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Chemistry and Biology of Non-Canonical Nucleic Acids
Chemistry and Biology of Non-Canonical
Nucleic Acids

Naoki Sugimoto
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 v

Contents

Preface xi

1 History for Canonical and Non-canonical Structures of Nucleic

Acids 1
1.1 Introduction 1
1.2 History of Duplex 1
1.3 Non-Watson–Crick Base Pair 5
1.4 Nucleic Acid Structures Including Non-Watson–Crick Base Pairs 7
1.5 Perspective of the Research for Non-canonical Nucleic Acid
Structures 8
1.6 Conclusion and Perspective 9
References 9

2 Structures of Nucleic Acids Now 11

2.1 Introduction 11
2.2 Unusual Base Pairs in a Duplex 11
2.2.1 Hoogsteen Base Pair 13
2.2.2 Purine–Pyrimidine Mismatches 13
2.2.3 Purine–Purine Mismatches 14
2.2.4 Pyrimidine–Pyrimidine Mismatches 16
2.3 Non-canonical Backbone Shapes in DNA Duplex 17
2.4 Branched DNA with Junction 19
2.5 Multi-stranded DNA Helices 20
2.6 Structures in RNA 20
2.6.1 Basic Structure Distinctions of RNA 20
2.6.2 Elements in RNA Secondary Structures 21
2.6.2.1 Hairpin Loop 22
2.6.2.2 Bulge Loop 22
2.6.2.3 Internal Loop 23
2.6.3 Elements in Tertiary Interactions of RNA 24
2.6.3.1 A-Minor Interactions 25
2.6.3.2 Ribose Zipper 25
2.6.3.3 T-Loop Motif 26
vi Contents

2.6.3.4 Kissing-Loop Interaction 26

2.6.3.5 GNRA Tetraloop Receptor Interaction 27
2.6.3.6 Pseudoknot Crosslinking Distant Stem Regions 29
2.7 Conclusion 29
References 30

3 Stability of Non-canonical Nucleic Acids 33

3.1 Introduction 33
3.2 Factors Influencing Stabilities of the Canonical Duplexes 34
3.2.1 Hydrogen Bond Formations 34
3.2.2 Stacking Interactions 35
3.2.3 Conformational Entropy 35
3.3 Thermodynamic Analysis for the Formation of Duplex 36
3.4 Factors Influencing Stabilities of the Non-canonical Nucleic Acids 39
3.4.1 Factors Influencing Stability of Triplexes 39
3.4.2 Factors Influencing Stability of Quadruplex 42
3.4.2.1 G-Quadruplexes 42
3.4.2.2 i-Motif 44
3.5 Thermodynamic Analysis for the Non-canonical Nucleic Acids 45
3.5.1 Thermodynamic Analysis for the Intramolecular Triplex and
Tetraplex 45
3.5.2 Thermodynamic Analysis for the Intermolecular Triplex 45
3.5.3 Thermodynamic Analysis for the Tetraplex 46
3.6 Conclusion 48
References 49

4 Physicochemical Properties of Non-canonical Nucleic

Acids 51
4.1 Introduction 51
4.2 Spectroscopic Properties of Non-canonical Nucleic Acids 51
4.2.1 Effect of Non-canonical Structure on UV Absorption 51
4.2.2 Circular Dichroism of Non-canonical Nucleic Acids 53
4.2.3 NMR Spectroscopy 56
4.2.4 Other Spectroscopic Characteristics of Non-canonical Nucleic Acids 57
4.3 Chemical Interactions on Non-canonical Nucleic Acids 59
4.3.1 Hydration 59
4.3.2 Cation Binding 61
4.3.3 pH Effect 62
4.3.4 Chemical Modification 63
4.4 Chemical Platform on the Non-canonical Structures 64
4.4.1 Specificity of a Ligand to Non-canonical Structures 64
4.4.2 Fluorescence Platform of Non-canonical Structures 67
4.4.3 Interface Between Proteins and Nucleic Acids 68
4.5 Physicochemical Property of Non-canonical Nucleic Acids in Cell 69
4.5.1 Molecular Crowding Condition that Reflects Cellular Environments 69
 Contents vii

4.5.2 Effects of Crowding Reagents on Non-canonical Nucleic Acid

Structures 70
4.5.3 Quantification of Physical Properties of Non-canonical Structures in
Crowding Condition 71
4.5.4 Non-canonical Structures Under Mimicking Organelle Environment 72
4.5.5 Insight for the Formation of Non-canonical Nucleic Acids in Cells 73
4.6 Conclusion 75
References 75

5 Telomere 79
5.1 Introduction 79
5.2 Structural Properties of Telomere 79
5.2.1 Structures of Telomere 79
5.2.2 Structural Properties of Human Telomeric G4s 81
5.2.3 Structure of Repeats of Human Telomeric G4s 84
5.3 Biological Relevance of Telomere G4 86
5.3.1 Telomerase Activity 86
5.3.2 Telomerase Repeated Amplification Protocol (TRAP) Assay 89
5.3.3 Alternative Lengthening of Telomere (ALT) Mechanism 89
5.4 Other Non-canonical Structures Related to Telomere Region 89
5.4.1 Telomere i-Motif 89
5.4.2 Telomere RNA 90
5.5 Conclusion 92
References 93

6 Transcription 95
6.1 Introduction 95
6.2 Transcription Process 96
6.2.1 Transcription Initiation 96
6.2.2 Transcription Elongation 98
6.2.3 Transcription Termination 99
6.3 Transcription Process Perturbed by Certain Sequences of DNA and
RNA 101
6.4 Transcription Process Perturbed by Non-canonical Structures of DNA
and RNA 103
6.5 Conclusion 110
References 110

7 Translation 113
7.1 Introduction 113
7.2 RNAs Involved in Translation Machinery 113
7.3 General Process of Translation 117
7.3.1 Translation Initiation 117
7.3.2 Translation Elongation 119
7.3.3 Translation Termination 119
viii Contents

7.4 RNA Structures Affecting Translation Reaction 121

7.4.1 Modulation of Translation Initiation in Prokaryotes 121
7.4.2 Modulation of Translation Initiation in Eukaryotes 123
7.4.3 RNA Structures Affecting Translation Elongation 126
7.4.4 RNA Structures Affecting Translation Termination 130
7.5 Conclusion 133
References 134

8 Replication 137
8.1 Introduction 137
8.2 Replication Machineries 137
8.3 Replication Initiation 138
8.3.1 Mechanism of Activation of Replication Origins 138
8.3.2 Activation Control of Origins by G4s 139
8.3.3 Control of Timing of Replication Initiation by G4s 142
8.4 DNA Strand Elongation 142
8.4.1 Mechanism of DNA Strand Elongation 142
8.4.2 Impact of G4 and i-Motif Formations on DNA Strand Synthesis 144
8.4.3 Relationship Between G4 and Epigenetic Modification 145
8.4.4 Expansion and Contraction of Replicating Strand Induced by Hairpin
Structures 147
8.5 Termination of Replication 148
8.6 Chemistry of the Replication and Its Regulation 148
8.6.1 Cellular Environments 148
8.6.2 Control of Replication by Chemical Compounds 150
8.7 Conclusion 151
References 152

