek, T., Peptoids as potential therapeutics. Curr. Opin.

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2007, 9 (4), 592.

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32. SCHNölzer, M.; ALEWOOD, P.; JONES, A.; ALEWOOD, D.; KENT, S. B., In situ neutralization in Boc‐ chemistry solid phase peptide synthesis. International journal of peptide and protein research 1992, 40 (3‐ 4), 180-193.

33. Olivos, H. J.; Alluri, P. G.; Reddy, M. M.; Salony, D.; Kodadek, T., Microwave- assisted solid-phase synthesis of peptoids. Organic letters 2002, 4 (23), 4057-4059.

34. Butterfoss, G. L.; Renfrew, P. D.; Kuhlman, B.; Kirshenbaum, K.; Bonneau, R., A preliminary survey of the peptoid folding landscape. Journal of the American Chemical

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3871-3886.

Chapter 3 : Small molecule recognition triggers secondary and tertiary interactions in DNA folding and

hammerhead catalysis

3.1 Overview

We have identified tris(2-aminoethyl)amine (tren)-derived scaffolds with two (t2M) or four

(t4M) melamine rings that can target oligo T/U domains in DNA/RNA. Unstructured T- rich DNAs cooperatively fold with the tren derivatives to form hairpin-like structures. Both t2M and t4M act as functional switches in a family of hammerhead ribozymes deactivated by stem or loop replacement with a U-rich sequence. Catalysis of bond scission in these hammerhead ribozymes could be restored by putative t2M/t4M refolding of stem secondary structure or tertiary bridging interactions between loop and stem. The simplicity of the t2M/t4M binding site enables programming of allostery in RNAs, recoding oligo-U domains as potential sites for secondary structure or tertiary contact. In combination with a facile and general method for installation of the t2M motif on primary amines, the method described herein streamlines design of synthetic allosteric riboswitches and small molecule-nucleic acid complexes.

3.2 A minimal binding motif for T/U rich nucleic acids

Most nucleic acid binding small molecules are natively derived1,1 obtained via library selection2, or designed to target pre-structured3-4, DNA and RNA or operate primarily via intercalation5. We report herein design and synthesis of small molecule tren derivatives that trigger the global folding of unstructured nucleic acid; further, these motifs mediate tertiary interactions between oligo-U domains in partially folded RNA. Binding is brokered by the same melamine base previously reported in bPNA, a family of peptoid6,

70 peptide7-11 and polyacrylates12-13 that triplex hybridize with DNA/RNA via base tripling melamine recognition14-18 with thymine/uracil. In contrast to artificial base pairs19-22, there are fewer examples of artificial base triples23, despite considerable interest in triplex structures24-26. Melamine base-triple docking to single TT/UU mismatches in structured nucleic acid is known to require linkage to an acridine intercalator27-28. Inspired by this design, we hypothesized that replacement of the non-specific intercalator with a second melamine ring would enable binding and folding of oligo T/U domains.

Figure 3.1. Overview of small molecule and RNA structures used in this chapter. (Top) Melamine-thymine/uracil triple in nucleic acid (NA) and tren scaffolds t2M, t3M and t4M. (a) Reductive alkylation of tren (R=Boc) with melamine acetaldehyde: 1) NaCNBH3, DMSO; 2) TFA. M=melamine. (Below) Schematic of minimal hammerhead ribozyme (HHmin) with tertiary AU contact indicated between enzyme (bold) and substrate (grey) strands (cleavage site=blue dashed). U-Stem (S-U4n) and U-loop (L-U4) enzymes were studied with “wild type” (wt) and U enriched (5U) substrates. Oligo-U / t4M binding sites are indicated.

Herein are described melamine-containing derivatives of tren, an open chain, sp3- hybridized scaffold, that can bind and trigger cooperative folding of unstructured oligo-T

DNAs. Further, these tren derivatives selectively bind oligo T/U bulges in partially folded DNA and RNA, functioning as allosteric triggers for hammerhead ribozyme catalysis via restoration of secondary and tertiary RNA structures29. While synthetic allosteric switches are well-established30-33, to our knowledge, this is the first report of de novo designed RNA-small molecule allostery that drives both secondary structure folding and formation of tertiary RNA interactions, effectively re-coding oligo-U domains as potential sites for structural turn-on.

A minimal oligo-T binding motif, t3M (Figure 3.1), is readily accessed in one step by triple arylation of tren with chloro-diaminotriazine. Incubation of t3M with dT10C4T10

DNA yielded a cooperatively melting complex (Tm=47°C), as observed by UV. This thermal signature only appeared with the complex, and disappeared upon methylation of the exocyclic melamine amines (t3MMe), supportive of specific melamine recognition.

Differential scanning calorimetry (DSC) indicated strongly exothermic t3M-DNA binding (-203 kcal/mole), consistent with prior studies on melamine-thymine8 and native19, 34-36 base-stacking (Figure 3.2). Binding occurs upon mixing, as evidenced by isothermal titration calorimetry (ITC, Figure 3.13, Figure 3.14) and circular dichroism, which revealed significant DNA folding upon addition of t3M (Figure 3.2).

Figure 3.2. Folding of dT10C4T10 DNA with t3M. (Top) Illustration of dT10C4T10 DNA complexation with 5 equivalents of t3M. (Left) CD of free dT10C4T10 DNA (┅) with increasing concentration of t3M from 10, 15, 20, 25, 50 (

┄) to 100 µM (—). (Right) Thermal denaturation of the dT10C4T10●t3M complex at 1:5 mole ratio followed by UV260 (---) and DSC (—). DNA concentration=2 µM (UV), 25 µM (DSC). All experiments run in 1X PBS, pH 7.4.

Titration of t3M against Fl-dT10C4T10-Dab DNA resulted in fluorescein (Fl) quenching by dabcyl (Dab), supportive of formation of a hairpin structure (Figure 3.12, Figure 3.18) with 5:1 t3M:DNA stoichiometry. Given the expected 2:1 thymine to melamine (T:M) ratio8, we hypothesized that only 2 melamine rings per t3M unit were needed to bind dT10C4T10. Indeed, t2M, with 2 melamine rings, exhibited the same 5:1 DNA stoichiometry, similar heat of binding (-226 kcal/mole) and has an available primary amine for labeling. Fluorescein-derivative t2M-Fl* bound to a tetrathymidine bulge in the

DNA structure 12-T2-12, a putative t2M binding site flanked by two 12mer duplex regions (Figure 3.3). Anisotropy measurements indicated micromolar affinity 1:1 binding to the tetrathymidine site for t2M-Fl* (Figure 3.3), confirmed by native gel-staining 12-

T2-12 with Cy5-labeled t2M (Figure 3.17). Alternative scaffolds were also prepared, but failed to improve upon binding or synthetic versatility of the tren derivatives (Table 3.1).

3.3 Extension of t2M motif: t4M

We hypothesized that increasing the number of bases while maintaining the t2M pattern would increase binding affinity. Thus, t4M, with four melamine rings on tren, was prepared by reductive alkylation of mono-protected tren (Figure 3.1). Gratifyingly, t4M exhibited enhanced DNA affinity, with an expected 2.5:1 stoichiometry of binding to a

Fl-dT10C4T10-Dab probe, and robust affinity to an octathymidine bulge in duplex DNA

12-T4-12 (Kd=50 nM). Methylation of the melamine rings in t2M and t4M abolished all

DNA binding, indicating that electrostatic interactions do not drive affinity (Figure 3.10,

Figure 3.11).

Figure 3.3. Binding ratio and affinity of t2M and t4M to T rich DNAs. (Left) Fluorescence quenching in a Fl-dT10C4T10-Dabcyl DNA is induced by t2M(●)/t4M(■). Formation of the higher order complex with t2M is shown schematically. (Right) Anisotropy binding isotherms of t2M-Fl* and t4M-Fl* to Tn-bulges (red) in DNA duplexes [12-T2-12] (●) and [12-T4-12] (■), respectively. Fits to a 1:1 binding model are shown (—) for t2M and t4M, with Kd’s ~4.2 µM and 44 nM, respectively. 74

3.4 Quaternary t3M : qt3M

In the process of looking for minimal binding motif for T/U rich nucleic acids, we also investigated the possibility of quaternation of tren scaffold, with the fourth arm on central nitrogen a synthetic handle for further modifications. It was found that qt3M alkyne was also able to fold unstructured dT10C4T10, with similar enthalpy to t3M and t2M (-199 kcal/mol), albeit lower thermal stability (46 °C by DSC).

3.5 Ribozyme rescue by small molecule triggers

To test the extent to which these molecules could be used to complement native nucleic acid function, we designed deactivated type I hammerhead U-ribozymes in which critical secondary and tertiary structural elements were ablated by replacement with oligo-U loops. Tertiary interactions between loop II and stem I are known to be essential in catalysis, with recent reports37 identifying a single essential A□○U Hoogsteen pair between loop (enzyme) and stem (substrate) in a binary hammerhead system. Despite the sensitivity of the hammerhead ribozyme to cations38-42, tetrabasic permethylated t4M

(t4MMe) did not activate RNA cleavage in any of the U-ribozymes studied. We first set out to test the extent to which t2M and t4M could restore catalysis by generating secondary structure alone. U(2,3) is a minimal ribozyme in which stems II and III are replaced with oligo-U loops. It therefore lacks secondary structure in stems II and III, and further, does not have the loop-stem sequences required for rate-enhancing tertiary interactions; bond scission is completely deactivated. Consistent with secondary

75 structural rescue, addition of t2M or t4M restored cleavage, with t4M exhibiting much greater efficacy (Figure 3.4).

Figure 3.4. U23 cleavage restored by t4M. (Left) U23 deactivated ribozyme, treated with t4M to restructure stems with oligo-U domains (red) as t4M binding sites. (Right) Cleavage of U23 (blue dashed) upon treatment with 10 µM t4M (■) or t4MMe (□) and RNA only (○) at 10 mM Mg2+ with 500 nM ribozyme, with 10 µM t4M in 1X Tris-Cl, pH=7.6 at 37℃.

We then test the extent to which t4M could simultaneously rescue secondary and tertiary interactions using S-U4n, a ribozyme in which stem II is replaced with 4 UU mismatches, but retains the GUGA sequence in loop II and U in position 1.7 in stem I, required for tertiary AU contact37, 43. The partially destructured S-U4n still cleaves itself at 10 mM magnesium, but was only weakly active at 0.1 mM Mg2+. Under low magnesium conditions, addition of t4M significantly enhanced S-U4n cleavage (Figure 3.5). Further, extension of stem I by two base pairs in S-U4n-2bp resulted in greatly diminished activity

(Figure 3.5). The two base extension in stem I is remote from the catalytic site, and thus the loss of activity likely indicates disruption of stem I tertiary interactions essential to

76 catalysis. Together, these data support the notion that t4M restructures stem II in such a way that facilitates both secondary and tertiary interactions within the RNA fold.

Figure 3.5. S-U4n and S-U4-2bp cleavage restored by t4M. Cleavage reactions (0.1 mM Mg2+, 500 nM ribozyme, with t4M in 1X Tris-Cl, pH=7.6. 37℃) of the indicated U-ribozymes is illustrated, with U4 domains indicated (red) as t4M binding sites. (A) S-U4n ribozyme, treated with t4M (■) at indicated concentrations, 10 µM t4MMe (-□-) and alone (○). (B) S-U4n-2bp ribozyme (extended by 2 bp on stem I) upon treatment with 10 µM t4M (▲) and alone (△).

We hypothesized that tertiary RNA interactions between loop II and stem I could be directly bridged by t4M upon replacement of discrete RNA contacts with U:M:U base triples. With all secondary structures intact in a binary hammerhead ribozyme system, tetraloop II (GUGA) was replaced with either GUUU or UUUU to yield L-GU3 and L-

U4, respectively. Substrates with U at positions 1.7 (native), 1.7-1.8 (UU), and 1.7-1.11

(UUUUU) in the unpaired region were studied under low magnesium conditions. While 77

L- GU3 was inactive with native substrate, considerable cleavage of the 2U substrate was observed, despite the absence of AU pairing. This suggests a plastic tertiary structure in this minimal hammerhead ribozyme, with other interactions, such as a GU wobble, possibly taking the place of the AU contact between loop II and stem I. Such a possibility is eliminated in L-U4, which exhibited greatly diminished cleavage activity with all substrates. Addition of t4M significantly increased cleavage of 2U and 5U substrates, while having no effect on the wt substrate, consistent with rescue of the loop-substrate tertiary interaction via t4M bridging of the U-domains (Figure 3.6). Interestingly, the reported structure44 of the full-length hammerhead ribozyme revealed a UAU base triple between the adenine of the enzyme loop and 2U’s in the substrate; similar triples have been observed in other functional RNAs26, 45-46. It is intriguing to speculate that t4M creates a similar type of tertiary interaction via bifacial melamine-uridine recognition.

