I Bifacial PNA in Nucleic Acid Folding, Peptide Ligation and in Vitro

Home , Ammeline, Melamine, Melamine cyanurate, Peptide nucleic acid

Bifacial PNA in Nucleic Acid Folding, Peptide Ligation and in vitro Selection

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

in the Graduate School of the Ohio State University

Xijun Piao, B.S.

Graduate Program in Chemistry

The Ohio State University

2016

Dissertation Committee:

Dr. Dennis Bong, Advisor

Dr. Jovica Badjic

Dr. Jonathan R. Parquette

Xijun Piao

2016

Abstract

This dissertation summarizes three research projects regarding the fundamental studies and applications of bifacial Peptide Nucleic Acid (bPNA) – nucleic acid hybrid triplex system. Chapter 2 describes the syntheses of a series of bPNAs with different length and the cooperative folding of thymine-rich deoxyoligonucleotides by bPNA into binary, ternary and quaternary complexes. Chapter 3 demonstrates that template effect exists in abiotic bPNA – nucleic acid triplex system in that nucleic acid templates acyl group transfer, bPNA ligation and extension. Chapter 4 presents the application of bPNA – nucleic acid triplex as a replacement of native duplex in constructing a reversibly-structured RNA library for in vitro selection against stapled highly helical HIV-1 Rev peptides and also explores the concept of target-directed evolution of RNA library.

In Chapter 2, we demonstrate melamine-displaying bPNA recognizes thymine-rich DNA in predictable and multifaceted ways that allow binding affinity, structure stability and stoichiometry to be tuned through simple bPNA length modification and matching with

DNA length. The longer recognition interface results in the increase of melting temperature and enthalpy change for dissociation, as well as decrease of dissociation constant. 10mer bPNA can tolerate a high number of “mismatches” on thymine tracts and still yields triplex formation whose melting temperature correlates directly with thymine content.

Interestingly, when a DNA host has more T − T sites than melamine sites on bPNA, two or three bPNAs can bind to a single DNA, resulting in ternary and quaternary complexes that have higher thermal stability than the binary (1:1) bPNA − DNA complex, suggestive of cooperative multisite binding. In contrast, when two bPNAs of different lengths bind to ii the same DNA host, a ternary complex is formed with two melting transitions, corresponding to independent melting of each bPNA component from the complex. This set of data serve as the foundation for our future research.

In Chapter 3, we demonstrate DNA- and RNA-templated chemical transformation of bifacial peptide nucleic acid (bPNA) fragments directed by an abiotic thymine/uracil – melamine – thymine/uracil triplex hybrid interface. Watson-Crick base-pairing is widely studied in nucleic acid-templated chemistry, however very few reports are about non-

Watson-Crick recognition. Triplex hybridization of reactive bPNA fragments with DNA template is shown to catalyze acryl group transfer, bPNA native chemical ligation and length-controlled bPNA extension. Gratifyingly, RNA-templated oxidative coupling of bPNA fragments is found to result in the emergence of engineered ribozyme cleavage.

These data demonstrate that nucleic acid template effect exists in abiotic melamine- thymine/uracil bifacial recognition and establish a connection between engineered and native reaction sites.

In Chapter 4, we report the use of bPNA – nucleic acid triplex hybridization to construct a reversibly-structured RNA library for in vitro selection against side chain-stapled HIV-1

Rev peptides. Bifacial PNA – nucleic acid triplex stem structurally replaces native duplex stem evolved from random region and forces the library into a stem-loop structure, leaving the precious random nucleotides for the evolution of binding motifs instead of non-binding duplex stem. This bPNA-assisted reversibly-structured RNA library also facilitates the problematic reverse transcription of selected RNA aptamers by loosening the extensive secondary structures. Although many peptide targets have been used for in vitro selection, a stapled peptide is not seen and known to enhance helicity and binding affinity. In vitro selection against stapled original Rev peptide yields RNA aptamers showing the conserved core element presented in native Rev Responsive Element (RRE) RNA. A key iii asparagine on the stapled Rev peptide is mutated to two synthetic triazine-displaying amino acids that direct the evolution of random oligonucleotides based on likely hydrogen- bonding preference. This ongoing project integrates engineered bPNA – nucleic acid triplex stem into in vitro selection and may solve the problem of reverse transcription of in vitro selection. Further single mutations on stapled peptide targets demonstrate the concept of directed evolution of RNA library.

Dedication

Dedicated to my wife and parents

Acknowledgements

I am very grateful to my PhD advisor, Dr. Dennis Bong. We have worked together for six years and he not only helped me enrich my knowledge in organic chemistry and biophysics, but also made me become a more confident and competent researcher. His patience, persistence and trust led us to complete our very challenging in vitro selection project, in which I learned nucleic acid chemistry from the very beginning and improved my problem- solving ability. His goal for research originality is highly admirable and everything I learned in his lab would become a great fortune for my future research.

I would also like to thank Dr. Badjic and Dr. Parquette for serving my candidacy and defense committee, as well as writing reference letters in support of my postdoctoral researcher application.

I also highly appreciate the helpful advice and discussions provided by friendly labmates, as well as productive collaborations.

Finally, I am very grateful and indebted to my family. My parents, Mingtao Piao and

Huiqing Gu, have been giving me unconditional love and tremendous support all the time throughout the years I was far away from home. My wife Ying Yu and my parents-in-law have been giving me endless love and indispensable trust since my marriage and I believe their love, understanding and support will make me become a better, more responsible and supportive husband to my family.

Vita

2003 – 2006……………………………………. Anshan No.1 Middle School, Anshan, China

2006 – 2010………………………………………… B.S., Fudan University, Shanghai, China

2010 – 2016...... ……Graduate Teaching / Research Assistant, The Ohio State University

Publications during PhD

(1) Xijun Piao, Xin Xia, Jie Mao, and Dennis Bong*, “Peptide Ligation and RNA

Cleavage via an Abiotic Template Interface” J. Am. Chem. Soc. 2015, 137, 3751-3754.

(2) Xijun Piao, Xin Xia, and Dennis Bong*, “Bifacial Peptide Nucleic Acid Directs

Cooperative Folding and Assembly of Binary, Ternary, and Quaternary DNA

Complexes” Biochemistry, 2013, 52, 6313-6323.

(3) Xin Xia, Xijun Piao, and Dennis Bong*, “Bifacial Peptide Nucleic Acid as an

Allosteric Switch for Aptamer and Ribozyme Function” J. Am. Chem. Soc. 2014, 136,

7265-7268.

(4) Xin Xia, Xijun Piao, Kurt Fredrick, and Dennis Bong*, “Bifacial PNA Complexation

Inhibits Enzymatic Access to DNA and RNA” ChemBioChem, 2014, 15, 31-36.

(5) Yingying Zeng, Yaowalak Pratumyot, Xijun Piao and Dennis Bong*, "Discrete

assembly of synthetic peptide-DNA triplex structures from polyvalent melamine -

thymine bifacial recognition" J. Am. Chem. Soc. 2012, 134, 832-835.

Fields of Study

Major field: Chemistry vii

Table of Contents

Abstract…………………………………………………………………………………………....ii

Acknowledgement…………………………………………………………………………….…vi

Vita………………………………………………………………………………………………..vii

Table of Contents………………………………………………………………………….…….vii

List of Figures…………………………………………………………………………………...xiv

List of Tables………………………………………………………………………………...... xviii

Chapter 1. Structures, Recognition and Template Effect of Nucleic Acids ...... 1

1.1 Nucleic acid structures ...... 2

1.1.1 DNA duplex ...... 3

1.1.2 DNA triplex ...... 4

1.1.3 Nucleic acid G-quadruplex ...... 6

1.1.4 RNA folding ...... 7

1.2 Modifications on nucleic acid to enhance triplex formation...... 8

1.3 Peptide Nucleic Acid (PNA) – Native nucleobases on pseudopeptide backbone . 10

1.3.1 Chemical structure of PNA ...... 10

1.3.2 PNA-native nucleic acid duplex and triplex ...... 11

1.3.3 Biological and medicinal applications of PNA ...... 12

1.4 Targeting nucleobase pairs with Janus-Wedge insertion strategy ...... 12

1.4.1 Janus-Wedge Insertion of a single nucleobase pair by small molecules ...... 13

1.4.2 Single Janus-Wedge insertion on biopolymer scaffold ...... 14 viii

1.4.3 Multivalent insertion of Janus-Wedge heterocycles into nucleobase pairs ..... 14

1.4.4 Triazine used to target nucleobase or nucleobase pairs and bifacial Peptide

Nucleic Acid (bPNA) ...... 16

1.5 Nucleic acid template effect on nonenzymatic reactions ...... 19

1.6 References ...... 22

Chapter 2. Bifacial Peptide Nucleic Acid Directs Cooperative Folding and Assembly of