9 Helicase 155
9.1 Introduction 155
9.2 Function and Structure of Helicases 155
9.3 Unwinding of Non-canonical DNA Structures by Helicases 158
9.4 G4 Helicases in Gene Expressions 162
9.5 G4 Helicases in Replication 163
9.6 G4 Helicases in Telomere Maintenance 164
9.7 Relation to Diseases by Loss of G4 Helicases 165
9.8 Insight into Specific Properties of Activities of G4 Helicase Under
Cellular Conditions 165
9.9 Conclusion 167
References 167

10 Dynamic Regulation of Biosystems by Nucleic Acids with

Non-canonical Structures 171
10.1 Introduction 171
10.2 Time Scale of Biological Reactions 171
10.2.1 Cell Cycle 172
10.2.2 Central Dogma 172
 Contents ix

10.2.3 Dynamic Structures of Nucleic Acids 174

10.3 Processes in the Central Dogma Affected by Dynamics of Nucleic Acid
Structures 176
10.3.1 Epigenetic Regulation Caused by Chemical Modification of DNA 176
10.3.2 Co-transcriptional Formation of Metastable RNA Structures 178
10.3.3 Co-transcriptional Translation and Transcription Attenuation 180
10.3.4 Co-transcriptional Ligand Binding and Gene Regulation 181
10.3.5 Translation Elongation and Co-translational Protein Folding 183
10.4 Conclusion 184
References 185

11 Cancer and Nucleic Acid Structures 189

11.1 Introduction 189
11.2 Detail Mechanism of Cancer 189
11.2.1 Cancer Incidence 189
11.2.2 The Relationship Between Genes and Cancer 192
11.3 Non-canonical Structures of Nucleic Acids in Cancer Cells 192
11.3.1 Structural Characteristics of Nucleic Acids in Cancer Cells 192
11.3.2 Non-canonical Structures Perturb Gene Expression of Cancer-Related
Genes 195
11.4 Roles of Non-canonical Structures of Nucleic Acids in Cancer
Cells 197
11.4.1 Monitoring of Non-canonical Structures in Cancer Cells 197
11.4.2 Regulation of Gene Expressions by the Non-canonical Structures in
Cancer Cells 198
11.5 Conclusion 199
References 199

12 Neurodegenerative Diseases and Nucleic Acid Structures 203

12.1 Introduction 203
12.2 Protein Aggregation-Induced Neurodegenerative Diseases 203
12.3 DNA Shows Key Role for Neurodegenerative Diseases 205
12.4 RNA Toxic Plays a Key Role for Neurological Diseases 210
12.5 Conclusion 212
References 212

13 Therapeutic Applications 215

13.1 Introduction 215
13.2 Oligonucleotide Therapeutics 216
13.2.1 Antisense Oligonucleotide 216
13.2.2 Functions of Antisense Oligonucleotide Therapeutics 217
13.2.3 Chemical Modifications in Therapeutic Oligonucleotides 220
13.2.3.1 Backbone Modified Oligonucleotides 220
13.2.3.2 Ribose Modified Oligonucleotides 221
13.2.3.3 Oligonucleotides with Unnatural Backbone 221
13.2.4 Oligonucleotide Therapeutics Other Than Antisense
Oligonucleotide 223
x Contents

13.2.4.1 Oligonucleotide Therapeutics Functioning Through RNA

Interference 224
13.2.4.2 Oligonucleotide Therapeutics Functioning Through Binding to
Protein 224
13.3 Non-canonical Nucleic Acid Structures as Therapeutic Targets 224
13.3.1 Traditional Antibiotics Targeting Structured Region of RNAs 225
13.3.2 Strategies for Constructing Therapeutic Materials Targeting Structured
Nucleic Acids 226
13.4 Non-canonical Nucleic Acid Materials for Inducing Non-canonical
Structures 230
13.5 Conclusion 231
References 232

14 Materials Science and Nanotechnology of Nucleic Acids 235

14.1 Introduction 235
14.2 Non-canonical Structure-Based Nanomaterials Resembling Protein
Functions 235
14.2.1 Aptamer 235
14.2.2 DNAzyme 238
14.2.3 Ion Channel 240
14.3 Protein Engineering Using G4-Binding Protein 240
14.4 Regulation of Gene Expression by G4-Inducing Materials 242
14.5 Environmental Sensing 246
14.5.1 Sensing Temperature in Cells 246
14.5.2 Sensing pH in Cells 248
14.5.3 Sensing K+ Ion in Cells 248
14.5.4 Sensing Crowding Condition in Cells 249
14.6 Conclusion 250
References 250

15 Future Outlook for Chemistry and Biology of Non-canonical

Nucleic Acids 253
15.1 Introduction 253
15.2 Exploring Potential: Properties of Non-canonical Structures in Unusual
Media 253
15.3 Systemizing Properties: Prediction of the Formation of Non-canonical
Nucleic Acids Structures 259
15.4 Advancing Technology: Applications of Non-canonical Structures Taking
Concurrent Reactions into Account 262
15.4.1 Co-transcriptional Dynamics of G-Quadruplex 263
15.4.2 Co-transcriptional Functionalization of Riboswitch-Like Sensor 263
15.4.3 Co-transcriptional RNA Capturing for Selection of Functional
RNAs 266
15.5 Conclusion 267
References 268

Index 271
 xi

Preface

In chemistry and biology, one of the most important and interesting research sub-
jects is nucleic acids: DNA and RNA. The nucleic acids consist of very simple mate-
rials: phosphate, sugar, and organic bases. Their structures are also very simple as
single strands or a double helix, in comparison with another biomolecules such as
proteins and carbohydrates; however the nucleic acids have very important genetic
information and functions.
As I mentioned in the Introduction in Chapter 1, there is close to 70 years his-
tory in nucleic acid research after the discovery of the double helix DNA structure
(B-form) as the canonical one by James Dewey Watson and Francis Harry Comp-
ton Crick in 1953 and chemical biology of nucleic acids are facing to new aspect
today, that is, non-canonical nucleic acids. Through this book, I expect that readers
understand how uncommon structure of nucleic acids became one of the common
structures as non-canonical nucleic acids that fascinate us now. This new research
field for non-canonical nucleic acids will soon big-spark at the interface of chemistry
and biology.
This book is comprised of 15 chapters covering various aspects of chemistry and
biology of non-canonical nucleic acids including not only their history, structures,
stabilities, and properties but also their functions on transcription, translation,
regulation, telomere, helicases, cancers, neurodegenerative diseases, therapeutic
applications, nanotechnology, and future outlook. This book is a valuable resource,
not only for graduate students but also researchers in the fields of physical
chemistry, organic chemistry, inorganic chemistry, analytical chemistry, biochem-
istry, biophysics, structural biology, computational biology, molecular medicine,
molecular biology, cell biology, and nanotechnology and who would like to learn
more about the potential important roles of non-canonical nucleic acids as well as
canonical ones.
I wish all readers enjoy this book and know the importance of not only
Watson–Crick double helical nucleic acids (B-form) but also non-canonical nucleic
acids like triplex and quadruplex. Instead of Hamlet by written by William Shake-
speare, please answer the question “To B or not to B, that is the question” in the
research field of nucleic acids.
I am deeply grateful to my colleagues in FIBER (Frontier Institute for Biomolecu-
lar Engineering Research), Konan University, for their excellent contribution to my
xii Preface