Figure 3.6. Binary ribozyme cleavage restored by t4M. (Left) L-U4 ribozyme (boxed) with the HHmin (wt) substrate (grey) with U in position 1.7 indicated (bold black) and ablated tertiary interaction indicated (---). L-U4 reacts with the 5U substrate (grey) with loop-stem binding mediated by t4M. (Right) L-U4 cleavage of 5U substrate with 10 µM t4M (■), 10 µM t4MMe (□), no additive (○) and cleavage of wt substrate with t4M (■). Reaction conditions: 4 µM enzyme, 320 nM Cy3 substrate, 10 µM t4M/t4MMe, 0.1 mM Mg2+ in 1X Tris-Cl buffer, pH=7.6, 27℃. 78

3.6 Conclusion

We have described herein design and synthesis of a minimal tren-based molecular motif for binding oligo T/U sequences in DNA and RNA, resulting in cooperative folding and structure-function turn-on9. While elegant systems are known in which small molecules trigger folding and chemistry in aptamers47-49, to our knowledge, this is the first report of synthetic small molecules that can trigger global folding of unstructured oligo T/U nucleic acid. The t4M derivative acts as a chemical inducer of dimerization50 for two short oligo-U domains, effectively generating tertiary interactions in RNA by serving as a synthetic molecular bridge. Binding sites for these small molecules are readily engineered into partially folded nucleic acids, potentially generalizing the design of compact, functional allosteric9, 51-52 switches sensitive to t2M derivatives in a wide range of RNAs.

Finally, the t2M motif may be easily installed on any primary amine, thus enabling versatile molecular targeting to nucleic acids for packaging, delivery, labeling and other biotechnology48-49, 53-54 applications.

3.7 Materials and general experimental procedures

All chemicals were used without further purification from commercial sources, unless otherwise noted. DNAs and labeled RNAs were purchased from Integrated DNA

Technologies (IDT) and used without further purification. SYBRⓇ gold was purchased from Thermo Fisher Scientific. DNA stock solutions were serially diluted in deionized water and concentrations were determined by measuring solution absorbance at 260 nm

79 by Thermo Fisher Nanodrop 2000. Sample fluorescence was measured on Thermo Fisher

Nanodrop 3300. RNA constructs were prepared by in vitro transcription except where noted. The promoter sequence in each DNA template is underlined and does not appear in the final RNA transcripts. 1X PBS buffer was prepared in house with a final

3- concentration of 137 mM NaCl, 2.7 mM KCl and 10 mM PO4 at pH 7.4. Tris-Cl buffer was prepared in house with tris(hydroxymethyl)aminomethane, HCl was used to adjust pH to 7.6, final concentration of 1X buffer was 50 mM.

3.7.1 Nucleic acid sequences used

12-Tn-4: 5’-CGC ATA TTT GCG-Tn-CCAG-3’ 3’-GCG TAT AAA CGC-Tn-GGTC-3’

12-T2-12: 5’-CGC ATA GCT CAG TTG ACT CGA TAC GC-3’ 3’-GCG TAT CGA GTC TTC TGA GCT ATG CG-5’

The following constructs were prepared by in vitro transcription, promoter sequence is underlined and will not appear in final RNA transcripts.

Transcription protocol. RNA constructs were made using T7 runoff transcription. RNA transcription buffer 10X: 1M HEPES-KOH pH 7.5, 100 mM MgCl2, 20 mM

Spermidine-HCl, 400 mM DTT. Transcription was performed in the following concentrations: 1x Buffer with the addition of 10 mM DTT, 10 mM MgCl2, 20 mM rNTP, 7.5% glycerol, 350 nM DNA template, and 2 μL of T7 polymerase stock (per 100

μL of transcription reaction solution, conditions subject to optimization based on sequences). Transcription took place at 37℃ for 2 h at which point 1.5 equivalents of

EDTA was added to quench the reaction and equal volume of 2X TBE/urea loading

80 buffer was added. Samples were heated at 95℃ for 5 minutes, cooled on ice and loaded onto acrylamide (19:1 acrylamide:bisacrylamide) denaturing gel (8.3cm X 7.3cm X

1.5mm) with 8M urea as the denaturant. The RNA was visualized using UV shadowing, cut out from the gel, crushed through a syringe, and soaked in water overnight while rotating at room temperature. The sample was then centrifuged at 5000 rpm for 2 min and the supernatant was removed, discarding the gel pieces. The RNA was precipitated by the addition of 10% volume of 5M NH4OAc pH 5.2 and 2.5 volume equivalents of 200 proof ethanol followed by incubation at -20°C for at least 2hrs and centrifugation at high speed for 10 minutes at 4℃. The RNA pellet was washed with 500 μL of ice-cold 70% ethanol in water and centrifuged again at high speed for 10 minutes at 4℃. RNA pellets were then dried in a speed-vac at room temperature for 20 minutes and suspended in water and stored at -20℃. Stock concentrations were determined by UV-vis spectroscopy, measuring absorbance at 260 nm. RNA purity was checked on a denaturing gel and visualized using SYBRⓇ gold staining.

• U-(2,3) ribozyme: Template: 5'-TCA CTG TAA AGA GGT GTT GGT TCT CTT AAT CTT TAA CTT AAA AGG TTA ATG CTA AGT TAG CTT TAC AGT GCG ACA AAA AAA AAA TCT CAA AAA AAA AAG TTT CGA AAA AAT CTC AAA AAA CTC ATC AGG CAC TGC CTA TAG TGA GTC GTA TTA ATT TC-3'

Transcript: 5'-GG CAG UGC CUG AUG AGU UUU UUG AGA UUU UUU CGA AAC UUU UUU UUU UGA GAU UUU UUU UUU GUC GCA CUG UAA AGC UAA CUU AGC AUU AAC CUU UUA AGU UAA AGA UUA AGA GAA CCA ACA CCU CUU UAC AGU GA-3'

• S-U4n ribozyme: Template: 81

5'-GCC GGG GGT GGG ATT TGA ACC CAC GTA AGG CGG ATC TGC AGT CCG CTG CCT AGC CCC TAG ACT ACC CCG GCT GGT AGA CTG TGA CCC AGT CTC CTG GGT TTC GAA AAC TCA CGA AAA CTC ATC AGA CAG TCT ATA GTG AGT CGT ATT A-3'

Transcript: 5'-G ACU GUC UGA UGA GUU UUC GUG AGU UUU CGA AAC CCA GGA GAC UGG GUC ACA GUC UAC CAG CCG GGG UAG UCU AGG GGC UAG GCA GCG GAC UGC AGA UCC GCC UUA CGU GGG UUC AAA UCC CAC CCC CGG C-3'

• S-U4n-2bp ribozyme: Template: 5'- GCC GGG GGT GGG ATT TGA ACC CAC GTA AGG CGG ATC TGC AGT CCG CTG CCT AGC CCC TAG ACT ACC CCG GCT GGT AGA GCC TGT GAC CCA GTC TCC TGG GTT TCG AAA ACT CAC GAA AAC TCA TCA GAC AGG CTC TAT AGT GAG TCG TAT TA-3'

Transcript: 5'-GAG CCU GUC UGA UGA GUU UUC GUG AGU UUU CGA AAC CCA GGA GAC UGG GUC ACA GGC UCU ACC AGC CGG GGU AGU CUA GGG GCU AGG CAG CGG ACU GCA GAU CCG CCU UAC GUG GGU UCA AAU CCC ACC CCC GGC-3'

• Binary HHR system. Enzyme strands: Wildtype: Template: 5'-TGG GTT TCG TCC TCA CGG ACT CAT CAG ACA GTC TAT AGT GAG TCG TAT TA-3' Transcript: 5'-G ACU GUC UGA UGA GUC CGU GAG GAC GAA ACC CA-3'

UU loop: Template: 5'-TGG GTT TCG TCC AAA CGG ACT CAT CAG ACA GTC TAT AGT GAG TCG TAT TA-3' Transcript: 5'-G ACU GUC UGA UGA GUC CGU UUG GAC GAA ACC CA-3'

4U loop: Template: 5'-TGG GTT TCG TCC AAA AGG ACT CAT CAG ACA GTC TAT AGT GAG TCG TAT TA-3' Transcript: 5'-G ACU GUC UGA UGA GUC CUU UUG GAC GAA ACC CA-3' 82

The following RNA sequences were purchased from IDT and used without further purification.

• Binary HHR system substrate strands: HHmin (wt): 5'-Cy3-UGGGUCAC--AGUCUCCAAUCC-3' UU: 5'-Cy3-UGGGUCAC--AGUCUUCAAUCC-3' 5U: 5'-Cy3-UGGGUCAC--AGUCUUUUU-3' Scissile bond: C--A

Figure 3.7. Secondary structure representation of the binary HHRs. Scissile bond shown in red between C17 and A1.1. Modified bases in the loop and stem I shown in red.

3.7.2 UV-melting

UV-melting curves were measured on Cary-100 UV-vis spectrophotometer equipped with an air-circulating temperature controller. All measurements were carried out with 83 temperature change rate of 1℃ /min and monitored at 260 nm. All samples were freshly annealed in 1X PBS buffer (pH=7.4) before measurement, concentration of DNAs is 2

μM, while the tren derivatives are held at 20 μM unless otherwise noted.

3.7.3 Circular Dichroism(CD) spectroscopy.

CD spectra were obtained from Jasco J815 Circular Dichroism Spectrometer equipped with Peltier device and water circulator. All measurements were taken at 25℃ in a

Hellma quartz cell (1mm path length) from 320-220 nm, data interval 0.5 nm, bandwidth

1 nm and D.I.T. 2s. For each sample, three scans were collected, averaged and corrected for blanks. All samples were freshly annealed in 1X PBS buffer (pH=7.4) before measurements.

3.7.4 Isothermal Titration Calorimetry (ITC).

ITC experiments were performed on a Nano ITC (TA instruments). DNA samples were diluted to 10 µM concentration in 1X PBS buffer (pH=7.4), small molecule were dissolved in 1X PBS to desired concentrations. Both samples were degassed and subjected to ITC experiment immediately. Reservoir contains 300 µl of DNA solution and micro syringe contains 50 µl of small molecule solution. Stirring rate was set at 400 rpm, injection volume at 2.02µl with 250 s between each injection.

3.7.5 Differential Scanning Calorimetry (DSC).

DSC experiments were carried out on Microcalorimeter VP-DSC. Samples were prepared to have 25 µM DNA and 250 µM tren small molecules in 1X PBS buffer (pH=7.4).

Samples were scanned from 25 ℃ to 90 ℃ with 60℃/h scanning rate, 16 s filtering period and low feedback. 1X PBS was used as reference. Background data was collected with only 25 µM DNA in the sample cell and was subtracted from all DNA/tren sample traces.

3.7.6 Fluorescence quenching assay.

FAM- T10C4T10 -Dab DNA, end-labeled with fluorescein (FAM) and DABCYL (4-(4’- dimethylaminophenylazo) benzoic acid) at the 5’ and 3’ ends, respectively, was purchased from IDT. DNA stock solutions were made by mixing 95 µM dT10C4T10 and 5

µM FAM-T10C4T10-Dab, due to limited amount of FAM-T10C4T10-Dab available.

Samples were prepared by annealing 50 µM DNA stock and the tren derivatives (0-2 mM) in 1X PBS buffer prior to the experiment. Fluorescence of each sample was measured on a ThermoFisher Nanodrop 3300.

3.7.7 Fluorescence anisotropy.

A series of samples were made by mixing various concentrations of T6C4T6 and constant concentration of FITC labelled tren derivatives (100 nM) in 1X PBS. All samples were annealed at 95℃ for 5min. Experiments were carried out on Molecular Devices

SpectraMax M5 with excitation wavelength at 495 nm and emission at 520 nm.

Fluorescence anisotropy was converted into complex concentration using equation 1:

퐹퐴 − 퐹퐴푚푖푛 [푐표푚푝푙푒푥] = ( ) ∗ [푡푟푒푛] 퐹퐴푚푎푥 − 퐹퐴푚푖푛

Where FA, FAmax, FAmin correspond to current fluorescence anisotropy, maximum fluorescence anisotropy (FITC labelled tren derivative fully bound to DNA) and minimum fluorescence anisotropy (FITC labelled tren derivative alone), respectively.

[complex] indicates the current complex concentration, [tren] indicates the concentration of FITC labelled tren derivatives (100 nM).

The concentration of complex was plotted against total DNA concentration in each sample, the data was fitted using equation 2:

[푐표푚푝푙푒푥] = (퐾푑 + [퐷푁퐴] + [푡푟푒푛])/2

− (√(퐾 + [퐷푁퐴] + [푡푟푒푛])2 − 4 ∗ [퐷푁퐴] ∗ [푡푟푒푛])/2

3.7.8 Electrophoretic mobility assay (EMSA)

A series of samples were made by mixing various concentrations of DNA duplexes and constant concentration of Cy5-t2M (1µM) in 1X PBS. All samples were incubated at room temperature for 30 min and then subjected to electrophoresis in a 15% TBE buffered native acrylamide gel at 120 V on ice. The gel was then scanned using Typhoon

FLA 9500 (GE Healthcare).

3.7.9 Additional scaffolds tested for DNA binding.

Alternate scaffolds based on the tren scaffold were tested for DNA binding. These compounds (Table S1.1) exhibited similar or diminished DNA binding as judged by preliminary thermal denaturation studies with dT10C4T10 DNA. With the exception of t2M and t4M, these compounds were not studied in further detail. Compounds with similar thermal stability as t3M and t2M (10 & 11) were also less versatile scaffolds in that additional functionalization of the scaffold was not straightforward. Regardless, these preliminary data indicate where the tren design is corroborated (similar atom spacing between base triples in 10 and 11) as well as the limits of this design. For instance, compounds 8 and 9 exhibit weak or undetectable thermal transitions, possibly due to increased steric interactions upon rigidification of the scaffold.

Table 3.1 DNA complexation dataa.