Binary, Ternary, and Quaternary DNA Complexes ...... 36

2.1 Introduction ...... 37

2.2 Results and Discussion ...... 41

2.2.1 1:1 bPNA-DNA complexes ...... 41

2.2.2 Binding affinity and enthalpy of bPNA-DNA complexes ...... 43

2.2.3 Tolerance of “mismatch” sites in (EM*)10 DNA complexation ...... 46

2.2.4 bPNA-DNA higher-order assembly ...... 51

2.3 Conclusion ...... 55

2.4 Materials and Equipments ...... 56

2.5 Synthesis ...... 57

2.5.1 Synthesis of Fmoc-Lys(M*)-OH ...... 57

2.5.2 Solid phase synthesis of bPNA ...... 63

2.5.3 HPLC and Maldi-TOF mass spectra of the synthetic peptides ...... 64

2.6 Experimental Methods ...... 70

2.7 Acknowledgement ...... 71

2.8 References ...... 71

Chapter 3. Peptide Ligation and RNA Cleavage via an Abiotic Template Interface ...... 78

3.1 Introduction ...... 79

3.2 Results and Discussion ...... 81

3.2.1 Acyl transfer catalyzed by bPNA-DNA hybridaztion...... 81

3.2.2 Native chemical ligation templated by bPNA-DNA hybridization ...... 82

3.2.3 Length-controlled bPNA extension ...... 85

3.2.4 Ribozyme-templated bPNA oxidative coupling and activation of ribozyme self-

cleavage function ...... 86

3.3 Conclusion ...... 89

3.4 Materials and Equipments ...... 89

3.5 Sequences ...... 90

3.6 Synthesis ...... 92

3.6.1 ABA-Gly thioester ...... 92

3.6.2 bPNA Synthesis ...... 101

3.7 Experimental Methods ...... 113

3.7.1 DNA Complexation ...... 113

3.7.2 UV melting ...... 113

3.7.3 Electrophoretic mobility shift assay (EMSA) ...... 113

3.7.4 Acyl small-molecule transfer ...... 114

3.7.5 Native chemical ligation ...... 114

3.7.6 Cysteine-free direct aminolysis ...... 114

3.7.7 Disulfide ligation ...... 115

3.7.8 Ellman’s test for dicysteine bPNA extension ...... 115

3.7.9 Product isolation from gel ...... 115

3.7.10 Gel quantification ...... 115

3.7.11 RNA transcription and purification ...... 116

3.7.12 Ribozyme cleavage with bPNA ...... 116

3.7.13 U3 Ribozyme-templated bPNA disulfide oxidation ...... 116

3.7.14 In situ bPNA oxidation and U3 Ribozyme cleavage ...... 117

3.8 Acknowledgement ...... 117

3.9 References ...... 117

Chapter 4. In vitro Selection of a bPNA-Assisted Reversibly-Structured RNA Library against Stapled HIV-1 Rev Peptides ...... 124

4.1 Introduction ...... 125

4.1.1 In vitro selection and aptamers ...... 125

4.1.2 Two major limitations in in vitro selection ...... 127

4.1.3 A novel strategy simultaneously overcomes the two limitations ...... 128

4.1.4 HIV-1 Rev peptide and it use in in vitro selection...... 129

4.1.5 Stapled peptides enhance α-helicity and binding affinity ...... 130

4.1.6 A bPNA-assisted reversibly-structured RNA library for in vitro selection against

stapled HIV-1 Rev peptides and directed evolution of aptamers ...... 131

4.2 Results and Discussion ...... 133

4.2.1 Stapled Rev peptides enhance helicity and binding affinity ...... 133

4.2.2 General procedure of in vitro selection ...... 136

4.2.3 Significant enrichment is observed after 6th round ...... 137

4.2.4 Sequences of interest are identified and share similarities with native RRE 139

4.2.5 Single mutation on Rev peptide directs the evolution of RNA library ...... 142

4.3 Conclusion ...... 143

4.4 Materials and Equipments ...... 143

4.5 Sequences ...... 144

4.5.1 Stapled Rev peptide sequences ...... 145

4.5.2 Nucleic acid sequences ...... 145

4.6 Synthesis ...... 146

4.6.1 Synthesis of Fmoc-azidoalanine ...... 146

4.6.2 Synthesis of Fmoc-Lys(Biotin)-OH ...... 147

4.6.3 Synthesis of Fmoc-Dap(M*)-OH ...... 149

4.6.4 Synthesis of Fmoc-Dap(N*)-OH ...... 150

4.6.5 Rev peptide synthesis and stapling ...... 153

4.7 Experimental Procedures ...... 157

4.7.1 Transcription of N25 RNA library ...... 158

4.7.2 Annealing of N25 RNA library with bPNA ...... 159

4.7.3 Pre-selection of annealed N25 RNA library ...... 160

4.7.4 In vitro selection against target ...... 161

4.7.5 Pull-down of binding aptamer ...... 161

4.7.6 Wash the beads with high salt buffer ...... 161

4.7.7 Denaturation and collection of binding aptamers ...... 161

4.7.8 Reverse transcription of selected RNA into complementary DNA ...... 162

4.7.9 PCR amplification of cDNA ...... 163

4.8 Acknowledgement ...... 164

xii

4.9 References ...... 164

Appendix A: Analysis of High-Throughput Sequencing Raw Data ...... 175

A.1 Installation ...... 176

A.2 Download raw data ...... 176

A.3 Removal of 3’-constant region on T-tract sequences ...... 176

A.4 Removal of 5’ constant region ...... 178

A.5 Removal of two T10 tracts ...... 179

A.6 Analysis of trimmed sequences ...... 181

A.7 References ...... 183

References ...... 184

Chapter 1 ...... 184

Chapter 2 ...... 197

Chapter 3 ...... 203

Chapter 4 ...... 209

Appendix A ...... 219

xiii

List of Figures

Figure 1.1 Chemical structures of nucleic acids………………………………….….………...3

Figure 1.2 Watson-Crick base pairing and DNA duplex………………………………..…....4

Figure 1.3 Nucleobase triplets and their orientations………………………………………...6

Figure 1.4 G-quadruplex structures………………………………………………………….....7

Figure 1.5 Secondary and tertiary structural elements in RNA……………………………...8

Figure 1.6 Examples of modifications on nucleic acid to enhance triplex formation………9

Figure 1.7 Peptide nucleic acid structure and its hybridization with native DNA………….10

Figure 1.8 PNA binding modes for targeting double-stranded DNA……………………….11

Figure 1.9 Janus-Wedge insertion into nucleobase pairs…………………………………..13

Figure 1.10 Examples of triazine-bearing sequences……………………………………….15

Figure 1.11 Bifacial melamine-thymine recognition…………………………………………18

Figure 1.12 Examples of nucleic acid template effect………………………………………20

Figure 2.1 Illustration of melamine-thymine interaction directing DNA and bPNA assembly and CD spectra………….……………………………………………………………………...38

Figure 2.2 Normalized fluorescence titration curve for Cbf-(EM*)n-dTnC4Tn……………..41

Figure 2.3 Characterization of binding affinity……………………………………………….43

Figure 2.4 Invasion of triplex (dT10C4T10+H2N-(EM*)10G) with dA10G4A10…………………44

Figure 2.5 Fluorescence titration curve for bPNA with mismatched DNA hairpin………..48

Figure 2.6 Normalized fluorescence titration curve for higher-order assembly…………..50

Figure 2.7 Illustration of higher-order bPNA-DNA assembly………………………………..52

Figure 2.8 Analysis of binary and ternary complexes of bPNA and dT15C4T15……………52

xiv

Figure 2.9 1H NMR of Fmoc-Lys(M*)-OH…………………………………………………….57

Figure 2.10 13C NMR of Fmoc-Lys(M*)-OH…………………………………………………..58

Figure 2.11 Characterization of 4mer bPNA Cbf-(β-A)-(EM*)4G……………………...... 60

Figure 2.12 Characterization of 5mer bPNA Cbf-(β-A)-(EM*)5G…….…………………….61

Figure 2.13 Characterization of 6mer bPNA Cbf-(β-A)-(EM*)6G………….……………….62

Figure 2.14 Characterization of 8mer bPNA Cbf-(β-A)-(EM*)8G………………..…………63

Figure 2.15 Characterization of 10mer bPNA Cbf-(β-A)-(EM*)10G…………………………64

Figure 2.16 Characterization of 10mer bPNA Aba-(β-A)-(EM*)10G…..……………………65

Figure 3.1 bPNA molecular recognition and nucleic acid templates………………………76

Figure 3.2 DNA-templated thioester exchange between bPNAs…………………………..78

Figure 3.3 DNA-templated native chemical ligation…………………………………………80

Figure 3.4 DNA-templated bPNA extension………………………………………………….82

Figure 3.5 Nucleic acid-templated oxidative coupling of bPNA thiols……………………..84

Figure 3.6 1H NMR of product 1………………………………………………………………..90

Figure 3.7 1H NMR of Product 2…………………………………………………………….…91

Figure 3.8 1H NMR for ABA-Gly thioester…………………………………………………….92

Figure 3.9 13C NMR of ABA-Gly thioester…………………………………………………….93

Figure 3.10 Characterization of bPNA 1………………………………………………………96

Figure 3.11 Characterization of bPNA 2………………………………………………………97

Figure 3.12 Characterization of bPNA 3………………………………………………………98

Figure 3.13 Characterization of bPNA 4………………………………………………………99

Figure 3.14 Characterization of bPNA 5…………………………………………………….100

Figure 3.15 Characterization of bPNA 6…………………………………………………….101

Figure 3.16 Characterization of bPNA 7…………………………………………………….102

Figure 3.17 Characterization of bPNA 8…………………………………………………….103 xv

Figure 3.18 Characterization of bPNA 9…………………………………………………….104

Figure 3.19 Characterization of bPNA 10…………………………………………………..105

Figure 4.1 General procedure of in vitro selection (SELEX)….…………………………..119

Figure 4.2 Illustration of bPNA-assisted reversibly-structured RNA library construction.121