writing as the co-authors at the following each chapter. They are Dr. Shuntaro Taka-
hashi (for Chapters 1, 4, 5, 8, 9, 14, and 15), Dr. Tamaki Endoh (for Chapters 2, 7,
10, 13, and 15), and Dr. Hisae Tateishi-Karimata (for Chapters 3, 6, 11, 12, and 15),
whose efforts have immeasurably improved the quality and accuracy of the infor-
mation. I am also deeply grateful to Ms. Miwa Inada for designing a lot of figures
and Ms. Katherine Wong and Dr. Lifen Yang in Wiley for their editing this book and
encouraging me.

Naoki Sugimoto
Frontier Institute for Biomolecular Engineering Research (FIBER)
Graduate School of Frontiers of Innovative Research
in Science and Technology (FIRST)
Konan University
Kobe, Japan
 1

History for Canonical and Non-canonical Structures of

Nucleic Acids

The main points of the learning:

Understand canonical and non-canonical structures of nucleic acids and think of
historical scientists in the research field of nucleic acids.

1.1 Introduction

This book is to interpret the non-canonical structures and their stabilities of nucleic
acids from the viewpoint of the chemistry and study their biological significances.
There is more than 60 years’ history after the discovery of the double helix DNA
structure by James Dewey Watson and Francis Harry Compton Crick in 1953, and
chemical biology of nucleic acids is facing a new aspect today. Through this book,
I expect that readers understand how the uncommon structure of nucleic acids
became one of the common structures that fascinate us now. In this chapter, I
introduce the history of nucleic acid structures and the perspective of research for
non-canonical nucleic acid structures (see also Chapter 15).

1.2 History of Duplex

The opening of the history of genetics was mainly done by three researchers.
Charles Robert Darwin, who was a scientist of natural science, pioneered genetics.
The proposition of genetic concept is indicated in his book On the Origin of Species
published in 1859. He indicated the theory of biological evolution, which is the basic
scientific hypothesis of natural diversity. In other words, he proposed biological
evolution, which changed among individuals by adapting to the environment and
be passed on to the next generation. However, that was still a primitive idea for
the genetic concept. After that, Gregor Johann Mendel, who was a priest in Brno,
Czech Republic, confirmed the mechanism of gene evolution by using “factor”
inherited from parent to children using pea plant in 1865. This discovery became
the concept of genetics. At the almost same time in 1869 as Mendel, Johannes
Chemistry and Biology of Non-Canonical Nucleic Acids, First Edition. Naoki Sugimoto.
© 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.
2 1 History for Canonical and Non-canonical Structures of Nucleic Acids

Friedrich Miescher, who was a biochemist in Swiss, discovered nucleic acids as a

chemical substance of the gene identity. He named it “nuclein” (later, it was named
“nucleic acid,” which exists acidic substance in nucleus) and made the opportunity
to study nucleic acid chemistry. However, it would be doubtful if he realized that
nucleic acid is the gene identity. After that, it was needed to take a lot of time to
conclude that the gene identity is proved the chemical substance.

Charles Robert Darwin (left), Gregor Johann Mendel (middle), and Johannes Friedrich
Miescher (right)

Erwin Rudolf Josef Alexander Schrödinger, who was a great physicist, pioneered
to go after the mystery of gene. He published a book titled What Is Life? in 1944 [1].
This book invited the study of the gene to many researchers. He mentioned in the
book that he believed a gene – or perhaps the whole chromosome fiber – to be an
aperiodic solid, although he also mentioned that gene is probably one big protein
molecule. After the 1950s, chemistry regarding nucleic acids had been developing.
One of the organic chemists was Erwin Chargaff, who was a professor at Colom-
bia University in the United States and born in Austria. He discovered that from the
result of paper chromatography targeted to the different types of DNA, the number of
guanine units equals the number of cytosine units and the number of adenine units
equals the number of thymine units [2]. It is called Chargaff’s rules. On the other
hand, analysis of the superstructure of nucleic acids was also proceeding. At the
beginning of the 1950s, at King’s College London, the results of X-ray crystal analysis
were accumulated by Maurice Hugh Frederick Wilkins, Rosalind Elsie Franklin, and
others. Finally, based on their result, Watson and Crick who worked at Cavendish
Laboratory in Cambridge and proposed the model of double helix structure of DNA
(Figure 1.1 and see Chapter 2), published as a single-page paper about DNA dou-
ble helix in Nature issued on 25 April 1953 [3]. By discovering DNA double helix
structure, Watson, Crick, and Wilkins were awarded the Nobel Prize in Physiology
or Medicine in 1962.
 1.2 History of Duplex 3

Figure 1.1 The diffraction pattern of the canonical DNA duplex and its chemical structure.
Source: Kings College London.

Erwin Rudolf Josef Alexander Schrödinger (left) and Erwin Chargaff (right)
4 1 History for Canonical and Non-canonical Structures of Nucleic Acids

Maurice Hugh Frederick Wilkins (left) and Rosalind Elsie Franklin (right)

James Dewey Watson and Francis Harry Compton Crick

 1.3 Non-Watson–Crick Base Pair 5

1.3 Non-Watson–Crick Base Pair

Although the discovery of Watson–Crick base pairs is famous, we need to make

sure that Watson and Crick initially “proposed” their model. Moreover, Watson and
Crick were not the first researchers who proposed the structure of nucleic acids.
The physicist Linus Pauling, who earned the Nobel Prize two times in his career,
first proposed the helix model of nucleic acids with his associate Robert Corey [4].
However, the structure was fault: it was a triple helix having negatively charged
phosphates located at the core of the helix, which could not exist in nature. After
the proposal of Watson–Crick base pairs, the race for determination of the helical
structure of DNA had been started using purine and pyrimidine monomers. The
first such study was reported in 1959, when Karst Hoogsteen – an associate of Robert
Corey at Caltech – used single-crystal X-ray analysis to determine the structures of
cocrystals containing 9-methyladenine and 1-methylthymine, where methyl groups
were used to block hydrogen bonding to nitrogen atoms otherwise bonded to sugar
carbons in DNA [5]. However, the structure was NOT Watson–Crick base pair, in
which the adenine base was flipped upside down. The different base pair was later
named Hoogsteen base pair (Figure 1.2 and see Chapter 2). After the discovery of