Name Structure Tm with dT10C4T10

t4M, 6 66°C

t3M, 1 47°C

10 46°C

t2M, 2a 35°C (Boc t2M)

11 34°C

8 17°C

9 Not detected

a Tm is measured with solution concentrations of 2µM DNA and 20µM synthetic compound in 1X PBS. 88

3.8 Additional data

Table 3.2 Summary of tren derivatives DNA binding data from DSC. Derivative DNA Tm (°C) ΔH (kcal/mol) Molar ratio

t3M T10C4T10 58 -203 5:1

Boc-t2M T10C4T10 51 -226 5:1

qt3M T10C4T10 46 -199 NA

Me Figure 3.8. UV-melting of t3M and t3M with dT10C4T10. Samples were prepared in 1X PBS, DNA concentrations were 2 µM, Tren derivatives concentrations were 20 µM. (Left) UV-melting traces show the change in absorbance at 260 nm with respect to temperature. (Right) First derivatives of the UV-melting traces with respect to temperature.

Figure 3.9. UV-melting of Tren derivatives with dT10C4T10. Samples were prepared in 1X PBS, DNA concentrations were 2 µM, Tren derivatives concentrations were 20 µM. (Left) UV-melting traces show the change in absorbance at 89

260 nm with respect to temperature. (Right) First derivatives of the UV-melting traces with respect to temperature.

Me Figure 3.10. UV-melting t2M and t2M with dT10C4T10. Samples were prepared in 1X PBS. DNA concentrations were 2 µM. Tren derivatives concentrations were 20 µM. (Left) UV-melting traces show the change in absorbance at 260 nm with respect to temperature. (Right) First derivatives of the UV-melting traces with respect to temperature.

Me Figure 3.11. UV melting of t4M and t4M with dT10C4T10. Samples were prepared in 1X PBS. DNA concentrations were 2 µM. Tren derivatives concentrations were 10 µM. (Left) UV-melting traces show the change in absorbance at 260 nm with respect to temperature. (Right) First derivatives of the UV-melting traces with respect to temperature.

Figure 3.12. Job plots of molecular beacon fluorescence quenching with t3M and t2M. Various amounts of tren derivatives (0-2 mM) were titrated into the 50 µM DNA solution of 19:1 dT10C4T10 : FAM-T10C4T10-Dab in 1X PBS. Fluorescence was measured on Thermo Fischer Nanodrop 3300.

Figure 3.13. ITC raw heat of t3M (600µM, 50µl, 1XPBS) injected into dT10C4T10 (10µM, 300µl, 1XPBS).

Figure 3.14. ITC raw heat of t3M (200µM, 50µl, 1XPBS) injected into 12-T2-12 (20µM, 300µl, 1XPBS).

Figure 3.15. DSC curves of 25 µM dT10C4T10 with Boc t2M or t3M. Tren derivatives concentration was 250 µM. Samples were prepared in 1X PBS buffer (pH=7.4) and scanned from 25 °C to 90 °C with 60 °C/h scanning rate, 16 s filtering period, and low feedback.

Figure 3.16. DSC trace of qt3M alkyne with dT10C4T10. DNA concentration is 25 µM while the small molecule concentration is 250 µM.

Figure 3.17.Gel shift of Cy5-t2M with 12-T2-12 (left) and 12-T0-4 (right). Cy5-t2M concentration is 1µM in each lane, while the DNA concentrations were 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100µM from left to right in each gel. The bands in the left gel indicate the stained DNA Cy5-t2M complex.

Figure 3.18. UV melting of 50 µM t3M with vaious concentrations of dT10C4T10. Samples were annealed at 95 °C in 1X PBS right before the experiment. (Left) UV- melting traces show the change in absorbance at 260 nm with respect to temperature. (Right) First derivatives of the UV-melting traces with respect to temperature.

3.9 Synthesis

Figure 3.19. Synthetic scheme for t3M.

N2-(2-(bis(2-((4,6-diamino-1,3,5-triazin-2-yl)amino)ethyl)amino)ethyl)-1,3,5-triazine-

2,4,6-triamine (t3M, 1). (Figure 3.19)

Tris(2-aminoethyl)amine (0.75 mL, 5 mmol) was dissolved in 30 mL H2O, followed by addition of diaminochlorotriazine(4.36g, 30 mmol) and NaHCO3(1.5 g, 18 mmol), the reaction was heated to 85°C and let stir overnight. After cooled down to room temperature, the solution was filtered, the solid was washed with water twice and dried 94 under vacuum (1.63g, 73%). The crude solid was further purified by prep HPLC using gradient of 0% to 15% acetonitrile over 40 minutes. 1H NMR: 3.68 (6H, t); 3.88 (6H, t);

13C NMR: 162.84; 159.11; 156.55; 52.61; 35.49; ESI: mass calculated[M+H]+=474.2770;

[M+2H]2+=237.6421; mass observed: 474.2788; 237.6426.

Figure 3.20. Synthetic scheme for t2M and t2MMe.

2-(tert-butoxylcarbonylamino)ethyl-bis-[(2-aminoethyl)]amine. (Figure 3.20)

Tris(2-aminoethyl)amine (6 mL, 40 mmol) was dissolved in 50 mL DCM and cooled in ice bath. A solution of Boc anhydride (1.09g, 5 mmol) in 20 mL DCM was added dropwise, followed by dropwise addition of triethylamine (0.7mL, 5 mmol) in 20 mL

DCM. The reaction was warmed up to room temperature slowly and stirred overnight.

After removal of solvent under reduced pressure, the crude oil was re-suspended in 50 mL H2O and extracted with DCM (50 mL*5). The organic phase was dried over Na2SO4 and solvent removed under reduced pressure. Chromatographic purification of the crude product (DCM:MeOH=5:1, 2% concentrated ammonium hydroxide) yielded 700 mg

(57%) as light yellow oil.

N2-(2-((2-aminoethyl)(2-((4,6-diamino-1,3,5-triazin-2-yl)amino)ethyl)amino)ethyl)-1,3,5- triazine-2,4,6-triamine (t2M, 2a). (Figure 3.20)

6-chloro-2,4-diamino-1,3,5-triazine(550 mg, 3.78mmol) and NaHCO3 (500mg,

5.95mmol) was added into 40 mL H2O solution of 2-(tert-butoxylcarbonylamino)ethyl- bis-[(2-aminoethyl)]amine (220mg, 0.89mmol). The slurry was heated to 85°C and reacted overnight. The reaction was then cooled down and filtered, the solid was washed with water twice and dried under vacuum. The crude was then purified by silica gel chromatography using DCM: MeOH: conc NH4OH=80:20:2 to yield Boc t2M as white solid (90 mg, 22%). 1H NMR: 1.32 (9H, s); 3.45 (8H, m); 3.62 (4H, m); 13C NMR: 27.52;

35.39; 35.97 54.04; 54.68; 81.82; 158.23; 164.98; 165.71; ESI: mass calculated:

[M+H]+=465.2905; mass observed: 465.2914.

Boc t2M was dissolved in TFA and reacted at room temperature for 10min, TFA was evaporated under N2 flow, the residue was dissolved in H2O and lyophilized to obtain the

TFA salt of t2M. 1H NMR: 3.43 (2H, m); 3.52 (4H, m); 3.66 (2H, m); 3.74 (4H, m); 13C

NMR: 166.73; 162.54; 159.25; 156.62; 52.76; 49.95; 35.58; 33.43; ESI: mass calculated[M+H]+=365.2381; [M+2H]2+=183.1227; mass observed: 365.2368; 183.1238.

N2-(2-((2-aminoethyl)(2-((4,6-bis(dimethylamino)-1,3,5-triazin-2- yl)amino)ethyl)amino)ethyl)-N4,N4,N6,N6-tetramethyl-1,3,5-triazine-2,4,6-triamine

(t2MMe, 2b). (Figure 3.20)

6-chloro-N2,N2,N4,N4-tetramethyl-2,4-diamino-1,3,5-triazine(80 mg, 0.4mmol) and

NaHCO3 (84mg, 1mmol) was added into 10 mL 1,4-dioxane:H2O=1:1 solution of 2-(tert-

96 butoxylcarbonylamino)ethyl-bis-[(2-aminoethyl)]amine (40mg, 0.162mmol). The slurry was heated to 85°C and reacted overnight. The reaction was then cooled down and extracted with DCM twice, organic layer was then dried and condensed to obtain a crude mixture, which was purified by silica gel chromatography using 2% MeOH in DCM with

2% concentrated ammonia in MeOH. The product was obtained as a white solid (45mg,

48.4%). 1H NMR: 1.41 (9H, s); 2.61 (6H, m); 3.05 (26H, m); 3.37 (4H, m); 5.29 (2H, t);

5.55 (1H, t); 13C NMR: 166.44; 165.92; 156.38; 78.99; 53.69; 38.93; 36.07; 28.54 ; ESI: mass calculated: [M+H]+=577.4157; [M+2H]2+=289.2115; mass observed:577.4148;

289.2105.

Boc t2MMe was dissolved in TFA and reacted at room temperature for 10min, TFA was evaporated under N2 flow, the residue was dissolved in H2O and lyophilized to obtain the

TFA salt of t2MMe. 1H NMR: 3.04 (24H, d); 3.33 (2H, m); 3.35 (6H, m); 3.78 (4H, m);

13C NMR: 162.25; 156.47; 156.62; 52.73; 51.07; 37.38; ESI: mass calculated:

[M+H]+=477.3633; [M+2H]2+=239.1853; mass observed: 477.3615; 239.1857.

Figure 3.21. Synthetic scheme for t2M alkyne.

97 t2M alkyne (3). (Figure 3.21)

N-(2-(bis(2-((4,6-diamino-1,3,5-triazin-2-yl)amino)ethyl)amino)ethyl)-hex-5-ynamide.

Di-tert-butyl (((2-aminoethyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate (di-Boc tren) was synthesized based on literature procedure55. 5-Hexynoic acid (0.1 mL, 0.97 mmol) was mixed with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (139 mg, 0.9 mmol) in DCM and reacted for 0.5 h. Di-Boc tren (175 mg, 0.5 mmol) was then added to the solution and reacted for 2h. The reaction was diluted to 50 mL DCM, washed with 30 mL 1N HCl (aq), 50 mL saturated NaHCO3 (aq), and 50 mL brine. The organic layer was then dried and condensed to obtain the crude product which was purified on silica gel column with a DCM/MeOH gradient from 20:1 to 10:1 to obtain 150 mg of di-Boc tren alkyne as a white solid (68%). Di-Boc tren alkyne was dissolved in 10 mL of 1:1

DCM/TFA and reacted at room temperature for 30 min. The solution was condensed to syrup under a stream of N2 before cold Et2O was added to form a white precipitate. The precipitate was centrifuged down, washed with cold Et2O and dried under vacuum to yield the TFA salt of tren alkyne. It was then dissolved in H2O and adjusted to pH=9 with

NaHCO3. 6-chloro-2,4-diamino-1,3,5-triazine was then added to the solution and additional NaHCO3 in order to keeping the pH at 9. The slurry was heated to 85 °C and reacted overnight. After cooling down to room temperature, a white precipitate formed which was centrifuged and washed with water to yield crude t2M alkyne 3. It was further purified by prep HPLC using gradient of 0% to 20% acetonitrile over 40 minutes. 1H

NMR: 1.77 (2H, m); 2.24 (2H, t); 2.38 (3H, m. terminal alkyne proton buried under);

3.56 (2H, t); 3.62 (6H, m); 3.85 (4H,t); ESI: mass calculated[M+2H]2+=230.1436; mass observed: 230.1452.

Figure 3.22. Synthetic scheme for FITC-t2M.

Typical procedure for installation of fluorescein on tren derivatives.

56 FITC-N3 was synthesized according to literature procedure .

Fl-t2M (Figure 3.22): To an aqueous solution of t2M alkyne (18 mM, 10 µl) was added

FITC-N3 in DMSO (300 mM, 10 µl) and 50µl of 1M Tris-Cl (pH=8.0). Then CuSO4(0.1

M, 10 µl) and sodium ascorbate (0.1 M, 20 µl) solution was added. The mixture was then reacted for 1h at room temperature before quenched by addition of 1N HCl. Product was purified by HPLC on C18 column to yield FITC-t2M (6.4 mM, 50 µl, 89%). ESI: mass calculated [M+H]+= 934.3750; [M+2H]2+=467.6911; [M+3H]3+=312.1299; mass observed: 934.3610; 467.6890; 312.1285.

Figure 3.23. Synthetic scheme for Cy5-t2M.

Cy5-t2M (Figure 3.21): Cy5 free acid was synthesized according literature procedure57.

The free acid (15 mg) was then dissolved in 5 mL DCM, followed by addition of 7 mg of

EDC and 8 mg of N-hydroxysuccinimide (NHS). The reaction was stirred at room temperature for 2 h. The reaction was then diluted in 50 mL DCM and washed with 20 mL 1N HCl and 20 mL brine. The organic layer was dried over Na2SO4 and condensed to yield crude product (12 mg). It was then dissolved in DMF to make a 15mM solution, to which 18mM aqueous solution of t2M-NH2 was added, 0.1 M Na2CO3 solution was used to adjust pH to 9. The reaction mixture was stirred for 2 h, then purified by HPLC to obtain t2M-Cy5. ESI: mass calculated [M+2H]2+=415.2641; mass observed: 415.2619.

100

Figure 3.24. Synthetic scheme for N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6- triamine.