Figure 4.3 Short Rev peptide adopts adaptive binding to its RNA aptamers…………...124

Figure 4.4 Structure of isolated short hairpin Rev responsive element (RRE), HIV-1 Rev peptide and their interaction………………………………………………………………….126

Figure 4.5 α-helix of Rev peptides…………………………………………………………..128

Figure 4.6 Procedure of in vitro selection of bPNA-assisted reversibley-structured RNA library.……………………………………………………………………………….………….129

Figure 4.7 UV melting of RNA library at different round with bPNA………………………131

Figure 4.8 Unique sequence percentage of each pool in three selections……………….131

Figure 4.9 Predicted folding of top aptamers of original stapled Rev peptide and native

RRE…………………………………………………………………………………………….134

Figure 4.10 Predicted folding of top sequences in Cluster 3 and possible bifacial recognition..……………………………………………………………………………………135

Figure 4.11 Characterization of stapled original HIV-1 Rev peptide…………………….146

Figure 4.12 Characterization of stapled melamine-displaying HIV-1 Rev peptide……..147

Figure 4.13 Characterization of stapled ammeline-displaying HIV-1 Rev peptide……..148

Figure 4.14 2% Agarose gel image for PCR duplexes in the 10th round of selection….156

Figure A.1 Removal of 3’ constant flanking region and adapter...... 169

Figure A.2 Removal of 5’ constant flanking region…………………………………………170

Figure A.3 Removal of one T-tract…………………………………………………………..171

Figure A.4 Removal of the other T-tract…………………………………………………….172

Figure A.5 Converting from fastq to fasta file……………………………………………….173 xvi

Figure A.6 Clustering the aptamers…………………………………………………………174

Figure A.7 Comparing the enrichment between different rounds………………………..175

xvii

List of Tables

a Table 2.1 Thermodynamic data for [(EM*)n-dTnC4Tn] ………………………………..…….42

Table 2.2 Tm of Cbf-10 complexes with T-rich 24-nt DNA…………………………………..46

Table 2.3 Tm’s of higher order bPNA-DNA assembly………………………………………..49

Table 4.1 Populated sequences identified from three in vitro selections………………..133

Table 4.2 In vitro selection condition.……………………………………………………….149

Table 4.3 Transcription condition for 1st round………..…………………………………….151

Table 4.4 Annealing condition………………………………………………………………..152

Table 4.5 Pre-selection condition..…………………………………………………………..152

Table 4.6 Reverse transcription condition…………………………………………………..154

Table 4.7 Manual hot start PCR condition…………………………………………………..155

Table A.1 Two representative sequences…………………………………………………..167

Table A.2 Representative sequence………………………………………………………...169

Table A.3 Representative sequence change……………………………………………….170

Table A.4 N25 region of interest………………………………………………………...…..172

xviii

Chapter 1. Structures, Recognition and Template Effect of Nucleic Acids

This dissertation describes and summarizes the fundamental studies and applications of hybrid triplexes formed by synthetic bifacial peptide nucleic acid (bPNA) and native nucleic acids (DNA, RNA). Related background of nucleic acid structures, enhanced triplex formed from modified oligonucleotides, peptide nucleic acid, Janus-Wedge insertion, bPNA and nucleic acid template effect is thus discussed in the introduction. A two-way communication is established in Chapter 2 and Chapter 3: bPNA folds DNA or RNA into a triplex stem-loop structure, while DNA or RNA catalyzes bPNA ligation. In Chapter 4, we test a novel strategy of constructing a bPNA-assisted reversibly-structured RNA library for in vitro selection against stapled original and point-mutated highly helical HIV-1 Rev peptides to overcome two major limitations exist in in vitro selection and explore the concept of directed evolution of nucleic acid.

1.1 Nucleic acid structures

Native nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Nucleic acid is also known as polynucleotides while each nucleotide is composed of one of the four nucleobases (cytosine, guanine, adenine and thymine in DNA; cytosine, guanine, adenine and uracil in RNA), a monosaccharide (2-deoxyribose in DNA; ribose in

RNA) and a phosphate group. Nucleobase forms N-glycosidic bond between nitrogen atom and 1’-OH of the sugar and phosphate group forms ester bond with 5’-OH of the sugar. Nucleotides are then bonded to each other linearly through 3’-5’ phosphodiester linkage and the name of polynucleotide indicates that nucleic acid is of highly repetitive covalent conjugation of nucleotides.1-3 The strand shows a 5’ to 3’ polarity.

Figure 1.1 Chemical structures of nucleic acids. Adapted from reference 3

1.1.1 DNA duplex

DNA tends to form helical duplex due to non-covalent Watson-Crick base pairing which states that adenine on one chain is specifically paired with thymine on the other chain, while guanine is always paired with cytosine on different chains.4 Thus, the two complementary strands are held together in an anti-parallel orientation. At least three forms of DNA duplex are found in nature, A-form, B-form and Z-form. And the most abundant B-form DNA is a right-handed helix with high periodicity. It is 23.7 Å wide and

34 Å per helical turn (~10.5 base pairs) about the axis in solution.5 A-form DNA helix is also right-handed but compressed along the axis, while Z-form DNA is left-handed and elongated along the axis. When forming a double-stranded helix, two types of grooves are

3 generated, major groove and minor groove. Major groove is wide and rich in chemical information which can be used to distinguish different base pairs. Minor groove is narrow and traditionally considered to contain less chemical information. However, Dervan and co-workers are able to differentiate base pairs using synthetic polyamides in DNA minor groove.6-9

Figure 1.2 Watson-Crick base pairing and DNA duplex. Base pairing with an exemplary

numbering (Left); DNA duplex of different forms (Right). Adapted from Molecular Biology

of the Gene, 5th edition, James D. Watson, published by Pearson Education, Inc.

1.1.2 DNA triplex

Nucleic acid triplex is formed when a third strand of oligonucleotide binds to a preformed

DNA duplex through Hoogsteen or reversed Hoogsteen base pairing in the major 4 groove.10 Hoogsteen base pairing is different from classic Watson-Crick base pairing in that N7 of the purine base is involved in base pairing instead of N1. A third strand of homopyrimidine can bind to a double-stranded helix composed of a homopurine strand and a homopyrimidine strand via Hoogsteen base pairing, based on T-A*T and C-G*C+ triplex base pairing (* denotes Hoogsteen base pairing; - denotes Watson-Crick base pairing; C+ denotes a protonated cytosine).11,12 A third strand of homopurine13,14 can also bind to a duplex composed of a homopurine strand and a homopyrimidine strand via reversed Hoogsteen base pairing pattern, based on T-A*A and C-G*G triplex base pairing.

In Hoogsteen base pairing, the third strand is parallel to the homopurine strand, whereas they are antiparallel in the reversed Hoogsteen base pairing.15 DNA triplex generally forms in non-physiological conditions, such as lower pH (<6).16 As seen in C-G*C+ triplet, cytosine on the additional strand needs to be protonated to form hydrogen bonds with guanine. Furthermore, divalent cation17 and polyamine18 are helpful to shield electrostatic repulsion between the three highly negatively-charged oligonucleotide strands.19,20

Although DNA triplex often forms at non-physiological conditions, some homopurine- homopyrimidine DNA duplex that is a potential targeting site for triplex formation is also found in vivo.21-23 Marky and co-workers found that formation of DNA triplex was accompanied by a favored enthalpic change and an unfavored entropic change, indicative of an enthalpy and entropy compensation. Comparison of the thermodynamic profiles of these triplexes yielded enthalpic contributions of -24 kcal/mol, -23 kcal/mol, and -22 kcal/mol for the formation of TAT/TAT, TAT/CGC+, and CGC+/CGC+ base triplet stack.24

Figure 1.3 Nucleobase triplets and their orientations. (Left) Hoogsteen base-pairing;

(Right) reverse Hoogsteen base-pairing. Solid lines, purine strands; stippled lines,

pyrimidine strands; vertical lines, Watson-Crick hydrogen bonds; diamonds, Hoogsteen

or reverse Hoogsteen hydrogen bonds. Adapted from reference 11.