Watson–Crick
base pair

Hoogsteen
base pair

Triplex structure G-quadruplex structure

HG bp HG bp

WC bp WC bp

Figure 1.2 Chemical structures of base pairs via Watson–Crick or Hoogsteen types.
6 1 History for Canonical and Non-canonical Structures of Nucleic Acids

Hoogsteen base pairs, many researchers looked for Watson–Crick base pairs. How-
ever, only Hoogsteen base pairs were identified. In 1973, Alexander Rich first dis-
covered Watson–Crick base pairs in the cocrystal of the AU and GC dinucleoside
phosphate complex [6]. And soon after, Richard E. Dickerson, who took over the
Pauling’s lab, first solved the single-crystal structure of a DNA dodecamer using
heavy atom X-ray crystallography in 1980 [7]. It takes more than 20 years after the
discovery of Watson–Crick base pairs. These results suggest that Watson–Crick base
pairs tended to stably form under the constraint of the helical structure of nucleic
acids, whereas Hoogsteen base pairs form in other structural conditions. Therefore,
there are canonical structures composed by Watson–Crick base pairs in the duplex
structures. On the other hand, non-canonical structures include non-Watson–Crick
base pairs such as Hoogsteen base pairs.

Linus Pauling (left), Robert Corey (middle), and Karst Hoogsteen (right)

Alexander Rich (left) and Richard E. Dickerson (right)

 1.4 Nucleic Acid Structures Including Non-Watson–Crick Base Pairs 7

1.4 Nucleic Acid Structures Including

Non-Watson–Crick Base Pairs

Behind the extensive efforts to identify the duplex structure of Watson–Crick base
pairs, Hoogsteen base pairs were also found in the structure of nucleic acids in the
1960s. Felsenfeld and Rich explained how poly(rU) strands might associate with
poly(rA)-poly(rU) duplexes to form triplexes [8]. From the chemical shift of NMR,
they identified evidence for triplex formation via protonated G–C+ Hoogsteen
base pairs at cytosine N3 in a poly(dG)-poly(dC) complex with dGMP at low
pH [9]. In 1962, it was found that short guanine-rich stretches of DNA could
assume unusual structures [10]. The diffraction studies of poly(guanylic acid) gels
suggested that if four guanines were close enough together, they could form planar
hydrogen-bonded arrangements now called guanine quartets (G-quartets). With a
stack of a few G-quartets, a tetraplex structure is formed called as G-quadruplex
(see Chapter 2). In the crystal structure, Hoogsteen base pairs of polynucleic
acids were first found in tRNA structure [11]. In the structure Watson–Crick base
pairs formed the secondary structure of tRNA, whereas Hoogsteen base pairs
supported the tertiary structure. Not only Hoogsteen base pairs but also other
types of non-Watson–Crick base pairs were found in tRNA structures. The tertiary
structure of nucleic acids is important especially for non-coding RNAs, which
do not code genetic information. The landmark of research of non-coding RNA
is the discovery of ribozyme (ribonucleic acid enzyme) by Thomas Robert Cech
in 1982 [12]. Ribozymes catalyze chemical reactions as well as protein enzymes.
Later structural studies revealed that there are a lot of non-Watson–Crick base
pairs to produce the active core of enzymatic reaction of ribozymes. Therefore,
non-canonical Watson–Crick base pairs including Hoogsteen base pairs have been
thought of as a tool for the tertiary structure of nucleic acids except for duplexes.

Thomas Robert Cech

8 1 History for Canonical and Non-canonical Structures of Nucleic Acids

With the progress of structural analysis technology in the 1990s, Hoogsteen

base pairs are gradually revealed to exist in DNA complexes with low molecular
weight compounds and proteins as well as transiently in Watson–Crick-type double
helix. Furthermore, another type of tetraplex structures was identified from DNA
sequence enriched in cytosine due to the cross intercalations of hemiprotonated
cytosine–cytosine (C–C+ ) base pairs under acidic conditions [13]. This tetraplex
is called as i-motif (see Chapter 2). Soon after, the roles of the non-canonical
structures have been gaining attention. Especially since the 2000s, research on the
G-quadruplex structure formed from Hoogsteen base pairs has made remarkable
progress. When a G-quadruplex is formed on DNA or RNA, the reactivity of the
protein involved in gene expression is affected (see Chapters 6–8). This means that
the central dogma proposed by Crick – that genetic information is determined
centrally by the flow of replication, transcription, and translation – is highly con-
trolled by the formation of a G-quadruplex structure. In general, it has been thought
that the regulation of gene information expression is due to protein functions.
However, the specific structure of Hoogsteen base pairs controls gene expression so
that the nucleic acid itself can function like a protein. That is, the roles of nucleic
acids might be properly used according to base pairs: Watson Crick base pair =
information, non-Watson Crick base pair = function. Many sequences that can
have a G-quadruplex structure are distributed in the telomere at the end of the
chromosome and the promoter region of the oncogene of the gene. Starting with the
2013 report, there have been many reports on the formation of G-quadruplexes and
i-motifs in cells. These reports point out that the oncogene may be activated by the
formation (or dissociation) of the G-quadruplex to cause cancer (see Chapter 11).
Furthermore, it has been suggested that the phase-separated structure formed by
the aggregation of RNAs with G-quadruplexes contributes to neurological diseases
such as amyotrophic lateral sclerosis (see Chapter 12).

1.5 Perspective of the Research for Non-canonical

Nucleic Acid Structures
As the regulation of gene expression by the specific structure of nucleic acids has
been clarified, the next important issue is knowing what specific structures are
formed where and when in cells. For example, Hoogsteen base pairs are affected
by the molecular environments such as ions, pH, and water activity. Cells are in an
environment crowded with molecules, so-called molecular crowding (see Chapters
3 and 4), and the molecular environment changes depending on the cell cycle [14].
For example, the nucleolus causes a change in the molecular density in the nucleus
by repeating formation and dissociation according to the cell cycle. This regulates
the timing of activation of rRNA transcription in each cell cycle, because the
transcription of rRNA specifically occurs in nucleolus. In addition, the environment
of mitochondria is particularly crowded (up to 500 mg ml−1 ) but heterogeneous
due to locally increased proton concentration by the proton gradient required for
ATP synthesis. Therefore, it is desirable to develop a technology that can predict
 References 9

physicochemical property of specific structures due to Hoogsteen base pairs in

each characteristic molecular environment [15]. In addition, there is a possibility
to make a new approach of drug development that treats diseases by changing the
molecular environments of cells, rather than targeting genes and proteins.