N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6-triamine (4). (Figure 3.24)

Aminoacetaldehyde dimethyl acetal (4 mL, 38.7 mmol) was dissolved in 30 mL H2O, diaminochlorotriazine( 4.35 g, 30 mmol) and NaHCO3( 2.8 g, 33 mmol) was added to the solution. The reaction was heated to 85°C and stirred overnight. After cooling down, the reaction was filtered and the solid was washed with water twice, after drying over vacuum,

N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6-triamine(4) was collected as white solid(4.5g, 70.1%) 1H NMR: 3.26 (6H,s); 3.28 (2H, m); 4.45 (1H,t); 5.99-6.14 (4H, br);

6.40 (1H, t); 13C NMR: 41.47; 52.93; 102.04; 150.04; 166.37; ESI: mass calculated

[M+H]+=215.1251; mass observed: 215.1262. Deprotection of 5 in 1N HCl for 2h at room temperature followed by drying solvent in vacuo yielded melamine acetaldehyde as white solid and was used without further purification.

Figure 3.25. Synthetic scheme for N2-(2,2-dimethoxyethyl)-N4,N4,N6,N6-tetramethyl- 1,3,5-triazine-2,4,6-triamine.

N2-(2,2-dimethoxyethyl)-N4,N4,N6,N6-tetramethyl-1,3,5-triazine-2,4,6-triamine (5). (Figure 3.25) 101

Aminoacetaldehyde dimethyl acetal (1.64mL, 15mmol) was dissolved in 10 mL H2O:1,4- dioxane=1:1 mixture, 6-chloro-N2,N2,N4,N4-tetramethyl-2,4-diamino-1,3,5-triazine

(2.01g, 10mmol) and NaHCO3( 2.8 g, 33 mmol) was added to the solution. The reaction was heated to 85°C and stirred overnight. After cooling down, the reaction was filtered and the solid was washed with water twice. After drying over vacuum, 5 was collected as white solid(2.1g, 77.8%) 1H NMR: 4.91 (1H, t); 4.45 (1H, t); 3.50 (2H, dd); 3.36 (6H, s); 3.05

(12H, s) ; 13C NMR:; 166.12; 165.77; 103.18; 54.04; 42.25; 35.89; ESI: mass calculated

[M+H]+=271.1877; mass observed: 271.1876. Deprotection of 5 in 1N HCl for 2h at room temperature followed by drying solvent in vacuo yielded tetramethyl melamine acetaldehyde as white solid and was used without further purification.

Figure 3.26. Synthetic scheme for t4M

N2-(2-((2-((2-aminoethyl)(2-(bis(2-((4,6-diamino-1,3,5-triazin-2- yl)amino)ethyl)amino)ethyl)amino)ethyl)(2-((4,6-diamino-1,3,5-triazin-2- yl)amino)ethyl)amino)ethyl)-1,3,5-triazine-2,4,6-triamine (t4M, 6). (Figure 3.26)

102

To a 1M DMSO solution of mono Boc-protected tren (15 mg, 0.061 mmol) was added two equivalents of melamine acetaldehyde as a 1 M DMSO solution and 2 equivalents of

NaBH3CN as a 1 M DMSO solution. After 2 hours an additional 2.5 equivalents of melamine acetaldehyde and NaBH3CN were added, both as 1 M solutions in DMSO and reacted for another 2 hours. The reaction process was monitored by HPLC and MS. The reaction mixture was then precipitated, washed in DCM, and purified by HPLC to obtain

Boc-t4M as a white solid (70% by HPLC). The solid was then dissolved in TFA and reacted at room temperature for 10min. The TFA was evaporated under N2 flow and the

1 residue was dissolved in H2O and lyophilized to obtain the TFA salt of t4M. H NMR:

2.86 (2H, t); 3.00-3.08 (6H, m); 3.43-3.52 (12H, m); 3.77 (8H, t); 3.78 (8H, t); 13C NMR:

165.68; 54.11; 51.10; 49.94; 47.86; 36.34; 36.05; ESI: mass calculated:

[M+H]+=755.4846; [M+2H]2+=378.2459; [M+3H]3+=252.4997; [M+4H]4+=189.6269. mass observed: 755.4799; 378.2437; 252.4988; 189.6269.

Figure 3.27. Synthetic scheme for t4MMe.

103

N2-(2-((2-((2-aminoethyl)(2-(bis(2-((4,6-bis(dimethylamino)-1,3,5-triazin-2- yl)amino)ethyl)amino)ethyl)amino)ethyl)(2-((4,6-bis(dimethylamino)-1,3,5-triazin-2- yl)amino)ethyl)amino)ethyl)-N4,N4,N6,N6-tetramethyl-1,3,5-triazine-2,4,6-triamine

(t4MMe, 7). (Figure 3.27)

To a 1 M DMSO solution of mono Boc-protected tren was added two equivalents of tetramethyl melamine acetaldehyde as a 1 M DMSO solution and 2 equivalents of

NaBH3CN as a 1 M DMSO solution. After 2 hours an additional 2.5 equivalents of tetramethyl melamine acetaldehyde and NaBH3CN were added, both as 1 M solutions in

DMSO and reacted for another 2 hours. The reaction mixture was then precipitated into 1

M NaOH solution, the solid was separated by centrifuge and washed with H2O. After drying, the solid was purified on silica gel column with 2% MeOH in DCM and 1%

NH4OH (aq) to obtain 35 mg (53.8%) white solid. The solid was then dissolved in TFA and reacted at room temperature for 10 min. The TFA was evaporated under N2 flow and

Me 1 the residue was dissolved in H2O and lyophilized to obtain the TFA salt of t4M . H

NMR: 2.77 (2H, t); 2.91-3.08 (56H, br); 3.31 (4H, t); 3.44 (8H, t); 3.78 (8H, t); 13C

NMR: 158.34; 156.46; 54.46; 53.85; 52.77; 49.92; 39.43; 39.13; 38.03; ESI: mass calculated: [M+2H]2+=490.3711; [M+3H]3+=327.2499; mass observed: 490.3701;

327.2495.

104

Figure 3.28. Synthesis scheme for qt3M alkyane

TriBoc Tren. (Figure 3.28)

Tris(2-aminoethyl)amine (1.5 mL, 10 mmol) was dissolved in 20 mL DCM and cooled in ice bath. A solution of Boc anhydride (6.97 g, 32 mmol) in 20 mL DCM was added dropwise, followed by dropwise addition of triethylamine (4.5mL, 32 mmol) in 20 mL

DCM. The reaction was warmed up to room temperature slowly and stirred overnight.

After removal of solvent under reduced pressure, the crude oil was resuspended in 50 mL

H2O and extracted with DCM (50 mL*5). The organic phase was dried over Na2SO4 and solvent removed under reduced pressure. Chromatographic purification of the crude product (DCM:MeOH=20:1) yielded 3.5g TriBoc Tren (78%) as white solid.

Quat TriBoc Tren nitrile. (Figure 3.28)

TriBoc Tren (1.65g, 3.7mmol) was dissolved in 2 mL MeCN, after addition of bromoacetonitrile (1.4mL, 20mmol), the mixture was transferred into a sealed tube and flushed with nitrogen gas. The reaction was incubated overnight at 80°C. After removal 105 of solvent under reduced pressure, the crude oil was purified with column chromatography (DCM:MeOH from 10:1 to 5:1) to yield 1.9g (85%) off-white solid 4 as bromide salt. 1H NMR: 1.42 (27H, s); 3.70 (6H, m); 4.02 (6H, m); 5.50 (2H, s); 6.25

(3H,t); 13C NMR: 28.51; 35.35; 60.84; 80.78; 156.54;

Quat TriBoc Tren alkyne . (Figure 3.28)

Quat TriBoc Tren nitrile (930 mg) was dissolved in 3 mL of anhydrous THF, purged with

N2 and cooled down to -78°C, then 4 mL of 1 M LiAlH4 solution in THF was added dropwise, the solution was slowly warmed up to 0°C and reacted for 1h. The reaction was quenched with 5 mL H2O under ice bath and extracted with DCM (50 mL*6). The organic phase was dried over Na2SO4, then solvent was removed under vacuum to yield

Quat TriBoc Tren amine (750mg, 80%). It was used for the next step without further purification. 5-Hexynoic acid (0.1 mL) was dissolved in 5 mL of DCM, to which

EDCI(139 mg) was added stirred for 30min, then Quat TriBoc Tren amine (140 mg) was added reacted further for 30min. The reaction was then washed with 1N HCl, saturated

NaHCO3 solution and brine. The organic layer was dried over Na2SO4, solvent was removed to yield Quat TriBoc Tren alkyne as chloride salt(5) (120 mg, 80%).

qT3M alkyne. (Figure 3.28)

Quat TriBoc Tren alkyne was dissolved in mL of 1:1 DCM/TFA and reacted at room temp for 30 min, the solution was condensed to syrup under a stream of N2 before cold

Et2O was added to form white precipitate. The precipitate was centrifuged down, washed

106 with cold Et2O and dried under vacuum to yield the TFA salt of Quat Tren alkyne. It was then dissolved in H2O and adjusted to pH 9 with NaHCO3, 6-chloro-1,3,5-triazine-2,4- diamine and NaHCO3 was then added to the solution. The slurry was heated to 85°C and reacted overnight. After cooling down to room temp, the white precipitate formed was centrifuged and washed with water to yield the crude Quat Tren 3M* alkyne. It was further purified by prep HPLC using gradient of 0% to 20% acetonitrile over 40 minutes.

1H NMR: 1.71 (2H, m); 2.16 (2H, t); 2.33 (3H, m. terminal alkyne proton buried under);

3.68 (10H, m); 3.85 (6H,t);

3.10 Ribozyme cleavage data

3.10.1 U-(2,3) ribozyme

A stock solution with U-(2,3) ribozyme (625 nM) and tren derivative (12.5 µM) in 1.25X

Tris-Cl buffer (pH=7.6) was annealed at 75 °C. An aqueous MgCl2 solution was added to the annealed solution to initiate the reaction at 37 °C. The final concentrations were: 500 nM ribozyme, 10 µM tren derivative and 10 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6).

Aliquots were taken at 0, 10, 45, 90, 150 min, quenched with urea-EDTA, and frozen immediately on dry ice. Samples were analyzed on 12% denaturing acrylamide gel and stained with SYBRⓇ gold. Cleavage rate was derived using the following equation:

퐹(푡) = 퐹푚푎푥 ∗ (1 − exp(−푘 ∗ 푡))

Where F(t) is the fraction cleaved at the time point t, Fmax is the maximum fraction cleaved, k is the cleavage rate, t represents time.

107

Figure 3.29. U-(2,3) ribozyme activity rescued by 10 µM tren derivative. Reaction conditions: 500 nM ribozyme, 10 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Top band indicates intact U-(2,3) ribozyme, bottom two bands indicate the cleavage products.

Figure 3.30. U-(2,3) ribozyme activity rescued by 10µM tren derivative. Reaction conditions: 500 nM ribozyme, 10 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Top band indicates intact U-(2,3) ribozyme, no obvious cleavage products were observed.

108

Figure 3.31. U-(2,3) ribozyme activity rescue by 100µM tren derivative. Reaction conditions: 500 nM ribozyme, 10 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Top band indicates intact U-(2,3) ribozyme, bottom two bands indicate the cleavage products.

Figure 3.32. U-(2,3) ribozyme cleavage quantification. Ribozyme activity rescued by 10 µM tren derivative with 500 nM ribozyme, 10 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Data quantified using equation described at the beginning of the section, R2>0.98.

109

3.10.2 S-U4n ribozyme

A stock solution with S-U4n ribozyme (625 nM) and tren derivative (12.5 µM) in 1.25X

Tris-Cl buffer (pH=7.6) was heated at 75 °C for 2 min and equilibrated at 37 ℃ for 5 min.

An aqueous MgCl2 solution was added to the annealed solution to initiate the reaction at

37 °C. The final concentrations were: 500 nM ribozyme, 10 µM tren derivative and 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6). Aliquots were taken at 0, 5, 15, 30, 60, 90 min, quenched with urea-EDTA and frozen immediately on dry ice. Samples were analyzed on

12% denaturing acrylamide gel and stained with SYBRⓇ gold. Cleavage rate was derived using the following equation:

퐹(푡) = 퐹푚푎푥 ∗ (1 − exp(−푘 ∗ 푡))

Where F(t) is the fraction cleaved at the time point t, Fmax is the maximum fraction cleaved, k is the cleavage rate, t represents time.

Figure 3.33. S-U4n ribozyme rescued by t4M. Reaction conditions: 500 nM ribozyme, 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Top band indicates intact S-U4n ribozyme, middle band indicates the tRNA cleavage product, bottom band indicates the ribozyme cleavage product.

110

Figure 3.34. S-U4n ribozyme rescue by t4M. Reaction conditions: 500 nM ribozyme, 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Top band indicates intact S-U4n ribozyme, middle band indicates the tRNA cleavage product, bottom band indicates the ribozyme cleavage product.

Figure 3.35. S-U4n ribozyme rescue control. Reaction conditions: 500 nM ribozyme, 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Top band indicates intact S-U4n ribozyme, middle band indicates the tRNA cleavage product, bottom band indicates the ribozyme cleavage product.

111

Figure 3.36. S-U4n-2bp ribozyme rescue by t4M. Reaction conditions: 500 nM ribozyme, 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Top band indicates intact S-U4n ribozyme, middle band indicates the tRNA cleavage product, bottom band indicates the ribozyme cleavage product.