1.1.3 Nucleic acid G-quadruplex

G-quadruplex is possible in both DNA and RNA when the sequence is rich in guanine.

Four guanine nucleobases can form a square planar structure through Hoogsteen hydrogen bonding, and several layers can stack on top of each other to form a G- quadruplex. This structure is further stabilized by the presence of potassium cation which is coordinated in the center of G-quadruplex.25,26 Different from nucleic acid triplex, G-

6 quadruplex forms at physiological conditions and is found in vivo.27,28 A well-known example is human telomeric repeat d(GGTTAG), which forms G-quadruplexes to decrease the activity of telomerase and fail the maintenance of telomere length.29 As shown in Figure 1.4, G-quadruplex can be either intramolecular or intermolecular.

Figure 1.4 G-quadruplex structures. Cited from reference 15.

1.1.4 RNA folding

Quite distinguished from DNA, a single-stranded RNA can actually fold itself into very complex structures. Indeed, a RNA single strand frequently folds back on itself to form paired regions due to base pairing. Several non-paired nucleotides may be looped out when adjacent sequences form complementary base pairs or wobble base pairs, resulting in hairpin, bulge and loop structures.30,31 Paired RNA region is similar with A-form DNA.32

In contrast to these stem-loop structures, pseudoknot33 indicates that non-contiguous regions form complex structures. Due to the flexibility (freedom of rotation) gained from unpaired region, RNA molecules have a number of complex tertiary structures that are not seen in DNA. Indeed, many functional forms of RNA single strands frequently require specific tertiary structures, such as tRNA34 and ribozyme.35

Figure. 1.5 Secondary and tertiary structural elements in RNA. Cited from reference 31.

1.2 Modifications on nucleic acid to enhance triplex formation

As discussed above, nucleic acid triplex structure is of interest, however, native nucleic acid triplex often forms at non-physiological conditions, which limits its in vivo applications.

Researchers thus modified nucleobase and backbone to favor the formation of triplex at physiological conditions. Modifications on sugar or phosphate reduce negative charges on RNA and DNA, lowering electrostatic repulsion between strands and stabilizing triplex.36,37 2’-aminoethyl (EA) introduces positive charges on sugar to neutralize negative charges on phosphate backbone, leading to a more stable triplex structure.38 Among these two types of modifications, locked nucleic acid (LNA) is quite famous, 2’ oxygen and 4’ carbon are linked within one ribose and locks ribose in the 3’-endo conformation.39 This preorganized conformation increases nucleobase stacking, resulting in more thermostable duplex and triplex with other oligonuleotide strands.40-42 Modifications on nucleobase can also enhance the formation of nucleic acid triplex. This usually remains

Watson-Crick or Hoogsteen recognition interface intact, but modifies the non-bonding side.

For example, by adding 8’ amino group, guanine and adenine can form more stable triplex with other native nucleobases.43 5’-methylated cytosine elevates the pKa of protonated

N3 and favors the formation of C-G*C+ triplet in DNA.44-46 6-thioguanine favors the formation of triplex over quadruplex and GA homodimer due to O→S modification which cripples the traditional trivalent hydrogen bonds and disfavors the formations of competing structures.47 In addition, a class of synthetic nucleobases interact with matched native nucleobase pairs simultaneously, thereby enhancing the stability of formed triplex.48

2-aminoethyl Locked Nucleic Acid (LNA)

8-aminoguanine Interaction with CG pair

Figure 1.6 Examples of modifications on nucleic acid to enhance triplex formation. Cited from reference 38, 39 and 48.

1.3 Peptide Nucleic Acid (PNA) – Native nucleobases on pseudopeptide backbone

1.3.1 Chemical structure of PNA

Different from modifications on phosphate group and sugar shown in last section, peptide nucleic acid (PNA) displays native nucleobases on a pseudopeptide chain, poly(N-(2- aminoethyl)-glycine).49,50 PNA thus mimics nucleic acid due to predictable base-pairing, and differs from nucleic acid in losing backbone enzymatic accessibility.51 All the base- pairing rules discussed in previous sections applies to PNA, including Watson-Crick base pairing and Hoogsteen base pairing.52,53 The native nucleobases are linked to secondary amine on the backbone and the repetitive structure mimics oligonucleotides greatly: three bonds between base and backbone while six bonds within a repeating monomer. PNA is found to bind to its complementary oligonucleotide stronger than its native counterpart, and this is attributed to the lack of negative charges on the backbone which reduces electrostatic repulsion upon hybridization.49,51 The poly(N-(2-aminoethyl)-glycine) chain is not easily accessible to nucleases and proteases, making PNA resistant to enzymatic degradation.54

Figure 1.7 Peptide nucleic acid structure and its hybridization with native DNA. Cited

from reference 51.

1.3.2 PNA-native nucleic acid duplex and triplex

The backbone and amide linker to the base show constrained flexibility that is important to base-pairing, and reduction of the amide is detrimental to nucleic acid binding. Due to the neutral backbone, PNA reduces the electrostatic repulsion between strands, resulting in a more thermostable duplex and triplex. Indeed, PNA can invade an existing DNA duplex, forming a hybrid duplex with its complementary strand. In PNA-DNA and PNA-

RNA duplexes, PNA adapts the A-form and B-form helices preferred by RNA and DNA, respectively.51 However, a homopyrimidine PNA usually invades double-stranded DNA,

55-58 forming PNA2-DNA triplex instead of an expected PNA-DNA2 adduct. This strand displacement occurs due to the very high stability of PNA2-DNA triplex.

Figure 1.8 PNA binding modes for targeting double-stranded DNA. Cited from reference

51.

1.3.3 Biological and medicinal applications of PNA

Since synthetic PNA can recognize DNA sequence of interest and invade the existing double-stranded DNA, it may be used to target a particular sequence,59,60 interfering with

11 biochemical processes, such as replication, transcription and translation.61 Once PNA invasion occurs, a PNA-DNA duplex or PNA2-DNA triplex will form, blocking enzymatic read-through of this DNA strand. This is called antigene strategy which mainly interferes with transcription process.53,62,63 If PNA forms stable complex with mRNA and inhibits the translation process, this is the antisense strategy.64-66 Other biological and medicinal applications of PNA include PCR amplification enhancement of small allelic products, determination of telomere size, and single base pair mutation analysis.61 Overall, the wide biological and medicinal applications of PNA originates from the higher thermostability of

PNA-nucleic acid complex which disables the enzyme activity of corresponding process.

1.4 Targeting nucleobase pairs with Janus-Wedge insertion strategy

1.4.1 Janus-Wedge Insertion of a single nucleobase pair by small molecules

To target a nucleobase pair, one needs to design a molecule bearing two recognition interfaces that will interact with each of the two nucleobases, Lehn and co-workers first developed the concept of wedge-like heterocycle that recognize cytosine and uracil mismatched pair in chloroform.67 However, a single insertion is not strong enough to be detected in water. Zimmerman and co-workers designed an intercalation-assisted melamine heterocycle that targets T-T or U-U mismatch in CTG or CUG trinucleotide repeat of myotonic dystrophy type 1 sequence, respectively. With the help of acridine, a strong nucleobase intercalator, melamine was able to recognize T-T or U-U mismatch with

68 a Kd at high nanomolecular range (390 nM) in aqueous buffers. This example demonstrates the potential use of such a strategy in diagnostics and therapeutics.69,70

Synthetic Janus compounds for the recognition of G-U mismatched nucleobase pair were also reported.71

b c

Figure 1.9 Janus-Wedge insertion into nucleobase pairs. (a) The concept of Janus-

Wedge insertion into matched A-T and C-G nucleobase pairs. (b) Example of C-U

mismatched nucleobase pair insertion. (c) Acridine-assisted melamine insertion into T-T

and U-U mismatched pairs. Cited from reference 67 and 68.

1.4.2 Single Janus-Wedge insertion on biopolymer scaffold

Researchers also insert a single synthetic nucleobase mimic that targets matched or mismatched nucleobase pair onto a biopolymer scaffold. Theoretically, a mutation may be detected by the slightly enhanced binding affinity caused by the bifacial recognition nucleobase analogue. Tor72 and Perrin73 designed bifacial nucleobase mimic and inserted it into a deoxyoligonucleotide, respectively. Instead of forming bifacial recognition with A

13 and T simultaneously, their synthetic nucleobases can only pair with A or T at one time, working as a surrogate for T and A in a DNA duplex. This may suggest that targeting a matched nucleobase pair with Janus-Wedge strategy at one single site may be difficult.

1.4.3 Multivalent insertion of Janus-Wedge heterocycles into nucleobase pairs

Different from insertion into a single nucleobase pair, assembly of multiple Janus-Wedge heterocycles onto a biopolymer backbone will provide dramatic increase of binding affinity in targeting nucleobase pairs by multivalent bifacial recognition. Two main categories of biopolymer backbone are DNA and PNA. As discussed in PNA section, PNA with a neutral backbone will give a higher stability of formed triplex hybrid, and this may enable a Janus-

Wedge insertion of perfectly matched nucleobase pairs (A-T and C-G) instead of mismatched pairs, as PNA provides extra energy to break preformed hydrogen bonds.