1.6 Conclusion and Perspective

According to Pauling’s personal communication revealed by the Nobel Foundation’s

disclosure, he considered Watson and Crick’s Nobel award to be premature. In spite
of his opinion, the Nobel Foundation decided to award the Prize to Watson and Crick.
This might suggest that Watson–Crick base pairs were very common and meaningful
at that time but non-Watson–Crick base pairs were artifact and meaningless. Nowa-
days, non-Watson–Crick base pairs are becoming common and significant as Pauling
perhaps predicted. Now, the day when the essence of nucleic acids becomes beyond
the concept of Watson and Crick is coming closer.

References

1 Schrödinger, E. (1944). What Is Life? The Physical Aspect of the Living Cell and
Mind. Cambridge: Cambridge University Press.
2 Tamm, C., Hodes, M., and Chargaff, E. (1952). J. Biol. Chem. 195: 49–63.
3 Watson, J.D. and Crick, F.H. (1953). Nature 171: 737–738.
4 Pauling, L. and Corey, R.B. (1953). Nature 171: 346–346.
5 (a) Hoogsteen, K. (1959). Acta Crystallogr. 12: 822–823. (b) Hoogsteen, K.R.
(1963). Acta Crystallogr. 16: 907–916.
6 (a) Day, R.O., Seeman, N.C., Rosenberg, J.M., and Rich, A. (1973). Proc. Natl.
Acad. Sci. U. S. A. 70: 849–853. (b) Rosenberg, J.M., Seeman, N.C., Kim, J.J.
et al. (1973). Nature 243: 150–154.
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8 Felsenfeld, G. and Rich, A. (1957). Biochim. Biophys. Acta 26: 457–468.
9 Kallenbach, N.R., Daniel, W.E. Jr., and Kaminker, M.A. (1976). Biochemistry 15:
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 11

Structures of Nucleic Acids Now

The main points of the learning:

(1) Learn interactions in nucleic acid structures.
(2) Understand structure polymorphisms of nucleic acids.
(3) Study differences in conformational properties between DNA and RNA.

2.1 Introduction
Nucleic acids are basically molecules with a high degree of structural flexibility and
polymorphic property. Phosphates in nucleic acids are negatively charged and cause
electrostatic repulsion in each phosphate moiety. This electrostatic repulsion is dis-
advantageous for nucleic acids to form a compact and ordered structure. Nucleic
acids form the higher-order structures by offsetting unfavorable entropy changes and
electrostatic repulsion by internal interactions such as hydrogen bonding and stack-
ing interactions and external factors such as interactions of nucleic acids with cations
and cosolutes. In other words, the canonical nucleic acid structure consisting of dou-
ble helix with Watson–Crick base pairs is a part of the possible structural forms, and
nucleic acids form various non-canonical structures depending on the internal and
external factors. This chapter shows basic elements that form non-canonical nucleic
acid structures including unusual base pairing, whose existence has been revealed
by structural analyses, and their properties of thermodynamic stabilities. Detailed
analyses of the stabilities of nucleic acid structures and factors that affect them are
explained in Chapter 3.

2.2 Unusual Base Pairs in a Duplex

As in Chapter 1, the canonical structure of nucleic acids is double-stranded helix

with Watson–Crick base pairs (Figure 2.1), which are almost identical in their

Chemistry and Biology of Non-Canonical Nucleic Acids, First Edition. Naoki Sugimoto.
© 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.
12 2 Structures of Nucleic Acids Now

N NH2 O N O H2N

N N HN N NH N
N N N N
O NH2 O

(a) (b)

H
N NH2 O H2N N O H2N

N N +
N HN N H N
N N N N
O O

(c) (d)

(e) (f)

Figure 2.1 Watson–Crick and Hoogsteen base pairs in double helix. Chemical structures
of A-T (a) and G-C (b) Watson–Crick base pairs. Chemical structures of A-T (c) and G-C+ (d)
Hoogsteen base pairs. N3 atom of cytosine nucleobase is protonated. (e) Structure of DNA
duplex consisting of all A-T Hoogsteen base pairs (PDBID: 1RSB). (f) Structure of DNA
duplex containing two consecutive G-C+ Hoogsteen base pairs (PDB ID: 1QN3). The DNA
duplex is bending due to interaction of TATA-box binding protein. Nucleobases forming the
Hoogsteen base pairs are emphasized dark. In (e) and (f), hydrogen bonds between the
Hoogsteen base pairs are shown in dashed lines.

geometric and dimensional arrangement in the helix. The sugar groups are both
attached to the bases on the same side of the base pair. The distance between C1′
atoms of the sugars on opposite strands is essentially the same. These geometric
features enable any sequence of Watson–Crick base pairs fit into the duplex. On
the other hand, a large number of other arrangements and hydrogen bonding
patterns of base pairs are possible, and many have been observed experimentally
such as using X-ray and NMR analyses. X-ray analysis can define the duplex
structures, which incorporate the non-Watson–Crick base pairs and provide details
of their hydrogen bonding scheme [1]. On the other hand, NMR analysis provides
information regarding the dynamics of the nucleic acid conformation such as
 2.2 Unusual Base Pairs in a Duplex 13

mismatched base pairs, in which transient tautomeric and anionic species form
Watson–Crick-type hydrogen bonds [2].

2.2.1 Hoogsteen Base Pair

Hoogsteen base pair (Figure 2.1) is one of the major non-Watson–Crick base pairs
that can be seen in several crystal structures of duplexes containing A⋅T base
pairs. For example, the crystal structure of AT-rich sequences adopted parallel
and antiparallel stranded duplexes with all Hoogsteen-type hydrogen bonding in
their A⋅T base pairs (Figure 2.1) [3]. In the case of antiparallel duplex with the
Hoogsteen base pairs, the overall structure features of the duplex such as diameter
of the duplex, number of base pairs per turn, and sugar pucker conformation
are similar to the canonical B-type DNA duplex. A unique characteristic is that
the adenine nucleobases in the duplex have syn conformation in their glycosidic
bond angles. Although the same pattern of hydrogen bonding is possible, A-type
RNA duplexes disfavor the A⋅U Hoogsteen base pair because the A-form geometry
disfavors the syn conformation in the adenine nucleobase due to sugar-backbone
rearrangements needed to sterically accommodate the adenine [4]. Formation
of Hoogsteen-type hydrogen bonding is also possible between guanine (G) and
cytosine (C+ ), in which N3 atom is protonated. Formation of transient Hoog-
steen base pairs including the G⋅C+ in diverse sequence composition has been
demonstrated by relaxation dispersion assay using NMR (Figure 2.1) [5]. It is
considered that the Hoogsteen base pairs play roles in modulating interaction
of proteins and biological reactions such as induction or repair of DNA damage
and replication of DNA by altering the structural and chemical properties of the
duplex [5].