Figure 3.37. S-U4n ribozyme cleavage data quantification. Ribozyme activity rescued by various concentrations of t4M with 500 nM ribozyme, 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Data quantified using equation described at the beginning of the section, R2>0.98.

112

Figure 3.38. S-U4n and S-U4n-2bp ribozyme cleavage data quantification. Ribozymes activities rescued by 10 µM t4M with 500 nM ribozyme, 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 37℃. Data quantified using equation described at the beginning of the section, R2>0.98.

3.10.3 Binary ribozyme

Samples for single turnover experiments were prepared by mixing 5 µM of enzyme strand with 400 nM of the Cy3 labeled substrate strand and 10 µM tren derivative in

1.25X Tris-Cl buffer (pH=7.6), mixture was heated at 75℃ for 2min and equilibrated at

27℃ for 10min. MgCl2 was then added to initiate the reaction at 27℃, the final concentration was 4 µM enzyme strand, 320 nM Cy3 labeled substrate strand, 10 µM tren derivative and 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6). Aliquots were taken at 0, 15,

30, 45s and 1, 2, 3, 5, 15, 30, 60 min, quenched with urea-EDTA and frozen immediately on dry ice. Samples were analyzed on 20% denaturing acrylamide gel. Cleavage rate was derived using the following equation:

퐹(푡) = 퐹푚푎푥 ∗ (1 − exp(−푘 ∗ 푡))

113

Where F(t) is the fraction cleaved at the time point t, Fmax is the maximum fraction cleaved, k is the cleavage rate, t represents time.

Figure 3.39. Binary ribozyme cleavage data quantification. L-U4/wt activity rescued by t4M. Reaction conditions: 4 µM enzyme strand, 320 nM Cy3 labeled substrate strand, and 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 27℃. Data quantified using equation described at the beginning of the section, R2>0.98.

Figure 3.40. Binary ribozyme cleavage data quantification. L-U4/UU activity rescued by t4M. Reaction conditions: 4 µM enzyme strand, 320 nM Cy3 labeled substrate strand, and 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 27℃. Data quantified using equation described at the beginning of the section, R2>0.98.

114

Figure 3.41. Binary ribozyme cleavage data quantification. L-U4/5U activity rescued by t4M. Reaction conditions: 4 µM enzyme strand, 320 nM Cy3 labeled substrate strand, and 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 27℃. Data quantified using equation described at the beginning of the section, R2>0.98.

Figure 3.42. Representative gel image for binary ribozyme cleavage. Top band is the intact Cy3 labelled substrate strand RNA, bottom band indicates the cleavage product. (Left) L-U4/5U; (right) L-U4/5U with 10 µM t4M, under 0.1 mM Mg2+ conditions.

115

Figure 3.43. Binary ribozyme cleavage data quantification. L-GU3 with HHmin(wt) and UU substrate. Reaction conditions: 4 µM enzyme strand, 320 nM Cy3 labeled substrate strand, and 0.1 mM Mg2+ in 1X Tris-Cl buffer (pH=7.6) at 27℃. Data quantified using equation described at the beginning of the section, R2>0.98.

Table 3.3 Summary of cleavage rates of binary ribozymes

Enzyme Substrate Small molecule [Mg2+] Cleavage rate

L-U4 wt None 0.1 mM 0.023±0.003 min-1

L-U4 wt t4M 0.1 mM 0.023±0.003 min-1

L-U4 UU None 0.1 mM 0.133±0.012 min-1

L-U4 UU t2M 0.1 mM 0.217±0.015 min-1

L-U4 UU t4M 0.1 mM 0.245±0.014 min-1

L-U4 5U None 0.1 mM 0.099±0.005 min-1

L-U4 5U t2M 0.1 mM 0.178±0.019 min-1

L-U4 5U t4M 0.1 mM 0.418±0.031 min-1

L-U4 5U t4MMe 0.1 mM 0.080±0.010 min-1

L-GU3 wt None 0.1 mM 0.049±0.005 min-1

L-GU3 UU None 0.1 mM 0.323±0.036 min-1

wt(L-GUGA) wt None 0.1 mM 0.603±0.026 min-1

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3.11 Compound characterization.

NMR spectra.

1 Figure 3.44. H NMR (400 MHz, D2O, pH=3) of t3M. TFA from HPLC.

13 Figure 3.45. C NMR (100 MHz, D2O) of t3M. Two d peaks are possibly due to different environments of the protonated triazine rings at pH 3 (TFA from HPLC). The peaks coalesce after basification to pH 6.0 (Figure 3.42). Similar phenomenon was seen in t2M and t4M NMR spectra, with the expected spectral signatures observed at pH 6.0 (below).

117

1 Figure 3.46. H NMR (400 MHz, D2O, pH=6) of t3M. TFA from HPLC.

13 Figure 3.47. C NMR (100 MHz, D2O+NaOD,pH=6) of t3M. TFA from HPLC.

118

1 Figure 3.48. H NMR (400 MHz, D2O) of Boc t2M.

13 Figure 3.49. C NMR (100 MHz, D2O) of Boc t2M.

119

1 Figure 3.50. H NMR (400 MHz, D2O, pH=3) of t2M. TFA from HPLC.

13 Figure 3.51. C NMR (125 MHz, D2O) of t2M. TFA from HPLC.

120

1 Figure 3.52. H NMR (400 MHz, D2O, pH=3) of t2M. TFA from HPLC.

13 Figure 3.53. C NMR (125 MHz, D2O+NaOD, pH=6) of t2M. TFA from HPLC.

121

1 Me Figure 3.54. H NMR (400 MHz, CDCl3) of Boc t2M .

13 Me Figure 3.55. C NMR (100 MHz, CDCl3) of Boc t2M .

122

1 Me Figure 3.56. H NMR (400 MHz, CD3CN:D2O=1:1) of t2M .

13 Me Figure 3.57. C NMR (125 MHz, CD3CN:D2O=1:1) of t2M .

123

1 2 Figure 3.58. H NMR (400 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)-1,3,5- triazine-2,4,6-triamine.

13 2 Figure 3.59. C NMR (100 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)-1,3,5- triazine-2,4,6-triamine.

124

1 2 4 4 6 6 Figure 3.60. H NMR (400 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)-N ,N ,N ,N - tetramethyl-1,3,5-triazine-2,4,6-triamine.

13 2 Figure 3.61. C NMR (100 MHz, DMSO-d6) of N -(2,2-dimethoxyethyl)- N4,N4,N6,N6-tetramethyl-1,3,5-triazine-2,4,6-triamine.

125

1 Figure 3.62. H NMR (400 MHz, D2O+NaOD, pH=6, water suppression) of t4M. TFA from HPLC. Peak assignments by analogy to t3M and t2M.

13 Figure 3.63. C NMR (125 MHz, D2O+NaOD, pH=6) of t4M. TFA from HPLC. Peak assignments by analogy to t3M and t2M.

126

1 Me Figure 3.64. H NMR (400 MHz, CD3CN:D2O=1:1) of t4M . TFA from deprotection of Boc.

13 Me Figure 3.65. C NMR (125 MHz, CD3CN:D2O=1:1) of t4M . TFA from deprotection of Boc.

127

1 Figure 3.66. H NMR (400 MHz, D2O, pH=3) of t2M alkyne.

13 Figure 3.67. C NMR (125 MHz, D2O, pH=3) of t2M alkyne.

128

1 Figure 3.68. H NMR (400 MHz, CDCl3) of tri Boc quat tren alkyne.

13 Figure 3.69. C NMR (100 MHz, CDCl3) of tri Boc quat tren alkyne.

129

1 Figure 3.70. H NMR (400 MHz, D2O, water suppression) of qt3M alkyne.

1 Figure 3.71. H NMR (400 MHz, MeOD) of FITC-N3.

130

HPLC and ESI Spectra.

HPLC traces are obtained on a reverse phase C18 analytical column with solvent A= 99%

H2O, 1% MeCN and 0.1% TFA and solvent B= 10% H2O, 90% MeCN and 0.07% TFA.

Flow rate of HPLC is 1 mL/min.

Figure 3.72. HPLC and ESI spectra of t3M. (Left) HPLC trace of t3M (1). Gradient: 0-5 min 0% solvent B, 5-15 min; linear gradient from 0 to 30% solvent B; 15-20 min, 30% solvent B; 20-22 min, linear gradient from 30 to 100% solvent B; 22-27 min, 100% solvent B; 27-29 min, linear gradient from 100 to 0% solvent B; 29-35 min, 0% solvent B. (Right) ESI spectra of t3M, peaks observed:474.2788, 237.6426 .

131

Figure 3.73. HPLC and ESI spectra of t2M. (Left) HPLC trace of t2M (2a). Gradient: 0-5 min 0% solvent B, 5-15 min; linear gradient from 0 to 30% solvent B; 15-20 min, 30% solvent B; 20-22 min, linear gradient from 30 to 100% solvent B; 22-27 min, 100% solvent B; 27-29 min, linear gradient from 100 to 0% solvent B; 29-35 min, 0% solvent B. (Right) ESI spectra of t2M, peaks observed:365.2368, 183.1238.

Figure 3.74. HPLC and ESI spectra of t4M. (Left) HPLC trace of t4M (6). Gradient: 0-5 min 0% solvent B, 5-15 min; linear gradient from 0 to 30% solvent B; 15-20 min, 30% solvent B; 20-22 min, linear gradient from 30 to 100% solvent B; 22-27 min, 100% solvent B; 27-29 min, linear gradient from 100 to 0% solvent B; 29-35 min, 0% solvent B. (Right)ESI spectra of t4M, peaks observed:755.4799, 378.2437, 252.4988, 189.6269.

132

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Chapter 4 : Small molecule G quadruplex sensor

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4.1 G quadruplex in aptamers

As discussed previously, G quadruplex is a common secondary structure motif in both

DNA and RNA. Due to their importance in chromosome protection and regulating gene expression1-4, G quadruplexes are widely studied. A number of DNA or RNA aptamers have potential G quadruplexes within their structure5, a few crystal structures were obtained to confirm such existence. For example, thrombin-binding 15mer

GGTTGGTGGTTGG forms two G tetrads, and is positioned in between two thrombin molecules6. Spinach aptamer, obtained through in vitro selection, also displays two G tetrads, which are critical in the binding pocket of DFHBI small molecule7. Interestingly,

Mango aptamer that binds with fluorogenic small molecule thiazole orange (TO) and shows enhance fluorescence also have G quadruplex8-9. Such common appearance of G quadruplexes lead us to think about the potential of developing a small molecule dye that can specifically target and image G quadruplexes.

Figure 4.1. G quadruplexes in Spinach (left) and Mango (right) aptamers. Figure is adapted from references 7 and 9.

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The dye-binding face of both Spinach and Mango aptamers are right above the G tetrads

(Figure 4.1), which presumably provide base stacking with the planar dye small molecule in addition to the hydrogen binding interaction within the binding pockets. Mutations of either layer of G tetrads into pyrimidines in Spinach completely abolished the fluorescence activation, most likely due to the disruption of G quadruplex structure7. A native T1 RNase protection assay with the Mango aptamer showed decreased level of digestion in the proposed G quadruplex region when titrate in the dye molecule (TO)8.

Both aptamers’ fluorescence activation showed strong potassium dependence, an ion known to stabilize G quadruplexes.

4.2 Selective imaging of G quadruplexes

G quadruplexes are structurally unique in nucleic acids, four guanines associated via

Hoogsteen hydrogen bonding to form a large planar aromatic face, with monovalent cations in the center of the tetrad or between multiple layers and sugar-phosphate backbone run along the outside of the G tetrad core. Selective targeting of G quadruplexes often involves large planar aromatic core four base stacking and cationic sidechains for electrostatic interactions, as discussed in chapter 1. Selective imaging of G quadruplexes has been studies extensively, current strategy involves modification on cyanine dye to increasing selectivity10, covalent linking of G quadruplex binder to dye molecules11 and FRET sensor with non-selective dye acceptor and G quadruplex selective donor12.

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4.3 Engineering the Spinach aptamers

Spinach aptamers binding pocket consist of two layers of G tetrads, additional G28 that complements the DFHBI small molecule on top the G tetrads, and a U29-A53-U50 triplex that seals the roof. When A53 is mutated to U, the fluorescence activation is abolished demonstrating the importance of the base triple. Based on these structure information, we proposed the replacement of U-A-U triplex to U-M*-U triplex, where

M* stands for melamine, a known artificial nucleobase able to form bifacial hydrogen bonding with thymine or uracil. We wish to activate the reconstructed aptamer with

DFHBI-melamine conjugates selectively.

Figure 4.2. Spinach aptamer-DFHBI interaction. Two layers of G tetrads and a U-A-U triplex forms the binding pocket of DFHBI, complemented by hydrogen bonding from G28 and A58.

Piccirilli and coworkers also demonstrated the truncation of P1 stem does not affect the fluorescence intensity even if only 5 base pairs remain. Thus, we based all our designs on the 5bp P1 version of Spinach, shown in Figure 4.3. Apart from the A53U mutant, we were also curious about the effect of completely deleting the whole U-A-U triplex and the

144

U turn. We inserted 1,2 and 4 pairs of U-U mismatch right next to the binding pocket, hoping to accommodate single melamine, t2M and t4M conjugates of dye molecules to increasing the selectivity of aptamer-small molecule binding.

Figure 4.3. Spinach U mutant sequences. Mutations are highlighted in red.