McLaughlin and co-workers integrated multiple synthetic Janus-Wedge bifacial recognition heterocycles onto a PNA backbone that successfully targeted C-T mismatch nucleobase pairs via triplex formation.74 A single matched C-G nucleobase pair may be wedged-like inserted when it was in the middle of unpaired C-T mismatches.75 Bong and co-workers assembled multiple melamine heterocycles on different scaffolds, such as peptide,76 peptoid77 and polymer.78 These synthetic melamine-displaying molecules have been demonstrated to target T-T or U-U mismatched nucleobase pairs efficiently, inhibit enzymatic activities79 and activate nucleic acid functions.80

1.4.4 Triazine used to target nucleobase or nucleobase pairs and bifacial Peptide

Nucleic Acid (bPNA)

Figure 1.10 Examples of triazine-bearing sequences. (a) cyanuric acid-displaying

PNA; (b) diaminotriazine-displaying peptide; (c) dioxotriazine-displaying peptide; (d)

melamine-displaying peptide; (e) melamine-displaying peptoid; (f) melamine-

displaying polyacrylate. Adapted from reference 76, 77, 78, 81 and 82.

Triazine can provide hydrogen bonds on three recognition interfaces and can be used in

Janus-Wedge insertion strategy. Triazine has been successfully attached to PNA,81 polyamide,82 peptide,76,82,83 peptoid77,82 and synthetic polymer78 backbone with different linker lengths ranging from 0 atoms to 4 atoms and at varied positions, including endocyclic carbon, endocyclic nitrogen and exocyclic nitrogen. Attaching triazine to a DNA backbone at exocyclic nitrogen is proved to be difficult due to isomerization and

15 hydrolysis.84 Upon covalently linked onto a backbone, one of the three recognition interfaces is blocked, leaving this heterocycle a bifacial recognition motif. Ganesh and co- worker replaced one or two thymine nucleobases of T8 PNA with cyanuric acid. Cyanuric acid was found a stabilizing effect when inserted in the middle of the sequence or at C- terminus.81 Krishnamurthy and Eschenmoser synthesized multiple triazine-bearing sequences on varied backbones with short carbon linkers (0 atoms to 2 atoms).

AcNH T Interestingly, they found 2,4-dioxotriazine-displaying oligomer AspGlu( OO)16 bound to corresponding pairing partner poly-adenine sequences dramatically weaker than that

AcNH T of poly-diaminotriazine sequence AspGlu( NN)16 bound to its corresponding partners.

They assumed the electronic property of 2,4-dioxotriazine nucleus disfavored such pairing system rather than a steric incompatibility. Missing one exocyclic amino group resulted in losing one recognition interface, thus their triazine-bearing sequences mainly formed 1:1 duplex with single-stranded oligonucleotide.82 Given the fact that diaminotriazine pairs well with thymine mismatch, Bong and co-workers successfully displayed multiple melamine

(2,4,6-triamino-1,3,5-triazine) heterocycles on different supporting backbones, including peptide, peptoid and polymers. This heterocycle clearly offered two recognition interfaces when attached to backbone. They found melamine-bearing chains formed triplex with unstructured polythymine oligonucleotides in a ratio of 1:2 with strong affinity. If two poly(dT) regions are connected by a non-intervening loop, it formed triplex stem-loop structure with a peptide-DNA ratio of 1:1. The well-studied melamine-displaying peptide system was thus termed bifacial Peptide Nucleic Acid (bPNA). Surprisingly, no duplex was detected during this process. This was further supported by the fact that monofacial triazine prepared by methylating one of the exocyclic amine did not pair with poly(dT) sequence. The linker length played a crucial role on the binding affinity, generally four carbon atoms and three carbon atoms showed much better than one or two carbon atom 16 linker. This may be explained by the decreased electrostatic repulsion between negative charges on the backbone when forming triplex. This is different from what Krishnamurthy and Eschenmoser reported in which 0 carbon atom to 2 carbon atom linkers resulted in moderate to robust duplex formation. Less repulsion in a duplex than that of a triplex could account for this different observations. This is also comparable to the native DNA duplex and triplex system and the latter one usually forms in relatively “harsh” conditions.

Surprisingly, given the same linker length and the same numbers of melamine units on one sequence, a peptoid backbone resulted in a significantly lower binding affinity, whereas peptide backbone yielded robust triplex formation. Despite of irregular structure, the synthetic polyacrylate-supported melamine sequence yielded a robust triplex formation, comparable to that of peptide backbone. Yet the origin of this backbone difference was unclear.76-78,83 Bifacial PNA has abiotic nucleobase and native α-peptide backbone, which is structurally different from conventional PNA that has native nucleobases and non-native polypeptide backbone. Bifacial PNA is also operationally distinguished from conventional PNA in that bPNA forms triplex with non-interacting and unstructured oligonucleotides in an associative fashion, whereas conventional PNA displaces pre-formed duplex in a dissociative manner. Unlike the modified oligonucleotides discussed in Session 1.2 that bind to a pre-formed native duplex and strengthen the triplex formation at physiological conditions, bPNA generates triplex from non-interacting single-stranded oligonucleotides under physiological conditions.

Figure 1.11 Bifacial melamine-thymine recognition. Synthetic bPNA shows a 1:2

binding of 10mer bPNA to (dT)10 and a 1:1 binding of 10mer bPNA to dT10C4T10. Cited

from reference 76.

1.5 Nucleic acid template effect on nonenzymatic reactions

Template effect exists widely in molecular biology, as evidenced by replication, transcription, reverse transcription and translation. In any of them, a single strand of nucleic acid serves as the template and relevant polymerase or ribosome reads through the template sequence and brings together the complementary nucleotide triphosphate or aminoacyl-tRNA based on Watson-Crick base pairing to synthesize the complementary strand of nucleic acid or make a chain of polypeptide directed by mRNA. These processes involve several indispensable enzymes that catalyze the ligation of nucleotides or amino 18 acids. Mimicking what nature does, researchers take the advantage of nucleic acid template effect and perform in vitro non-enzymatic oligonucleotide ligation,85,86 PNA ligation,87-89 small-molecule synthesis,90,91 peptide ligation,92,93 reporter transfer94 for the purpose of prebiotic study,85,95,96 drug screening,90,91 mutation diagnosis97 and so on. Von

Kiedrowski studied a self-replicating system in which a hexadeoxynucleotide assembled two trideoxynucleotides through base-pairing and catalyzed the ligation of the two trideoxynucleotides via nucleophilic attack of a pre-activated phosphate group, resulting in a native phosphate linkage.98,99 Orgel and co-workers demonstrated the feasibility of template effect and information transfer between PNA, RNA and DNA.100-102 Szostack and co-workers pioneered non-enzymatic primer extension to study RNA self-replication.103,104

Liu and co-workers developed a method for reaction discovery105 and selection of macrocycles90 encoded by DNA library. Liu’s work is an excellent combination of nucleic acid template effect and in vitro selection of nucleic acid library.

In most examples of nucleic acid-templated reactions, one single strand of oligonucleotide serves as the template and the recognition is promoted by Watson-Crick base-pairing.

However, other recognition patterns can also be used for templated reactions. For example, Nicolaou and co-workers employed DNA duplex as the template for a self- replication system.106,107 Ladame and co-workers designed DNA-templated fluorogenic reaction for the synthesis of trimethine cyanine dyes to monitor the formation of G- quadruplex.108 A number of aqueous compatible chemical reactions can be used for nucleic acid-templated reactions and thus the resulted linkers are versatile, such as native phosphate109,101 and amide formed by direct aminolysis110 or native chemical ligation,111 as well as phosphorothioate112 / phosphoramide113 resulted from nucleophilic substitution, secondary amine prepared by reductive amination,114,115 olefin yielded by Wittig reaction.116,117 19

Figure 1.12 Examples of nucleic acid template effect. (a) Templated ligation in DNA,

RNA and PNA. (b) DNA-templated group transfer to facilitate the synthesis of

macrocycle library and in vitro selection of macrocycle library. Cited from reference 98

and 100.

These nucleic acid-templated reactions usually occur efficiently at low micromolar to nanomolar range, while regular organic reactions occur at milimolar range. The reason

20 behind nucleic acid template effect could be accounted by effective molarity: the template assembles reactants onto itself, arranging to the favored orientations and proximity, as well as increasing the effective molarity of each reactant.118 Therefore, reactions take place efficiently at significantly low absolute concentrations.

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Chapter 2. Bifacial Peptide Nucleic Acid Directs Cooperative Folding and

Assembly of Binary, Ternary, and Quaternary DNA Complexes

2.1 Introduction

Triplex formed from nucleic acid or its analogue has inspired decades of research aimed at understanding triplex formation1 and targeting the information presented in the nucleic acid duplex.2,3 Nucleic acid triplex formation usually involves Hoogsteen base-pairing of a single-stranded oligonucleotide to a preformed duplex paired by Watson-Crick recognition.