2.2.2 Purine–Pyrimidine Mismatches

Mismatched base pairs between purine and pyrimidine nucleobases are known as
transition mismatches. G⋅T and G⋅U mismatches can form two hydrogen bonds,
which are usually known as typical “wobble” base pairs, by shifting the nucleobase
geometry from that of Watson–Crick base pairs (Figure 2.2). These mismatches
are one of the most stable mismatches in DNA and RNA duplexes (Tables 2.1
and 2.2). G⋅U base pair is frequently observed in natural RNAs. It is because that
both guanine and uracil take anti conformation in their glycosidic bond angles
that are the same with Watson–Crick base pairs and the distance and geometric
arrangement of C1′ atoms of the sugars do not largely change compared with the
canonical duplex. Thus, there is no significant worsening of base stacking and
littler perturbation of the duplex conformation. Adenine and cytosine also form
two hydrogen bonds as similar way as the G⋅T(U) mismatch when the adenine
nucleobase is protonated (Figure 2.2). Formation of the A+ ⋅C mismatched base
pair is demonstrated by X-ray diffraction analysis using dodecamer oligonucleotide
14 2 Structures of Nucleic Acids Now

O O
NH2
NH O N NH O N N H2N
N N N
N O N N
O HN HN +
O H N N
N N
H2N H2N N
(a) (b) (c)

(d) (e) (f)

Figure 2.2 Wobble base pairs in duplexes. Chemical structures of G-T (a), G-U (b), and A+ -C
(c) wobble base pairs. N1 atom of adenine nucleobase is protonated. (d) Structure of B-form
DNA duplex containing G-T wobble base pairs (PDB ID: 113D). (e) Structure of A-form RNA
duplex containing two consecutive G-U wobble base pairs (PDB ID: 433D). (f) Structure of
B-form DNA duplex containing A+ -C wobble base pairs (PDB ID: 1D99). Nucleobases
forming the wobble base pairs are emphasized dark. Hydrogen bonds in the wobble base
pairs are shown in dashed lines.

strand [7]. However, neutral A⋅C mismatches are in equilibrium between the
wobble and reverse wobble forms, each of which only forms one hydrogen bond.
Thus, A⋅C mismatch is much less stable than G⋅T(U) mismatch in a physiological
condition (Tables 2.1 and 2.2).

2.2.3 Purine–Purine Mismatches

Purine–purine and pyrimidine–pyrimidine mismatches are known as transversion
mismatches. There are G⋅A, G⋅G, and A⋅A mismatches in the purine–purine mis-
match. Among them, G⋅A mismatch forms relatively stable unusual base pairs in
both DNA and RNA duplexes (Tables 2.1 and 2.2). It is highly polymorphic depend-
ing on sequence compositions. In DNA duplexes containing G⋅A mismatches that
form two hydrogen bonds, various combinations of anti and syn were observed in
their glycosidic bond angles (Figure 2.3). G⋅G mismatch potentially adopts a base
paring with two hydrogen bonds, in which two guanosines are symmetrically or
asymmetrically oriented with anti and syn conformation in their glycosidic bond
angles (Figure 2.3). When the asymmetric G⋅G base pairs face each other in rota-
tion, four guanines form a symmetric quartet as described later (Chapter 3). Recent
X-ray diffraction analyses also demonstrated polymorphic feature of the G⋅G mis-
match by showing that with syn–syn combination in the glycosidic bond angles in
 2.2 Unusual Base Pairs in a Duplex 15

Table 2.1 Thermodynamic parameters for duplex formations in 1M NaCl by DNA

oligonucleotides containing mismatchesa).

−𝚫H ∘ −𝚫S ∘ −𝚫G37

o
Tm
Sequence XY (kcal (cal mol ) −1
(kcal (∘ C at
mol−1 ) K−1 ) mol−1 ) 10−4 M)

5′ CAAA X AAAG CG 64.5 183 7.7 42.9

3′ GTTT Y TTTC GC 62.8 179 7.3 40.8
AT 68.0 196 7.2 40.1
TA 58.6 168 6.5 36.8
GG 53.5 158 4.5 25.6
TG 55.6 165 4.4 25.7
GA 52.6 156 4.2 23.9
GT 46.7 137 4.2 22.3
AG 39.9 116 3.9 18.0
AA 36.9 107 3.7 15.0
CT 53.2 161 3.3 19.1
TC 50.0 151 3.2 17.5
CA (40.3)b) (120)b) (3.1)b) (13)b)
TT (54.6)b) (167)b) (2.8)b) (17)b)
AC (35.8)b) (106)b) (2.9)b) (9)b)
CC (55.3)b) (171)b) (2.3)b) (15)b)

5′ CAACTTGATATTAATA Mismatch
+

3′ GTTGAACTATAATTAT – 102.1 289 12.4 55.8

3′ GTTGAGCTATAATTAT TG 92.6 266 10.1 49.4
3′ GTTGAACTATAGTTAT TG 95.5 274 10.5 50.5
′
3 GTTGAACTCTAATTAT TC 98.4 286 9.7 47.3
3′ GTTGAATTATAATTAT GT 91.3 264 9.4 47.1
′
3 GTTGAACCATAATTAT AC 90.9 265 8.7 44.6
3′ GTTGAACAATAATTAT AA 92.0 267 9.26 46.2

a) Values are summarized ones in a reference [6].

b) Values in parenthesis are significantly less accurate estimated using flat lower baselines in
their melting analyses.

the presence of chromomycin A3, which binds minor groove of the mismatched
place and supports the structure analysis [8]. Detailed structure of A⋅A mismatch
is rarely determined by X-ray diffraction analysis. It is considered that the mismatch
is dynamically fluctuated and not able to be a particular structural state.
Another random document with
no related content on Scribd:
girl at the wheel was showing disturbing signs of putting the motor into
reverse and seeking to back off the heavy piece of bridge-railing that,
jammed in between their rear mud-guard and the side of the car, was the
only thing preventing the machine from plunging off into eternity.

"I say, leave the motor alone!" Rodrigo shouted at once and scrambled
hurriedly out from behind the wheel of the sedan, his companions following.

"And whose motor is it, may I ask?" the pretty blonde in the driver's seat
came back promptly, at the same time jabbing furiously at levers.

Rodrigo was by this time at her side and, horrified, was clutching for her
wrist. "Lady, lady," he cried half in fear and half in mockery. "Shut off the
motor and get out quick. You're on the brink of eternity."

"Yes, Sophie, do," the other girl, slightly older and a brunette, agreed.

At first inclined to be stubborn, Sophie at length permitted herself to be

helped down from her precarious perch and her companion followed,
Terhune and Bond re-inforcing Rodrigo.