4.4 DFHBI derivatives with modified Spinach aptamers

We first tested the fluorescence activation of DFHBI melamine conjugate (DFHBI-4C-

M*) (Table 4.1) on the wildtype 5bp Spinach. Strikingly compared to DFHBI small molecule, the melamine conjugate is almost twice as bright, while the tretramethylated melamine conjugate (DFHBI-4C-M*4M) has the same fluorescence intensity, suggesting that melamine participate in the structuring of binding pocket through hydrogen bonding, possibly by replacing the U-A-U triplex with U-M*-U.

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Table 4.1. Summary of DFHBI derivatives tested. Name Structure

DFHBI-4C-M*

DFHBI-4C- M*4M

To further confirm this hypothesis, we tested the fluorescence turn on with A53U mutant of Spinach, where the U-A-U triplex is disrupted. The background fluorescence of A53U mutant with DFHBI is barely detectable, while upon binding of DFHBI-4C-M*, strong fluorescence was observed. Methylated control again gave completely abolished fluorescence. The significant drop of fluorescence intensity of the A53U mutant compared to the wildtype Spinach aptamer, albeit similar binding affinity (Figure 4.16) towards the small molecule, is probably due to the unknown structural effect of A53 base once it’s displaced by melamine.

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Figure 4.4. Selective fluorescence turn on by DFHBI-4C-M*. RNA and small molecules (1 µM each) were incubated in 1X Spinach buffer (40 mM HEPES pH=7.3, 5 mM MgCl2, 125 mM KCl) at room temperature for 30 min before measurements. Fluorescence was measured on ThermoFisher Nanodrop 3300 with blue light source with emission wavelength at 505 nm. (Left) Fluorescence turn on of wt Spinach aptamer (5bp Spinach). (Right) Fluorescence turn on of 5bpS A53U mutant.

4.5 Thiazole orange with modified Spinach aptamers

Compared with Spinach-DFHBI system, Mango-TO system has much higher binding affinity (3.2nM versus 500nM)8, 13, making it an attractive candidate for RNA detection.

The binding face of TO with Mango is strikingly similar with Spinach, both consists of G tetrads, thus we hypothesize that with proper engineering, Spinach would also turn on TO fluorescence.

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Table 4.2. Summary of TO derivatives tested. Name Structure

TO-4C-M*

TO-4C-M*4M

TO-t2M

TO t2MMe

Metal ion dependence, especially potassium ion, is a strong indication of G quadruplex structure. To test whether thiazole orange binds to G quadruplex in the Spinach aptamer pocket, fluorescence was measured when various concentration of K+ and Na+ were

148 added to the buffer solution. Gratifyingly, the Spinach aptamer fluorescence turn on by

TO-t2M was strongly influenced by K+ concentration, while Na+ have little effect.

Figure 4.5. Metal ion dependence of 5bp S U2 aptamer with TO-t2M. RNA and small molecule concentrations are held at 1 µM in 40 mM HEPES pH=7.3, 5 mM MgCl2 and various concentrations of NaCl or KCl. Samples with were incubated at room temperature for 30 min before measurements. Fluorescence was measured on ThermoFisher Nanodrop 3300 with blue light source with emission wavelength at 532nm.

To further probe the TO binding in the Spinach aptamer, we prepared melamine conjugates of TO. If TO and DFHBI shared the same binding pocket in the Spinach aptamer, we should observe selectivity in fluorescence turn on of the Spinach U mutants by TO melamine conjugates over its methylated control. We speculate that restructuring of the U stretch next to the G quadruplex in Spinach aptamer could be achieved by covalently linking TO with t2M. Indeed, significant turn on was observed on U2 and U4 mutants of Spinach aptamer by TO-t2M, while TO alone and the methylated control of

TO-t2M showed minimal fluorescence (Figure 4.6). Similar with DFHBI-4C-M*, TO-

149 t2M could also enhance the fluorescence turn on of intact Spinach aptamer. We speculate that it was due to the displacement of U-A-U triplex to a more stable U-M*-U triplex by the melamine base in t2M.

Figure 4.6. Selective turn on of Spinach aptamers by TO-t2M. RNA and small molecules (1 µM each) were incubated in 1X Spinach buffer (40 mM HEPES pH=7.3, 5 mM MgCl2, 125 mM KCl) at room temperature for 30 min before measurements. Fluorescence was measured on ThermoFisher Nanodrop 3300 with blue light source with emission wavelength at 532 nm.

Among all the Spinach mutants, 5bpS U2 has the best selectivity in fluorescence turn on with TO-t2M, marked by the almost ten-fold increase in fluorescence intensity over TO and TO-t2MMe. In other words, we designed a new aptamer for TO-t2M based purely on structural analysis and point mutation of existing RNA aptamer. We believe this strategy could be generalized for aptamer de novo design.

To confirm this hypothesis, single melamine conjugates of thiazole orange (TO-4C-M*) was made as an analogue to DFHBI-4C-M*. The fluorescence intensity of single U mutants of Spinach (5bpS U1) are almost twice as much with TO-4C-M* compared to 150 with TO-4C-M*4M. As the binding pocket become more and more unstructured (from

U1 to U4 mutant), the fluorescence intensity with TO-4C-M*4M decreases to the minimal, while TO-4C-M* still showed significant fluorescence turn on. Surprisingly, the intact Spinach aptamer (5bp Spinach) also showed the same difference between the melamine conjugate and its control. This difference could again arise from the displacement of A with melamine in the U-A-U base triple. Another interesting fact is that 5bpS U1 mutant was turned on more than the intact Spinach and A53U mutant, probably due to the enthalpic again from forming U-M*-U triple without the need to compensate for disruption of U-A-U hydrogen bonding. Indeed, we saw the best selectivity of TO-4C-M* fluorescence turn on in 5bpS U1, proving the generality of the de novo design strategy.

Figure 4.7. Selective turn on of Spinach aptamers by TO-4C-M*. RNA and small molecules (1 µM each) were incubated in 1X Spinach buffer (40 mM HEPES pH=7.3, 5 mM MgCl2, 125 mM KCl) at room temperature for 30 min before measurements. Fluorescence was measured on ThermoFisher Nanodrop 3300 with blue light source with emission wavelength at 532 nm.

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4.6 De novo design of G quadruplex containing aptamer for selective targeting.

G quadruplex is a very important feature in vivo, much effort has been devoted in the sensing G quadruplexes in vivo14. However, no gold standard has been found to date, draw backs often involve low brightness, low affinity and lack of selectivity over other secondary structures of nucleic acids. The successful de novo design of aptamers for TO derivatives led us believe that similar strategy could be applied to in vivo G quadruplexes sensing. We chose the common repeating sequence of GGGTTA in human telomere

DNA as our target. Structural information obtained by X-ray crystallography and NMR showed a G quadruplex structure with flanking TTA loops15-18 (Figure 4.8). TO was chosen as the ideal dye due to its brightness and high affinity with Mango aptamers, which possess three layers of G tetrads.

Figure 4.8. Two relevant structures of human telomere G quadruplex core sequence (GGGTTA)3GGG, TT mimatches are highlighted in red. Figure is adapted from reference 15 and 16.

We hypothesize that a TO-t2M conjugate could increase the selectivity and affinity towards this human telomere G quadruplex, due to the t2M targeting of TT-TT mismatch. 152

We set out to test the fluorescence turn on of TO-t2M conjugate with (GGGTTA)3GGG

DNA. Fluorescence of the complex was increased about seven times at 1:1 DNA/small molecule ratio (Figure 4.9) with TO-t2M versus TO t2MMe, the methylated control (Table

4.2). This result suggests that hydrogen bonding of the melamine moiety is responsible for the increase in fluorescence, in line with our hypothesis. Due to the common appearance of the (GGGTTA)3GGG sequence in human telomere, TO-t2M could potentially be used to selectively image the human telomere.

Figure 4.9. Fluorescence turn on of (GGGTTA)3GGG with TO-t2M. All samples were prepared with 1 µM TO derivatives with various amounts of pre- annealed DNA in 10 mM sodium cacodylate buffer (pH=7.3) and 100 mM KCl. Samples were incubated at room temperature for 30 min before measurements. Fluorescence was measured on ThermoFisher Nanodrop 3300 with blue light source with emission wavelength at 532 nm.

4.7 Conclusions.

In summary, we found that melamine conjugate of fluorogenic dye DFHBI could selectivity turn on fluorescence when bound to U mutants of Spinach aptamers, due to

153 potential U-M*-U triplex formation. Interestingly, these U mutants could also be turned on by melamine derivatives of thiazole orange dye, which is not the native ligand for

Spinach aptamers. These results and the structural analysis of both Spinach and Mango aptamers led us think that covalent linking of melamine derivatives to fluorogenic dyes could be used as a selective probe for sensing G quadruplexes. To this end, we tested the turn on of human telomere G quadruplex core sequence (GGGTTA)3GGG with t2M derivative of TO. Indeed, a seven-fold increase in fluorescence intensity was observed over the methylated control, proving the utility of our approach. Hydrogen bonding between the TTA loop and the melamine in t2M motif is believe to contribute to the structural stability of the G quadruplex-dye molecule complex, thus leading to the enhancement in fluorescence turn on. Moreover, we believe these results point out a new pathway to obtain new aptamers, that is, through rational de novo design of known aptamers, instead of through in vitro selection. Further optimization and application for these small molecules are currently underway.

4.8 Compound preparations.

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Figure 4.10. Synthetic scheme for DFHBI-4C-M* and DFHBI-4C-M*4M.

DFHBI melamine conjugates were synthesized via the scheme shown above. Briefly, diaminobutane was selectively protected by Boc group at one end while the other end was functionalized with diaminotriazine or N2,N2,N4,N4-tetramethyl diaminotriazine, after deprotection of Boc group and neutralization, the free amino was coupled to (Z)-4-

(3,5-difluoro-4-hydroxybenzylidene)-2-methyloxazol-5(4H)-one in ethanol reflux to obtain DFHBI-4C-M* or DFHBI-4C-M*4M.

(Z)-3-(4-((4,6-diamino-1,3,5-triazin-2-yl)amino)butyl)-5-(3,5-difluoro-4- hydroxybenzylidene)-2-methyl-3,5-dihydro-4H-imidazol-4-one (DFHBI-4C-M*)

To a mixture of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyloxazol-5(4H)-one

(150mg, 0.54mmol) and N2-(4-aminobutyl)-1,3,5-triazine-2,4,6-triamine (230mg, 1.16 mmol) was added 2mL EtOH and K2CO3 solid (165mg), the mixture was heated at reflux for overnight. After cooling down, 15mL of EtOH was added to the reaction, solid was removed by centrifugation, the remaining solution was condensed with rotary 155 evaporation collected 84 mg of orange solid. The crude was then purified by silica gel chromatography using DCM: MeOH=10:1 and 1% acetic acid. 1H NMR: 1.66 (4H, m);

2.41 (3H, s); 3.36 (2H, t); 3.68 (2H, t); 6.89 (1H, s); 7.76 (2H, d). ESI: Mass calculated:

[M+H+]=419.1750; Mass observed: [M+H+]=419.1737.

(Z)-3-(4-((4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)amino)butyl)-5-(3,5-difluoro-4- hydroxybenzylidene)-2-methyl-3,5-dihydro-4H-imidazol-4-one (DFHBI-4C-M*4M)

To a mixture of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyloxazol-5(4H)-one

(42mg, 0.15mmol) and N2-(4-aminobutyl)-N4,N4,N6,N6-tetramethyl-1,3,5-triazine-2,4,6- triamine (100mg, 0.395mmol) was added 1mL EtOH and K2CO3 solid (50mg), the mixture was heated at reflux for overnight. Solvent was removed by rotary evaporator and crude was purified by silica gel chromatography using 1% MeOH in DCM and 1% acetic acid to collected 40mg product as yellow solid (56%). 1H NMR: 1.69 (4H, m);

2.41 (3H, s); 3.11 (12H, s); 3.44 (2H, t); 3.68 (2H, t); 6.87 (1H, s); 7.77 (2H, d). ESI:

Mass calculated: [M+H+]=475.2376; Mass observed: [M+H+]=475.2368.

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Figure 4.11. Synthetic scheme for TO derivatives.

Melamine conjugates of TO was synthesized based on the procedures from reference 8.

Briefly, Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU,

15.2 mg, 40.1 µmol, 14 equiv.) and N,N-diisopropylethylamine (DIPEA, 14.2 µl, 81.5

µmol, 28 equiv.) were added to a solution of TO-Acetate (1.0 mg, 2.9 µmol, 1.0 equiv.) and the amine (5 equiv.) in N,N-dimethylformamide (250 µl). The reaction mixture was then stirred overnight at ambient temperature. The final products were isolated and purified directly by reverse-phase HPLC and identified by mass spectrometry. HPLC solvent A is 99% H2O and 1% MeCN with 0.1% TFA, solvent B is 10% H2O and 90%

MeCN with 0.07% TFA.

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Figure 4.12. HPLC spectra of TO-t2M. Gradient: 0-5 min 10% solvent B, 5-15 min; linear gradient from 0 to 35% solvent B; 15- 20 min, 35% solvent B; 20-22 min, linear gradient from 35 to 100% solvent B; 22-27 min, 100% solvent B; 27-29 min, linear gradient from 100 to 10% solv1ent B; 29-35 min, 10% solvent B. ESI: mass calculated: [M+H+]=695.3208; [M+2H2+]=348.1640; [M+3H3+]=232.4451. mass observed: [M+H+]=696.3229; [M+2H2+]=348.6650; [M+3H3+]=232.7805.