In particular, a homopurine or homopyrimidine oligonucleotide is inserted into the major groove of a duplex formed by a homopurine and homopyrimidine oligonucleotides. Typical forms of nucleobase triplet include T-A*T, C-G*C+, T-A*A and C-G*G. Triplex formation often occurs under non-physiological pH and salt conditions, because cytosine in the third strand needs to be protonated under acidic pH to form Hoogsteen hydrogen bonds with guanine. Higher salt concentration and divalent salts4,5 are usually necessary to shield the electrostatic repulsion of the highly negatively-charged backbones. We recently reported an alternative strategy for a new class of DNA triplex formation involving synthetic melamine-displaying peptide and polythymine oligonuclotide that did not require any prior

6 secondary structures. Two unstructured (dT)10 were assembled onto a bridging melamine-displaying peptide to form a 2:1 DNA-peptide heterotriplex via melamine- thymine bifacial recognition. When the two (dT)10 strands were connected by a non- interacting loop, a 1:1 DNA-peptide triplex stem-loop structure formed with higher affinity compared to its non-looped counterpart. We therefore term it bifacial peptide nucleic acid

(bPNA). Unlike DNA triplex or triplex involving native nucleobases, bPNA does not self- assemble, use Hoogsteen base-pairing or require direct interaction between DNA strands and instead targets unstructured T-tracts for binding. Bifacial PNA is an artiﬁcial peptide strand that binds and folds a DNA partner through the use of melamine as a synthetic nucleobase mimic that hydrogen bonds with two thymine bases on their Watson-Crick binding faces. This bifacial recognition concept is similar to intramolecular DNA triplexes3 36 and synthetic Janus-wedge bifacial nucleobase mimics,7-9 as well as the targeting of short, single-stranded oligopurines with pyrimidine “clamps” or cyclic DNA10 with the distinction that no prior secondary structure, covalent cyclization, or Mg2+ is needed. Different from conventional PNA with a non-native polypeptide backbone and native nucleobase content, bPNA has an α-peptide structure derived from native amino acids and a non-native bifacial melamine nucleobase mimic. Bifacial PNA is also operationally distinguished from conventional PNA in that bPNA is an associative agent that brings noninteracting oligothymidine DNA strands together in 1:2 bPNA-DNA triplex, whereas PNA is often used as a dissociative agent that invades existing nucleic acid (NA) duplexes to produce 2:1

PNA-NA triplex hybrids.11-14

The melamine bPNAs are easily prepared via reaction of the ε-amino group of lysine with diaminochlorotriazine to yield an amino acid derivative for standard solid-phase peptide synthesis.15 The facility of direct amino substitution on chlorotriazine rings has led to considerable investigation of melamine as a diversity platform for combinatorial chemistry and dendrimer synthesis,16,17 and triazines themselves have been suggested as products of prebiotic chemistry,18-21 forming readily from cyano derivatives.22-26 The synthetic triaminotriazine (melamine) provides a divalent site for thymine recognition when monoderivatized on an exocyclic nitrogen. Melamine and its derivatives have been extensively studied in organic solvents and the solid-state as an assembly partner for cyanuric acid derivatives,27-29 and more recently in aqueous-phase assemblies.30-41

Heterocycle recognition of melamine and cyanurates is directed by hydrogen bond recognition, but driven by exothermic base-stacking akin to DNA-recognition.39 Co-

37 a b

Figure 2.1 Illustration of melamine-thymine interaction directing DNA and bPNA assembly and CD spectra. a) The structure of bPNA with N- and C- capping groups shown. Cbf = 5(6)-carboxyfluorescein, ABA = 4-acetamidobenzamide. b) Schematics of

1:1 bPNA-DNA hairpin triplex structures. c) CD spectra of single-stranded dTnC4Tn and the corresponding bPNA-DNA complex. The concentration is 5 μM.

38 crystallization of melamine with thymine, uracil, and 5-ﬂuorouracil (5-FU)37 from aqueous solution provided structural evidence of the expected multifacial hydrogen bonding patterns between triazine and nucleobase in the solid state. Furthermore, Zimmerman and co-workers had demonstrated that melamine-acridine conjugates selectively intercalated into T-T and U-U mismatch sites in solution,42,43 indicative of bifacial melamine recognition.

Robust exothermic binding of 5-ﬂuorouracil to polyacrylate-displayed melamine was found to trigger condensation of soluble polymer into 5-FU loaded nanoparticles, suggesting that melamine-cyanurate type recognition may be generally extended to include interactions with thymine/uracil.37 Indeed, Eschenmoser and Krishnamurthy prepared triazine- displayed peptide/peptoid that interacts with native DNA/RNA to form duplexes.23,24 They have demonstrated that diaminotriazine-displaying α-peptides function as novel PNAs that form stable peptide-DNA/RNA duplexes. We speculated that replacement of the aliphatic

CH2 linkage to the heterocycle in diaminotriazine with an NH to yield a triaminotriazine

(melamine) side chain derivative would create a bifacial recognition nucleus for thymine and switch the assembly from duplex to triplex structures. This was demonstrated in our

6 earlier study using 10mer bPNA and (dT)10, as discussed above. In this study, we focus our examination on melamine-thymine driven assembly of bPNA-DNA complexes as a function of interface length in the context of the hairpin-triplex stem motif. The dependence of bPNA-DNA binding on the length of the interface was investigated by synthesis of

(EM*)n peptides with n = 4, 5, 6, 8, and 10. These peptides bound to their dTnC4Tn partners with the expected 1:1 hairpin triplex stoichiometry, and complex stability decreased with n. Notably, length matching of the DNA and peptide was critical in determining stoichiometry; longer DNA strands were found to host more than one peptide, supporting the notion that a single melamine repeat unit (EM*) is required to interact with each T-T pair. The recognition interface tolerated a high number of “mismatches” and indicated half- 39 site, or monofacial, recognition between melamine and thymine may occur if only 1 complementary nucleobase was available.

2.2 Results and Discussion

2.2.1 1:1 bPNA-DNA complexes

Bifacial Peptide Nucleic Acid (bPNA) was prepared by standard solid phase synthesis with the general form (EM*)n, wherein M* denotes melamine-lysine residue and E represents glutamic acid. N indicates the number of melamine heterocycles. Glutamic acid was chosen to occupy alternate positions to achieve similar sequence pattern shown by

Eschenmoser23,24 and Ghadiri21, respectively. In addition, acidic side chains are deprotonated in physiological conditions, improving solubility of bPNA and minimizing nonspecific electrostatic interaction when hybridizing with nucleic acid. DNA complexation of 5(6)-Carboxyfluorescein-labeled bPNA (Cbf-bPNA) showed fluorescence quenching and increased fluorescence anisotropy. (EM*)n showed a clear 1:1 complexation with dTnC4Tn by fluorescence titration with the exception of (EM*)4 which exhibited weakest binding and was difficult to determine a clear stoichiometry under the same conditions

(Figure 2.2). Hybridization with bPNA induced a similar structural change in DNA as qualitatively judged by circular dichroism (CD). A large positive Cotton effect at ~280 nm corresponding to unstructured DNA was transformed into a strong negative peak at ~260 nm (Fig. 2.1c). Bifacial PNA itself showed a negligible signal on CD.

Figure 2.2 Normalized fluorescence titration curve for Cbf-(EM*)n-dTnC4Tn. The concentration of fluorescent peptide was 1 µM in each titration. (a) n=4, (b) n=5, (c) n=6,

(d) n=8, (e) n=10. Excitation: 492 nm, emission: 525 nm. Titration was done at 25 C.

2.2.2 Binding affinity and enthalpy of bPNA-DNA complexes

a Table 2.1 Thermodynamic data for [(EM*)n-dTnC4Tn]

Longer recognition interface generally results in stronger binding as quantitatively judged by UV melting, Kd determination and differential scanning calorimetry (DSC). In particular,

(EM*)n-dTnC4Tn triplexes were melted at different temperatures ranging from 19 ºC to 57

ºC along with the increase of (EM*) units. This melting temperature trend corresponded to enthalpy change of dissociation. A heat of 293 kcal/mol was needed to break (EM*)10- dT10C4T10 complex, whereas the ΔHd of (EM*)4-dT4C4T4 was undetectable under the same conditions. Fluorescence anisotropy was used to obtain dissociation constant (Kd) by fitting to a 1:1 binding model. The longest bPNA (n=10) yielded a Kd of ~2.6 nM. This bPNA-DNA hybridization is robust to survive dA10G4A10 invasion as evidenced by the fact that the intensity of (EM*)10-dT10C4T10 complex did not change despite of increasing amount of dA10G4A10. This is also supported by the distribution of (EM*)10-dT10C4T10 triplex and dT10C4T10-dA10G4A10 duplex when equal amounts of bPNA and two oligonucleotides were denatured and allowed to cool slowly (Lane 1, Figure 2.4). As summarized in Table

1, melting temperatures of bPNA-DNA triplexes drop significantly from n = 10 to 4, though 42 a b

c d

Figure 2.3 Characterization of binding affinity. a) Normalized first derivatives of thermal melts (UV) of the 1:1 (EM*)n-dTnC4Tn complexes at 5 μM. b) Binding isotherms of complex formation fit to a 1:1 binding model (R2 ≥ 0.96) with 25 nM bPNA concentration. c) DSC upscans of the 1:1 (EM*)n-dTnC4Tn complexes at 25 μM. d) Thermodynamic parameters for the complex dissociation (as labeled) and Tm (●) on the right axis as a function of repeat unit.