Thus the Oxonians made the acquaintance of Sophie Binner and Adele
Du Bois, ladies of the chorus in "The Golden Slipper," the current revue at
the Gayety. On the promise of stopping at the nearest garage and having the
wrecked machine sent for, the girls consented to enter the sedan and be
driven back to London. By the time the outskirts of the city were reached,
the party was a very gay one and Sir William Newbold's Treasure Hunt was
quite forgotten.

Rodrigo was especially interested in Sophie. He had at that time met

very few ladies of the stage informally. The frankness, sharp tongue and
cream-and-gold beauty of Sophie intrigued him. Rodrigo was rather adroit
with all types of women, even at twenty. He flattered Sophie half seriously,
half banteringly, exchanged bon mots, made an engagement in a low voice to
see her again. Bill Terhune told her on the quiet that Rodrigo was the son of
a real Count, thus increasing many fold the force of the impression the good-
looking Latin had made upon her.
 The Oxonians had dinner with Sophie and Adele, saw the show at the
Gayety, and took the girls later to a supper club. It was the first of several
parties in which Rodrigo's and Sophie's friends took part.

Having, following this adventure, made his apologies to his uncle and
aunt for having left the Treasure Hunt flat, the excuse being the necessity of
rescuing an automobile party in distress, Rodrigo proceeded to cultivate the
further acquaintance of Miss Binner assiduously and without the knowledge
of the Newbolds.

He was her constant cavalier. She taught him much—for instance, that a
baby-faced blonde can possess a wicked tongue, a sudden and devastating
temper and a compensating tenderness that made up for both defects. He was
thoroughly infatuated at first. Then his ardor cooled as he realized that
Sophie was professing to take his wooing seriously. The idea of contracting
an alliance with a future nobleman seemed to appeal to her. Rodrigo did not
think of her in that regard at all, and he was alarmed. He began looking for a
loophole.

The climax came at a party arranged for after the show in Sophie's
Mayfair apartment. Rodrigo had recruited Bill Terhune, Bond and three or
four other Oxford friends for the fun. They had accumulated Sophie, Adele
and a quartet of their sister coryphees at the theatre after the evening
performance and whirled them through the London streets in a fleet of
taxicabs. At two o'clock in the morning the party was in full swing. The
tinpanny piano crashed out American jazz under the nimble fingers of
Sophie. Leslie Bond numbered drumming among his numerous avocations
and had brought along the clamorous tools of his hobby. His hysterical
efforts on drums, cymbals and cowbells augmented the din and broke both
drums.

The revelers sang, danced, drank and made love. Bill Terhune, under the
impetus of spirits, was especially boisterous.

There was a sharp knocking upon the door. A corpulent, red-faced

Englishman in a frayed and gaudy bathrobe announced that he occupied the
apartment below, had been awakened by plaster falling upon his bed and his
person, and that "this Donnybrook Fair must cease at once." He was set upon
joyously by three burly Oxonians and good-naturedly propelled down the
stairs.

Sophie, from the piano, however, did not share their enthusiasm. "It may
interest you impetuous lads to know that our killjoy friend is a magistrate
and will probably have a couple of bobbies here in five minutes," she
warned them. They laughed at her and the party went on.

In twenty minutes there was another knock. Two bobbies, each built like
Dempsey, confronted Rodrigo when he opened the door. The policemen
entered with that soft, authoritative tread that London police have. One of
them laid hands upon Bill Terhune. Bill, former intercollegiate boxing
champion, was in a flushed and pugnacious mood. He promptly struck the
officer in the face and sent him reeling to the floor.

Immediately the party grew serious. Englishmen respect the police. An

American may attack a Broadway policeman, but hitting a London bobby is
something else again. The other bobby swung into action with his club.
There was a concerted rush for the door. Rodrigo could have easily escaped.
But he chose instead to stand by Sophie, who, he knew, was due for trouble
as the tenant of the apartment. When the tumult and the shouting died, the
room contained Sophie, Rodrigo, one angry bobby with pencil raised over
his book, and one still bobby recumbent upon the floor.

"The names now—the right ones," commanded the erect bobby.

"First, don't you think we'd better revive your friend on the floor?"
Rodrigo suggested.

When they had brought the fallen one back to life, Rodrigo soothingly
and skillfully persuaded the officers to let Sophie alone, to allow him to
assume sole responsibility for the trouble. He asked only permission to
telephone his uncle, Sir William Newbold. The bobbies generously
consented to take him, without Sophie, to jail for the rest of the night, but
they declined to allow him the use of the telephone.

The jail cell was cold, cramped and dirty. Rodrigo's cellmate was a hairy
navvy recovering from a debauch. Rodrigo had to listen to the fellow's
alternate snoring and maudlin murmurings until dawn. When, around ten
o'clock in the morning, he did succeed in getting in touch with his uncle, the
latter's influence was sufficient to secure his release.

Sir William delivered to his nephew a severe lecture. Then he telephoned

the newspaper offices with the idea of having any possible news of his
nephew's incarceration suppressed. In this endeavor he was unsuccessful.
Two papers contained an account of the arrest, and the more sensational
sheet of the two declared that Rodrigo, son of Count Angelo Torriani and
nephew of "London's leading merchant-knight, Sir William Newbold," was
the fiancé of Sophie Binner and that they were to be married shortly.
Rodrigo denied this vehemently to his uncle and was indeed just as angry
about it as was Sir William. He saw in it evidence that Sophie had
prevaricated to the newspapers, had used his ill fortune as a means of
securing notoriety and possibly of binding him publicly to an alliance that
did not exist.

He resolved to call upon her and break off any possible entanglement
with her.

He confronted her in her apartment in the middle of the next afternoon.

She looked especially lovely, her spun-gold tresses in informal disarray and
her beauty encased in a silken lounging gown. But Rodrigo was firm. He
accused her of exploiting last night's episode in the papers, of giving out
news of an engagement that was false. Though she denied this, at first
poutingly, then coyly and finally with considerable vehemence not unmixed
with vulgarity, Rodrigo insisted. He worked her into a tempest and, at the
climax, dramatically walked out of the room and, as he thought, of her life.

During the two years following his graduation from Oxford, Rodrigo had
vague ambitions to become a painter and spent considerable time browsing
about the galleries of England, Spain, France and his native Italy. He had a
workroom fitted up in the palace of the Torrianis and did some original work
in oil that was not without merit. But he worked spasmodically. His heart
was not in it. He knew good painting too well to believe that his was an
outstanding talent, and he lacked ambition therefore to concentrate upon
developing it.
 In the pursuit of pleasure and the spending of money he was more
whole-hearted. He skied and tobogganed at St. Moritz, gambled at Monte
Carlo, laughed at Montmartre's attempts to shock him, and flirted in all three
places. Upon the invitation of the bobby-assaulting American Rhodes
scholar, Terhune by name, now squandering his South Dakotan father's
money in New York under the pretence of making a career in architecture,
Rodrigo visited America. America, to Rodrigo, was represented by the
Broadway theatre and nightclub belt between dusk and dawn. Having in a
few weeks exhausted his funds and finding his cabled requests for more
greeted with a strange reticence, Rodrigo started for home. Three days out
from New York he received the cable announcing to him Count Angelo
Torriani's sudden death.