Figure 4.13. HPLC spectra of TO-t2MMe. Gradient: 0-5 min 20% solvent B, 5-15 min; linear gradient from 20 to 40% solvent B; 15-20 min, 40% solvent B; 20-22 min, linear gradient from 40 to 100% solvent B; 22-27 min, 100% solvent B; 27-29 min, linear gradient from 100 to 20% solv1ent B; 29-35 min, 20% solvent B. ESI: mass calculated: [M+H+]=807.4464; [M+2H2+]=404.2266. mass observed: [M+2H2+]=404.2249. 158

Figure 4.14. HPLC spectra of TO-4C-M*. Gradient: 0-5 min 20% solvent B, 5-15 min; linear gradient from 20 to 40% solvent B; 15-20 min, 40% solvent B; 20-22 min, linear gradient from 40 to 100% solvent B; 22-27 min, 100% solvent B; 27-29 min, linear gradient from 100 to 20% solv1ent B; 29-35 min, 20% solvent B. ESI: mass calculated: [M+H+]=528.2289; [M+2H2+]=264.6181. mass observed: [M+H+]=528.2299; [M+2H2+]=264. 6179.

Figure 4.15. HPLC spectra of TO-4C-M*4M. Gradient: 0-5 min 20% solvent B, 5-15 min; linear gradient from 20 to 40% solvent B; 15-20 min, 40% solvent B; 20-22 min, linear gradient from 40 to 100% solvent B; 22-27 min, 100% solvent B; 27-29 min, linear gradient from 100 to 20% solv1ent B; 29-35 min,

159

20% solvent B. ESI: mass calculated: [M+H+]=584.2915; [M+2H2+]=292.6494. mass observed: [M+H+]=584.2882; [M+2H2+]=292.6483.

4.9 Fluorescence turn on experiment.

Samples for fluorescence experiments were prepared by incubating 1 µM dye molecules with 1 µM RNA in 1X Spinach buffer (40 mM HEPES pH=7.3, 5 mM MgCl2, 125 mM

KCl) for 30 min unless otherwise noted. All fluorescence was measured on ThermoFisher

Nanodrop 3300 with blue light source, emission was measure at 505 nm for DFHBI derivatives and 532 nm for TO derivatives.

4.10 Apparent Kd determination.

DFHBI derivatives: Procedure adapted from reference 7. Samples of 1 µM RNA and various amounts of DFHBI-4C-M* were incubated in 1X Spinach buffer (40 mM HEPES pH=7.3, 5 mM MgCl2, 125 mM KCl) for 30 min at room temperature, followed by fluorescence measurements on ThermoFisher Nanodrop 3300 with blue light source, emission wavelength at 505 nm.

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Figure 4.16. Apparent Kd determination of DFHBI-4C-M* with Spinach RNAs. (Left) DFHBI-4C-M* with wt Spinach, Kd=1.43±0.87 µM. (Right) DFHBI-4C-M* with 5bpS A53U mutant, Kd=3.71±1.55 µM. A single representative plot is shown for each set, Kd value is the average of three independent experiments.

TO derivatives: Procedure adapted from reference 9. Samples of 100 nM TO derivatives and various amounts of RNA were incubated in 1X Spinach buffer (40 mM HEPES pH=7.3, 5 mM MgCl2, 125 mM KCl) for 30 min at room temperature, followed by fluorescence measurements on ThermoFisher Nanodrop 3300 with blue light source, emission wavelength at 532 nm.

Figure 4.17. Apparent Kd determination of TO derivatives with Spinach RNAs. (Left) TO-t2M with 5bpS U2, Kd=53.67±7.36 µM. (Right) TO-4C-M* with 5bpS U1, Kd=140.06±1.86 µM. A single representative plot is shown for each set, Kd value is the average of three independent experiments.

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4.11 References.

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6. Padmanabhan, K.; Padmanabhan, K.; Ferrara, J.; Sadler, J. E.; Tulinsky, A., The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer. Journal of Biological Chemistry 1993, 268 (24), 17651-17654.

7. Huang, H.; Suslov, N. B.; Li, N.-S.; Shelke, S. A.; Evans, M. E.; Koldobskaya,

Y.; Rice, P. A.; Piccirilli, J. A., A G-quadruplex–containing RNA activates fluorescence in a GFP-like fluorophore. Nat Chem Biol 2014, 10 (8), 686-691.

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Jeng, S. C. Y.; Unrau, P. J.; Ferre-D'Amare, A. R., Structural basis for high-affinity fluorophore binding and activation by RNA Mango. Nat Chem Biol 2017, advance online publication.

10. Yang, Q.; Xiang, J.; Yang, S.; Zhou, Q.; Li, Q.; Tang, Y.; Xu, G., Verification of specific G-quadruplex structure by using a novel cyanine dye supramolecular assembly:

I. Recognizing mixed G-quadruplex in human telomeres. Chemical Communications

2009, (9), 1103-1105.

11. Yang, P.; De Cian, A.; Teulade‐ Fichou, M. P.; Mergny, J. L.; Monchaud, D.,

Engineering Bisquinolinium/Thiazole Orange Conjugates for Fluorescent Sensing of G‐

Quadruplex DNA. Angewandte Chemie International Edition 2009, 48 (12), 2188-2191.

12. Allain, C.; Monchaud, D.; Teulade-Fichou, M.-P., FRET templated by G- quadruplex DNA: a specific ternary interaction using an original pair of donor/acceptor partners. Journal of the American Chemical Society 2006, 128 (36), 11890-11893.

13. Paige, J. S.; Wu, K. Y.; Jaffrey, S. R., RNA Mimics of Green Fluorescent Protein.

Science 2011, 333 (6042), 642-646.

14. Vummidi, B. R.; Alzeer, J.; Luedtke, N. W., Fluorescent probes for G‐ quadruplex structures. ChemBioChem 2013, 14 (5), 540-558.

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15. Parkinson, G. N.; Lee, M. P. H.; Neidle, S., Crystal structure of parallel quadruplexes from human telomeric DNA. Nature 2002, 417 (6891), 876-880.

16. Dai, J.; Punchihewa, C.; Ambrus, A.; Chen, D.; Jones, R. A.; Yang, D., Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation. Nucleic Acids Research 2007, 35 (7), 2440-2450.

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(T2AG3) 3] G-tetraplex. Structure 1993, 1 (4), 263-282.

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2009, 131 (12), 4301-4309.

164

Chapter 5 : bPNA(+)

165

5.1 Minimal T-T/U-U recognition motif: t2M

Triazines have been studied extensively in assembly1-5 and as base-pairing surrogates for adenine in PNA6. While much is known regarding artificial base pairs7-9, there are fewer examples of artificial base triples10, despite interest in native triple stranded structures11-

13. Triaminotriazine (melamine) forms a base triple with two thymine or uracil bases when presented on small-molecule5, 14-17 or macromolecular scaffolds18-24. Previously, we have described a compact DNA recognition element t2M: two melamines on a linear, 5- atom, sp3-hybridized scaffold. This small molecule could drive triplex hybridization with

DNA with micromolar affinity. Furthermore, reductive alkylation on the tren scaffold yielded t4M, with two t2M motif linked by a tertiary nitrogen. t4M has much high binding affinity to oligothymine motifs, with a 50 nM Kd to the octathymine bulge within

DNA duplex. We were pleasantly surprised by the dramatic increase that came from the multiple display of t2M and want to test the strategy on the peptide backbone.

5.2 Integration of t2M motif on peptide backbone: bPNA(+)

We have previously reported on bifacial peptide nucleic acid (bPNA), an α-peptide with displaying melamine derivatized lysine (M*) at alternate positions. This family of peptides forms triple-stranded hybrids with two oligo-T/U tracts18-19. Low nanomolar to micromolar affinity to T-rich DNA is observed for bPNAs with 10 and 4 melamine bases, respectively19. Due to alternating solubilizing groups, bPNA could be as long as 20 residues, which lead to entropy cost as much as 946 cal/(mol▪K) during the hybridization with DNA19. We hypothesized that installation of t2M motif on the peptide side chain

166 would decrease the length of peptide needed for triplex formation, thus reducing the entropy cost during the hybridization process, yielding a tighter binding to T/U rich nucleic acids. To test the utility of the t2M motif in multivalent recognition, a family of hexapeptides was prepared with amino side chains at alternate positions and either glutamic acid (E) or serine (S) at the remaining positions. Dendritic DNA-binding motifs were installed on side chain amines by reductive dialkylation with a melamine-bearing aldehyde (Scheme 1)25-26. Unlike previously reported bPNAs, the dendritic side chain displays two melamine bases on a tertiary amine like t2M, which bears a positive charge on protonation. We call this new family of peptide nucleic acid bPNA(+).

Figure 5.1. Structure and synthesis scheme for bPNA(+)

5.3 Biophysical study of bPNA(+)

We tested the ability of bPNA(+) to form triplex structure with T rich DNAs. Circular dichroism showed a clear transition from single stranded dT6C4T6 into folded DNA 167

+ structure when complexed with (EK )3, with a positive Cotton peak at 280nm diminished upon complexation, while the negative Cotton peak at 250nm was redshifted and increased in magnitude. These findings are consistent with the triplex formation of single stranded T rich DNA by bPNA18-19 and peptoid23.

Figure 5.2. Circular dichroism spectra showing 1:1 bPNA(+)-DNA complexation + (10µM each) of dT6C4T6 alone (---) and dT6C4T6 complex with (EK )3 (─)

Additionally, single melting transitions were observed with all bPNA(+)-DNA complexes by UV-melting experiments, indicating cooperative binding interaction between bPNA(+) and DNA. UV-melting traces and their first derivatives traces were shown in Figure 5.3 and melting temperatures were summarized in Table 5.1.

168

Figure 5.3. UV melting traces (left) and first derivatives (right) of hexamer bPNA(+) with dT6C4T6 and pentamer bPNA(+) with dT10C4T10. Tm values are summarized in Table 5.1.

The serine bPNA(+) form DNA complexes that are generally more thermally stable than their glutamic acid analogs by 5-6°C, presumably due to more favorable electrostatic interactions with DNA for the cationic S-series bPNA(+) relative to the zwitterionic E- 169 series bPNA(+). The greater DNA affinity of the S bPNA(+) relative to the E bPNA(+) was reflected in equilibrium binding isotherms derived from fluorescence anisotropy and gel shift. While the E-series 6mer bPNA(+) bound dT6C4T6 DNA with low nanomolar

DNA affinity (Kd ) when fit to a 1:1 binding model, the S-series bPNA(+) binding curves were sharply saturated even at 10 nM DNA, suggesting that DNA concentration is likely much greater than Kd (Figure 4). Further substrate dilution was beyond the detection limit of our method.

Figure 5.4. Hexapeptide bPNA(+) hybridization with dT6C4T6 DNA. (Left) Binding isotherms of fluorescently labeled (R1=Fl*) bPNA(+) and dT6C4T6. DNA + + 2 concentration is 50nM with (EK )3 (○) and 10nM with (SK )3 (●). R >0.96. (Right) DSC traces of bPNA(+) complexes with DNA at 1:1 stoichiometry, as labeled, at 25µM DNA + and bPNA(+). (SK )5 is complexed with dT10C4T10. All experiments carried out in 1XPBS pH 7.4.

Overall, the bPNA(+) family of peptides exhibits remarkable DNA affinity given the minimal hexapeptide footprint. Indeed, DNA hybridization for 6 residue bPNA(+) peptides rival or improve upon those observed for 20 residue bPNAs19. Notably, the 170 enthalpy of binding to dT6C4T6 for bPNA(+) hexapeptides is similar to that of bPNA

(EM*)6 , which also has 6 total melamine bases, but on a 12 residue peptide backbone (-

127kcal/mole). However, the bPNA(+) hybrids are 15-28°C more thermally stable than

+ the (EM*)6 bPNA hybrid (Table 1). Extension of bPNA(+) to 10-residue yields (EK )5

+ and (SK )5, which can form 1:1 complexes with dT10C4T10 with remarkably high Tm’s of

68 and 78 °C, respectively. To further probe these effects, the basic residue was varied from diaminobutyric acid (B+) to ornithine (O+) and to lysine (K+), which have 2, 3 and 4 atom linkages from base to the α- carbon, respectively. All bPNA(+) cleanly formed 1:1 triplex stem-loop hybrids with dT6C4T6 DNA. Though binding enthalpies were similar,

+ + + the B bPNA(+) hybrid Tm's were 7-10°C lower than that of the O and K bPNA(+) hybrids, even though charge is constant within an E or S bPNA(+) family (Table 5.1).

This suggests that all bPNA and bPNA(+) maintain similar base stacking, while lower hybrid Tm derives from greater entropic cost of DNA binding. If base stacking geometry is maintained, the shorter side chains of B+ bPNA(+) bring the peptide and DNA backbones closer together than the O+ /K+ bPNA(+), resulting in greater backbone restriction. Likewise, bPNA has a longer backbone than bPNA(+), and greater backbone entropic cost for binding DNA. Together, these studies support the notion that improved bPNA(+) hybridization properties relative to bPNA derive from a decrease in peptide backbone length and commensurate reduction in backbone entropy loss upon hybridization with DNA. The improvements over bPNA combine decreased entropic cost with favorable electrostatic interactions between DNA and bPNA(+).