1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 2.4 Invasion of triplex (dT10C4T10+H2N-(EM*)10G) with dA10G4A10. Gel was

stained with SYBR Gold. Lane 1) H2N-(EM*)10G+dT10C4T10+dA10G4A10, 250 nM for

each. 2) dA10G4A10, 500 nM. 3) dA10G4A10+H2N-(EM*)10G, 500 nM for each. 4)

dT10C4T10, 500 nM. 5) DNA duplex (dT10C4T10 + dA10G4A10), 250 nM. 6) Triplex

(dT10C4T10+H2N-(EM*)10G), 500 nM. 7) Triplex (500 nM) + dA10G4A10 (125 nM). 8) Triplex

(500 nM) + dA10G4A10 (250 nM). 9) Triplex (500 nM) + dA10G4A10(375 nM). 10) Triplex

(500 nM) + dA10G4A10(500 nM). 11) Triplex (500 nM) + dA10G4A10(750 nM). 12) Triplex

(500 nM) + dA10G4A10(1000 nM). 13) Triplex (500 nM) + dA10G4A10(2500 nM). The

sample in Lane 1 and the DNA duplex were heated at 95 C for 5 min followed by slowly

cooled to room temperature, respectively. Triplex(dT10C4T10+H2N-(EM*)10G) and all

other samples were heated at 37 C for 1 h, then slowly cooled to room temperature.

Kd increases are less substantial, suggestive of strong enthalpy-entropy compensation.

This was borne out by differential scanning calorimetry analysis, which indicated enthalpically driven44,45 bPNA-DNA base-stacking became markedly less exothermic as the recognition interface decreased. Near perfect enthalpy-entropy compensation was observed as the length of the interface varied, with both heat of binding and entropic cost 44 of assembly sharply decreasing with the number of (EM*) repeat units46,47 The enthalpic benefit of bPNA-DNA binding vanished with an (EM*) repeat of four, as reﬂected by the signiﬁcant loss of DNA affinity as bPNA length is decreased from 5 to 4.

2.2.3 Tolerance of “mismatch” sites in (EM*)10 DNA complexation

Although the selectivity of (EM*)10 bPNA for polythymidine DNA relative to the other native

DNA nucleobases has been previously established, the tolerance of non-thymine nucleobases at the recognition interface was unknown. We systematically replaced selected thymine nucleobases with cytosine and tested the affinity of Cbf-10 for a series of 24-nt oligonucleotides based on dT10C4T10, as a function of thymine content. Eighteen

DNA binding partners for Cbf-10 with thymine content decreasing relative to dT10C4T10 were evaluated by Tm measurement to assay the binding affinity (Table 2.2). Notably, complex formation was detectable even with 40% replacement of T for C, though thermal stability dropped to ~19 ºC with this high number of mismatches. Thermal stability of the complexes showed good linear correlation with thymine content (Figure 2.5d). Selected low melting complexes were studied by ﬂuorescence quenching titration to confirm the 1:1 bPNA-DNA stoichiometry of a hairpin triplex, despite the high number of mismatches

(Figure 2.5a-c). Inspection of complex Tm as a function of T→C substitution demonstrates bPNA can bind to single thymine half-sites, presented as T-C or C-T mismatch sites formed when the 24-nt DNA is folded into a hairpin structure. Indeed, two T→C substitutions that yield half-sites (entry 3, Tm = 54.1 ºC) are more thermally stable than substitutions that do not (entry 5, T m = 49.8 ºC). This effect may also be observed when comparing entries 6 and 11, which have identical thymine fraction (67%) yet are separated by 6 ºC in melting temperature. Again, the higher melting complex formed in entry 6

45 distributes the T→C substitutions exclusively into half-sites (T-C, C-T pairs), whereas entry 11 presents two contiguous C-C mismatch sites, effectively dividing the

Table 2.2 Tm of Cbf-10 complexes with T-rich 24-nt DNA

46 original dT10 tract into two T4 tracts separated by CC linkers. Similarly, the favorable presence of T-C, C-T half-sites is seen when comparing entries 12 and 14, both of which are 58 % T. Thus, it appears that clustered T→C substitutions are more damaging to bPNA recognition than distributed substitutions; the latter pattern allows the entire peptide to participate in full site (T-T) or T-C and C-T half-site recognition. With a total of six T-T sites, Cbf-10 binding to entry 16 is analogous to the [Cbf-6 · dT6C4T6] complex

(Table 2.1), which in fact has much higher thermal stability (Tm= 38 ºC). Lower thermal stability relative to [6 · dT 6C4T6] may be a result of the greater entropic cost of complexing larger macromolecules with more degrees of freedom, as well as a

“dangling end” effect in which the terminal bases are not buried on both sides of the T- tract in entry 16.

Figure 2.5 Fluorescence titration curve for bPNA with mismatched DNA hairpin. The concentration of fluorescent bPNA 10 was 1 µM, excitation: 492 nm, emission: 525 nm.

Titration was done at 25 ºC. (a) dTTCTTCTTCTC4TCTTCTTCTT, Tm was 33.4 ºC, (b) dTTTTCCCTTTC4TTTCCCTTTT, Tm was 29.9 ºC, (c) dTCTTCTTCTTC4TCTCTTCTTT,

Tm was 34.0 ºC, (d) Melting temperature dependence on thymine content.

2.2.4 bPNA-DNA higher-order assembly

Table 2.3 Tm’s of higher order bPNA-DNA assembly

The clear dependence of complex stability on the melamine-thymine interface length suggested that higher-order bPNA-DNA assembly may be possible by altering the interface length of bPNA or DNA. We set out to test this notion via two different models:

1) Inserting multiple copies of the same bPNA into a long DNA host; 2) Inserting bPNA fragments of different length into a long DNA host. In the first type of assembly, a long oligonucleotide, dT18C4T18, assembled two copies of Cbf-bPNA when n = 6-10 into ternary complexes, while three Cbf-4 and Cbf-5 were inserted into the same DNA host, resulting in quaternary complexes (Figure 2.6 and 2.7). This type of higher-order assembly showed a single transition with up to 10 ºC increase on melting temperature compared to length- matched binary bPNA-DNA complexation. These stability increases are indicative of strongly cooperative multiplex binding that releases all bound bPNA at once without detectable intermediate complexes.

Figure 2.6 Normalized fluorescence titration curve for higher-order assembly. The concentration of Cbf-10 was 1 µM. (a) n=4, dT18C4T18 (b) n=5, dT18C4T18 (c) n=6, dT18C4T18 (d) n=8, dT18C4T18 (e) n=10, dT18C4T18 (f) n=4, dT10C4T10.

However, this conclusion does not fit the second type of higher-order assembly in which

(EM*)4 and (EM*)10 were inserted into dT15C4T15. The reason to use dT15C4T15 rather than dT18C4T18 was that dT15C4T15 was not long enough to host two (EM*)10, but only one.

Replacement of the fluorephore with ABA on bPNA 10 permitted the formation of a 1:1 complex between ABA-10 and dT15C4T15 that would be spectroscopically invisible in a

fluorescence quenching titration with Cbf-4. The [ABA-10 · dT15C4T15] complex would thus be preorganized to accept an additional bPNA binding partner in the five unbound T-T sites. Gratifyingly, fluorescence quenching titration of Cbf-4 against preformed [ABA-

10 · dT15C4T15] revealed a clear transition to a 1:1 stoichiometry of interaction with the complex, providing evidence for formation of a 1:1:1 complex of [ABA-10 · Cbf-

4 · dT15C4T15]. This heterotriplex showed two distinct melting temperatures, corresponding to the separate thermally driven dissociations of the two bPNA fragments. A similar titration with Cbf-4 and dT15C4T15 indicated that a 3:1 bPNA-DNA binding. Further investigation by DSC revealed that dissociation of [ABA-10 · dT15C4T15] required 281 kcal/mol, similar to what was observed on analysis of [Cbf-10 · dT10C4T10] by DSC (293 kcal/mol). The heterotrimer [ABA-10 · Cbf-4 · dT15C4T15] exhibited two melting transitions with absorption of 6 kcal/mol in the ﬁrst transition, followed by 294 kcal/mol in the second transition. These ﬁndings are again consistent with the binary, length-matched