171

Table 5.1. DNAa hydridization data for bPNAb and bPNA (+)

a + + DNA was for dT6C4T6 all samples except (EK )5 and (SK )5 which used dT10C4T10. b(EM*)6 is bPNA while all others are bPNA(+). cData fit well to a 1:1 model but DNA concentration was well above fitted Kd (10nM).

5.4 Applications of bPNA(+)

All bPNA(+)●DNA hybrid complexes are more thermally stable (10-20 °C) than calculated Tm of an analogous fully complementary DNA AT-rich duplexes. Thus bPNA(+) are expected to be competent in duplex strand invasion, unlike bPNA. To test

+ this notion, we incubated DNA duplex [dT6C4T6●dA6G4A6], (Tm=49 °C), with (SK )3 at room temperature. Full displacement of the dA6G4A6 strand and formation of the hybrid was observed after room temperature incubation for 2 hours. Similarly, the thermal denaturation profile of the sample shifted from 49 °C to 61 °C transition temperature, consistent with in situ formation of a bPNA(+)●DNA hybrid.

172

Figure 5.5. Strand invasion of DNA dupelx by bPNA(+). (Top) Illustration of DNA duplex strand displacement by (SK+)3 bPNA(+). (Left) Native gel of (a) dA6G4A6, (b) dT6C4T6, (c)duplex DNA [dT6C4T6●dA6G4A6] and (d) duplex after room temperature incubation with 2, 4, 8 μM (SK+)3 and 8 μM (SK+)3 after annealing at 96°C and cooling to RT. (Right) First derivative of thermal denaturation curves followed by UV (260 nm) of the DNA duplex [dT6C4T6●dA6G4A6] alone (---) and after 2 hours room temperature incubation with 1 equivalent (SK+)3 (—). DNA concentration was 2 μM in all experiments.

Overall, the multivalent presentation of melamine bases on bPNA(+) side chains delivers a significant entropic benefit to hybridization over standard bPNA. Applications such as nucleic acid imaging and structural probing, allosteric triggers for non-coding RNAs and nucleic acid delivery are currently underway.

173

5.5 Synthesis and characterization of bPNA(+)

5.5.1 Monomer synthesis.

Figure 5.6. Synthesis of N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6-triamine

N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6-triamine (1).

N2-(2,2-dimethoxyethyl)-1,3,5-triazine-2,4,6-triamine(1) was collected as white solid(4.5g, 70.1%) 1H NMR: 3.26 (6H,s); 3.28 (2H, m); 4.45 (1H,t); 5.99-6.14 (4H, br);

6.40 (1H, t); 13C NMR: 41.47; 52.93; 102.04; 150.04; 166.37; ESI: mass calculated

[M+H]+=215.1251; mass observed: 215.1262.

174

1 2 Figure 5.7. H NMR(400 MHz, d6-DMSO) of N -(2,2-dimethoxyethyl)-1,3,5-triazine- 2,4,6-triamine.

13 2 Figure 5.8. C NMR(100 MHz, d6-DMSO) of N -(2,2-dimethoxyethyl)-1,3,5- triazine-2,4,6-triamine.

175

5.5.2 Typical procedure for synthesis of bPNA(+).

214 mg of 1 was dissolved in 5mL of 1N HCl and reacted at room temperature for 1h, the solvent was then removed by rotary evaporator. The solid was dissolved in 1 mL of

DMSO to make a stock solution of melamine aldehyde (1M).

+ ABA-βAla-(SK )3-G. 50µl of 16.6mM of ABA-βAla-(SK)3-G were lyophilized to dryness, then re-dissolved in 28.4µl of DMSO and 5µl of aqueous solution of 1M acetate buffer (pH=4.5). Stock solution of melamine aldehyde in DMSO (1M, 8.3µl, 10 eq) and stock solution of NaBH3CN in MeOH (1M, 8.3µl, 10 eq) were then added. The final reaction mixture has 16.6 mM peptide, 166 mM of melamine aldehyde and 166 mM of

NaBH3CN in DMSO:MeOH:H2O=7.34:1.66:1. The mixture was reacted at 65°C for 1hr, then treated again with an additional 10 equivalence of both aldehyde and NaBH3CN.

After an additional 1 hour reaction, HPLC analysis showed target peptide as the major peak (Figure 5.9). Peak area of the target peptide was integrated, and divided by the total peak area of the trace to yield a 65% conversion. The product was then purified by

HPLC.

176

+ Figure 5.9. HPLC traces for reductive alkylation to bPNA(+) (SK )3 (red) from aminopeptide and a separate injection of the starting material peptide, (SK)3 (black). Peptides are N-terminated with ABA and ABA absorbance (270 nm) is shown. 65% of ABA-labeled material is the desired hexaalkylated product.

5.5.3 Synthesis and characterization of bPNA(+)

Peptide purity was checked by HPLC on a reverse phase analytical C18 column using the following gradient: 5% solvent B in 0-5min, 5-25% solvent B in 5-20min, 25% solvent B in 20-25min, 25-100% solvent B in 25-27min, 100% solvent B in 27min-32min, 100-5% solvent B in 32-34min, 5% solvent B in 34-40min (solvent A=0.1%TFA in water, solvent

B=0.1% TFA in 80% acetonitrile, 20% water).

177

+ Figure 5.10. HPLC trace of ABA- Ala-(EK )3-G, using the gradient described at the beginning of the section.

+ Figure 5.11. MALDI spectrum of ABA- Ala-(EK )3-G. Mass found: 1990.901, mass calculated: 1991.039. Y-axis is intensity, X-axis is mass charge ratio (m/z).

178

+ Figure 5.12. HPLC trace of ABA- Ala-(EO )3-G, using the gradient described at the beginning of the section.

+ Figure 5.13. MALDI spectrum of ABA- Ala-(EO )3-G. Mass found: 1947.588, mass calculated: 1948.992. Y-axis is intensity, X-axis is mass charge ratio (m/z).

179

+ Figure 5.14. HPLC trace of ABA- Ala-(EB )3-G, using the gradient described at the beginning of the section.

+ Figure 5.15. MALDI spectrum of ABA- Ala-(EB )3-G. Mass found: 1907.147, mass calculated: 1906.945. Y-axis is intensity, X-axis is mass charge ratio (m/z).

180

Figure 5.16. HPLC trace of ABA- Ala-(SK+)3-G, using the gradient described at the beginning of the section.

+ Figure 5.17. MALDI spectrum of ABA- Ala-(SK )3-G. Mass found: 1864.726, mass calculated: 1865.136. Y-axis is intensity, X-axis is mass charge ratio (m/z).

181

+ Figure 5.18. HPLC trace of ABA- Ala-(SO )3-G, using the gradient described at the beginning of the section.

+ Figure 5.19. MALDI spectrum of ABA- Ala-(SO )3-G. Mass found: 1822.973, mass calculated: 1822.973. Y-axis is intensity, X-axis is mass charge ratio (m/z). 182

+ Figure 5.20. HPLC trace of ABA- Ala-(SB )3-G, using the gradient described at the beginning of the section.

+ Figure 5.21. MALDI spectrum of ABA- Ala-(SB )3-G. Mass found: 1781.088, mass calculated: 1780.913. Y-axis is intensity, X-axis is mass charge ratio (m/z).

183

+ Figure 5.22. HPLC trace of ABA- Ala-(EK )5-G, using the gradient described at the beginning of the section.

+ Figure 5.23. MALDI spectrum of ABA- Ala-(EK )5-G. Mass found:3113.235, mass calculated: 3113.638. Y-axis is intensity, X-axis is mass charge ratio (m/z).

184

+ Figure 5.24. HPLC trace of ABA- Ala-(SK )5-G, using the gradient described at the beginning of the section.

+ Figure 5.25. MALDI spectrum of ABA- Ala-(SK )5-G. Mass found: 2903.621, mass calculated: 2903.585. Y-axis is intensity, X-axis is mass charge ratio (m/z). 185

5.6 General experimental procedures

5.6.1 Materials and general experimental procedures.

Tris(2-aminoethyl)amine was purchased from Chem-Impex International, Inc.

Oligonucleotides were purchased from Integrated DNA technologies. SYBRⓇ gold was purchased from Thermo Fisher Scientific. DNA stock solutions were serial diluted in deionized water, concentrations were determined by measuring absorbance at 260 nm by

Thermo Fisher Nanodrop 2000. Sample fluorescence was measured on Thermo Fisher

Nanodrop 3300.

DNA sequences used:

12-Tn-4: 5’-CGC ATA TTT GCG-Tn-CCAG-3’

3’-GCG TAT AAA CGC-Tn-GGTC-3’

12-T2-12: 5’-CGC ATA GCT CAG TTG ACT CGA TAC GC-3’

3’-GCG TAT CGA GTC TTC TGA GCT ATG CG-5’

AT duplex for strand invasion: 5’-AAA AAA GGG GAA AAA A-3’

3’-TTT TTT CCC CTT TTT T-5’

5.6.2 UV-melting

UV-melting curves were measured on Cary-100 UV-vis spectrophotometer equipped with an air-circulating temperature controller. All measurements were carried out with

186 temperature change rate of 1°C/min and monitored at 260 nm. All samples are freshly annealed in 1X PBS buffer (pH=7.4) before measurements, concentration of DNAs and bPNA(+) are held at 2 μM each.

5.6.3 Differential Scanning Calorimetry (DSC).

DSC experiments were carried out on Microcalorimeter VP-DSC. Samples were prepared to have 25 µM DNA and 25 µM bPNA(+) in 1X PBS buffer (pH=7.4). Samples were scanned from 25 °C to 90 °C with 60°C/h scanning rate, 16s filtering period and low feedback. 1X PBS was used as reference. Background data was collected with only 25

µM DNA in the sample cell and was subtracted from all DNA/bPNA(+) sample traces.

5.6.4 Fluorescence anisotropy.

A series of samples were made by mixing various concentrations of T6C4T6 and constant concentration of FITC labelled peptides (25nM or 50nM) in 1X PBS. All samples were annealed at 95°C for 5min. Experiments were carried out on Molecular Devices

SpectraMax M5 with excitation wavelength at 495 nm and emission at 520 nm.

Fluorescence anisotropy was converted into complex concentration using equation 118:

퐹퐴 − 퐹퐴푚푖푛 [푐표푚푝푙푒푥] = ( ) ∗ [푝푒푝푡푖푑푒] 퐹퐴푚푎푥 − 퐹퐴푚푖푛

Where FA, FAmax, FAmin correspond to correspond to current fluorescence anisotropy, maximum fluorescence anisotropy (FITC labelled peptides fully bound to DNA) and minimum fluorescence anisotropy (FITC labelled peptides alone), respectively.

187

The concentration of complex was plotted against total DNA concentration in each sample, the data was fitted using equation 227:

[푐표푚푝푙푒푥] = (퐾푑 + [퐷푁퐴] + [푝푒푝푡푖푑푒])/2

− (√(퐾푑 + [퐷푁퐴] + [푝푒푝푡푖푑푒])2 − 4 ∗ [퐷푁퐴] ∗ [푝푒푝푡푖푑푒])/2

5.6.5 Electrophoretic mobility shift assay (EMSA).

A series of samples were made by mixing various concentrations of T6C4T6 and constant concentration of FITC labelled peptides (50 nM or 25nM) in 1X PBS. All samples were annealed at 95℃ for 5min. Samples were then subjected to electrophoresis in 15% native acrylamide gel at 120V under ice. The gel was then stained with SYBRⓇGold and scanned using Typhoon FLA 9500 (GE Healthcare).

5.6.6 Strand invasion.

AT duplex was annealed at 95℃ prior to the experiment. For strand invasion, 2 µM AT

+ duplex was incubated with various amount of (SK )3 peptide at room temperature for 2 hrs. Samples were then subjected to 15% native acrylamide gel electrophoresis at 120V for 1.5 hrs. The gel was stained with SYBRⓇ gold and imaged using Typhoon FLA 9500

(GE Healthcare).

188

5.7 Additional UV-melting data

+ Figure 5.26. UV-melting of bPNA(+) (SK )3 and control peptides with T6C4T6 DNA. Only bPNA(+) exhibits a strong cooperative thermal transition, while the methylated control and the native SK3 peptide do not, proving that charge has a minimal effect on the strong binding of bPNA(+) with T6C4T6 DNA. (Left) UV-melting traces: absorbance at 260nm with respect to temperature. (Right) First derivatives of the UV-melting traces with respect to temperature.

5.8 Kd fitting from fluorescence anisotropy

Figure 5.27. Fluorescence anisotropy assay of T6C4T6 titrating into FITC-βAla- + (EK )3-G. Peptide concentration is 50 nM. Data fitted using 1:1 binding model. A single representative plot is shown, Kd value is the average of three independent experiments. 2 Kd=16.87±2.24 nM. R >0.96.

189

Figure 5.28. Fluorescence anisotropy assay of T6C4T6 titrating into FITC-βAla- + (EO )3-G. Peptide concentration is 50 nM. Data fitted using 1:1 binding model. A single representative plot is shown, Kd value is the average of three independent experiments. 2 Kd=4.77±2.04 nM. R >0.96.

Figure 5.29. Fluorescence anisotropy assay of T6C4T6 titrating into FITC-βAla- + (EB )3-G. Peptide concentration is 50 nM. Data fitted using 1:1 binding model. A single representative plot is shown, Kd value is the average of three independent experiments. 2 Kd=13.56±1.26 nM. R >0.96.

190

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