[(EM*)n · dTnC4Tn] systems, wherein [Cbf-4 · dT4C4T4] melting did not yield detectable heat release; thus, a modest but detectable improvement in Cbf-4 binding could account for the

6 kcal/mol absorbance in the DSC spectrum. This was due to the pre-organization of [ABA-

10 · dT15C4T15] that lowered the entropic cost for binding to Cbf-4. Calorimetric analysis of

[(Cbf-4)3 · dT15C4T15] yielded the most striking ﬁnding: 330 kcal heat per mole of DNA was absorbed on complex dissociation! This value was nearly the same as a full-length bPNA binding with dT15C4T15 (an estimated dissociation enthalpy change of 341 kcal/mol). 51

Figure 2.7 Illustration of higher-order bPNA-DNA assembly

Figure 2.8 Analysis of binary and ternary complexes of bPNA and dT15C4T15. bPNA

Cbf-4 and ABA-10 were used, labeled as follows: (---) = [(Cbf-4)3 · dT15C4T15], (□) = [Cbf-

/ABA-10 · dT15C4T15], and (·) = [Cbf-/ABA-10 · Cbf-4 · dT15C4T15]. (Left) Normalized

ﬂuorescence quenching plots indicating stoichiometry of binding of Cbf-4 as a function of increasing dT15C4T15 (---) and as a function of increasing preformed [ABA-

10 · dT15C4T15] complex (·). (Center) UV melts of dT15C4T15 complexes, as labeled.

(Right) Differential scanning calorimetry (upscans only) of the binary and ternary complexes of dT15C4T15.

This indicated that a very strong cooperative assembly of ternary and quaternary bPNA−DNA complexes exhibit enthalpy and thermal stability gains relative to the length- matched bPNA-DNA heterodimers.

2.3 Conclusion

We ﬁnd optimal bPNA-DNA complexation when the ratio of melamine to thymine bases is

1:2, consistent with interface length-matching selection. Although enthalpy-entropy compensation yielded a slow increase in Kd as the recognition interface was shortened, dissociation enthalpy change and thermal stability exhibited strongly linear positive correlations with the number of melamine binding sites in bPNA and thymine content of the DNA interface. Complexation may thus be easily tuned by the length of the designed interface. Maximum binding efficacy was observed when the numbers of T-T hairpin sites and melamine peptide residues were the same, though a high number of mismatch sites and T-C / C-T half-sites were tolerated before complex Tm dropped below 37 ºC. As both

DNA and bPNA partner develop secondary structure on binding, complex formation is generally cooperative, with the entropic cost of folding paid for by the enthalpic beneﬁt of base-stacking. A striking feature of this system is the high degree of cooperative multisite binding observed in the complexation of long T-tracts with short melamine peptides. This may be rationalized because the ﬁrst peptide folds thymine-rich DNA into a hairpin structure, aligning additional T-T sites for further melamine peptide binding, resulting in highly cooperative binding. Similar to turn nucleation,48-55 the greatest entropic cost is expected to be associated with the initial formation of the dC4 loop structure to allow antiparallel presentation of the two T-rich hairpin strands; base-stacking fails to compensate for this cost around four triplex repeat units of bPNA-DNA. More structured synthetic multivalent systems that have decoupled folding from binding are known to bind 53 monovalent ligands independently rather than cooperatively.56,57 Overall, the melamine- thymine recognition motif may be utilized to predictably prepare stable binary, ternary and quaternary macromolecular bPNA-DNA complexes with thymine-rich single-stranded

DNA. The structural simplicity and synthetic accessibility of the bPNA strand, coupled with its robust polythymidine recognition properties, raises the intriguing possibility that triazine- bearing macromolecules may be used as biotechnology tools to direct the chemistry of native nucleic acids.

2.4 Materials and Equipments

Chemicals for amino acid derivative synthesis, solid phase peptide synthesis, purification and characterization were purchased from Sigma-Aldrich, Chem-Impex and AAPPTec without further purification unless otherwise specified. Rink Resin LS (100-200 mesh, 0.28 mmol/g) was purchased from Advanced ChemTech. All DNA oligomers were obtained from Sigma-Aldrich. Dulbecco’s Phosphate-Buffered Salines (DPBS 1×, no calcium, no magnesium) used as the binding buffer was purchased from Invitrogen. 40% acrylamide and bis-acrylamide solution (19:1) was purchased from Bio-Rad. TBE Buffer (10× solution) for gel electrophoresis was purchased from American Bioanalytical. SYBR Gold (10000×) was purchased from Invitrogen.

All DNA oligomers and synthetic peptides were quantified on UV-Vis HP 8543. MALDI-

TOF Mass spectra were acquired on Bruker Microflex MALDI-TOF instrument.

Electrospray mass spectroscopy was acquired on Bruker MicroTOF equipped with an electrospray ionization source. Mass spectrometry instruments were provided by a grant from the Ohio BioProducts Innovation Center. NMR spectra were acquired on Bruker

Advance DPX 400 instrument. Solid phase peptide synthesis was performed on AATTPec

Apex 396 peptide synthesizer. All peptides were purified on RP-C18 preparative HPLC 54 column and the purity was confirmed on RP-C18 analytical column. UV melting was performed on Varian Cary-100 UV-Vis Spectrophotometer equipped with Cary

Temperature Controller. Fluorescence titration was performed on Perkin Elmer

Luminescence Spectrometer LS 50B equipped with PTP 1 Temperature Programmer.

Fluorescence anisotropy was performed on Molecular Devices SpectraMax M5. Circular

Dichroism spectra were obtained on Jasco J815 Circular Dichroism Spectrometer.

Differential scanning calorimetry was performed on Microcalorimeter VP-DSC.

Fluorescence gel images were acquired using a Typhoon Trio Variable Mode Imager

(Amersham Biosciences).

2.5 Synthesis

2.5.1 Synthesis of Fmoc-Lys(M*)-OH

Scheme 2.1 Synthesis route of Fmoc-Lys(M*)-OH

Fmoc-Lys(M*)-OH was synthesized based on slight modification of our recent publication.

Briefly, Boc-Lys-OH (4.94 g, 20 mmol) was stirred with 6-chloro-2,4-diamino-1,3,5-triazine

(3.49 g, 24 mmol) and NaOH (1.60 g, 40 mmol) in 35 ml of water at 85 ºC overnight. Solid

55 was removed from the solution by centrifuge and the solution was extracted with EtOAc six times. The aqueous solution was then dried under reduced pressure. Boc protecting group was removed in 15 ml TFA for 2 hours. Most TFA was removed under reduced pressure and the resulting sticky oil was diluted in 75 ml water and neutralized with

Na2CO3 solid carefully to pH 7.5-8, followed by adding 75 ml THF solution of Fmoc-OSu

(7.42 g, 22 mmol) and stirring overnight. The reaction was washed with EtOAC six times to remove unreacted Fmoc-OSu and then acidified with 1 M HCl solution to precipitate product. Off-white solid product (5.60 g, 58% yield) was collect through filtration and washed by water and EtOAc. 1H NMR (400 MHz, d6-DMSO) δ (ppm) 1.35 (d, J=2.8Hz,

2H), 1.50 (t, J=5.4Hz, 2H), 1.62-1.71 (m, 2H), 3.25 (d, J=2.4Hz, 2H), 3.93 (m, 1H), 4.22 (t,

J=5.4Hz, 1H), 4.27-4.30 (m, 2H), 7.31 (t, J=5.8Hz, 2H), 7.40 (t, J=6Hz, 2H), 7.63 (d, J =

8Hz, 1H), 7.72 (m, 2H), 7.88 (m, 6H), 8.09 (s, 1H). 13C NMR (100 MHz, d6-DMSO) δ (ppm)

23.48, 28.80, 30.95, 47.18, 54.30, 66.10, 120.61, 125.78, 127.56, 128.15, 141.23, 144.34,

156.69, 158.96, 174.45. Calculated Mass [M+H]: 478.2205. Found: 478.2382.

Figure 2.9 1H NMR of Fmoc-Lys(M*)-OH

Figure 2.10 13C NMR of Fmoc-Lys(M*)-OH

2.5.2 Solid phase synthesis of bPNA

Standard Fmoc chemistry was employed. DIC/HOBt were used as the coupling reagents.

And 20% piperidine in NMP was used for Fmoc deprotection. 95% TFA was used to cleave peptide from resin and remove the protecting groups. Resin was removed by filtration through cotton and the peptide was precipitated and washed by cold diethyl ether. Crude peptides were then dissolved in solvent A and purified by HPLC on a C18 reversed phase column using a gradient of 10–50% solvent B in 50 min (solvent A= 0.1% TFA in water, solvent B = 0.01% TFA in 45% acetonitrile, 45% isopropanol, 10% water). The UV detector was set at 238 nm. Purified peptides were lyophilized to dryness.

2.5.3 HPLC and Maldi-TOF mass spectra of the synthetic peptides

Figure 2.11 Characterization of 4mer bPNA Cbf-(β-A)-(EM*)4G. (a) HPLC trace on a