Rodrigo had adored and respected his quiet, high-minded English

mother, from whom he had inherited the thin vein of pure gold concealed
deep down below the veneer of selfishness and recklessness that coated his
character. He loved his father, from whom he drew the superficial and less
desirable traits of his personality. Loved him and, without respecting him
particularly, treated him as he would an older brother of kindred tastes and
faults.

His father's death shook Rodrigo down considerably for a while. It

sobered him, made him suddenly aware of his appalling aloneness in a world
of many acquaintances but not an understanding relative nor close friend.
The secondary calamity of having been, out of a clear sky, left penniless and
in debt did not at first impress itself upon him. When the late Count
Torriani's will was read, revealing the surprisingly devastated condition of
the Torriani finances, and debtors began to present their claims, Rodrigo,
now Count Rodrigo faced the realization that his whole mode of life must be
changed.

He dismissed the servants, keeping Maria because she refused to go,

even after being informed that she would probably have to serve without pay
if she stayed. He finally brought himself to talking with an agent at Naples
about renting the palace and selling some of the works of art which it
contained. The agent was very brisk and business-like. He jumped up and
down from his chair and rubbed his hands continually, like an American.
Rodrigo was irritated by the vulgarian. He abruptly left the matter and the
realtor up in the air and jumped into his car outside. As he swung along the
shore of the bay he was in very low spirits, lonesome and as nearly
depressed with life as he had ever been. In his preoccupation he paid only
subconscious attention to the road ahead and the swift speed at which his car
was traveling. He heard suddenly a shriek and flashed his eyes in its
direction just in time to avoid killing a girl.

In the flash he saw that the girl was dark, and beautiful in a wildflower-
like manner. She was also very dusty from walking. In the torrent of oaths
which she poured after him, she furthermore revealed herself as charmingly
coarse and unrestrained. Rodrigo cheered up. After the weeks of grief and
loneliness, and particularly after the Naples realtor, he found himself
wanting ardently to talk to a woman, any woman. He stopped the car and
slowly backed up even with the approaching girl. She continued to swear at
him. He smiled. When she had gradually quieted, he apologized and offered
her a seat beside him. Her angry face relaxed, she pouted, and ended by
accepting.

In a few days he had drifted into a fast ripening friendship with Rosa
Minardi, who was childlike, was no tax upon his conversational charms or
ingenuity, and who liked him very much. Her mother was dead, her father
was away in Rome on some mysterious errand. Rodrigo badly needed any
sort of companionship, and Rosa filled the need.

CHAPTER III

Maria's gnarled knuckles beat vigorously upon her young master's door.
When her tattoo failed to bring results, she opened the door and walked
boldly in. Waddling to the floor-length windows, she flung aside the heavy
draperies, drenching the room with sunlight. With a guttural exclamation that
was half disgust and half tenderness, she turned toward the dark, recumbent
form upon the canopied bed, still undisturbed by her activities. She
approached Rodrigo and shook him.
 When at last he blinked up at her, she said sharply, "Get up, lazy one.
Your American has already breakfasted and is downstairs waiting for you."

Rodrigo's face screwed itself interrogatively, American? Then his

drowsy, somewhat fuddled brain remembered Dorning, of Dorning and Son.
Rodrigo frowned. Bother Americans. So full of restless energy, such early
risers. He looked languidly at the watch upon his wrist. Eleven o'clock. He
sat upright in bed and indulged in a prodigious yawn. With a grimace at the
ample back of Maria, just disappearing out of the door, he slid out of bed.

Half an hour later, having bathed and breakfasted, Count Rodrigo,

looking as fresh and bright of eye as a trained athlete, walked briskly
downstairs to find that his guest had apparently not missed him in the least.
Dorning was standing in front of the expansive canvas of an oil painting in
the great entrance hall of the Torrianis. He had just donned a pair of tortoise-
rimmed glasses and was bending over to read the metal plate set in the
elaborate frame of the painting. The plate read: "Francesca Torriani, 1527-
1562." Dorning realized the likeness between the ruffled-collared,
sardonically smiling aristocrat on the canvas and his host, whom he now
turned to greet.

"I see you are making the acquaintance of my ancestors," said Rodrigo.
"This one, like the others, you will observe, led a short life and, so I
understand, a merry one." Rodrigo noted curiously how glasses added at
least five years to the age of John Dorning. Having at the instant of their first
encounter at the Café Del Mare set the American down as an innocent and
probably a prig, Rodrigo had, during their discourse and drinking of the
previous night, changed his mind and conceived a mild liking for the man.
Dorning was honest, outspoken, and possessed of considerable culture. He
was, Rodrigo vaguely felt, the sort of person whom he should cultivate, the
type that develops into a staunch and worth-while friend.

"Your ancestor has at least had the good fortune to have been perpetuated
by an excellent artist," said Dorning.

"Here is something that will interest you," offered Rodrigo, walking over
to a low, ornately carved cabinet set against an adjacent wall. "This is the
best example of Early Renaissance cabinet work anywhere around here."
Dorning bent a grave, interested head and ran expert fingers over the
carving. His host tugged at the doors of the cabinet. As he wrenched them
apart, a shelf inside, unbalanced by his effort, slid out upon the floor, spilling
its contents as it came. The two young men looked at each other, and
Rodrigo grinned sheepishly. Two bundles of letters and a feminine lace fan
lay at Dorning's feet.

Rodrigo dropped to his knees and, replacing the souvenirs, closed the
cabinet. He rose, dusted his hands, said suavely, "The cabinet was made by
Beniti, in Genoa, around 1627. The contents are slightly more modern."

"So I judged," said John Dorning dryly. Then with more enthusiasm, "I
only wish I knew Italian antiques as well as you do, Count Torriani—and
antiques are my business."

Both turned as Maria came toward them in considerable agitation. "A

man named Minardi and a girl are here to see you," she announced in rapid
Italian to Rodrigo. "I do not like his looks. I refused to admit him, but he has
pushed his way into the outer hall." She indicated the draperies on the other
side of the room.
 "STOP! DO YOU WANT TO BE ARRESTED? THIS GENTLEMAN IS
AN AMERICAN."

Rodrigo's face clouded. Damn the fellow's persistence. "Tell him to go

away. I will not see him. Tell him I shall have him arrested if he continues to
bother me," he instructed Maria.

She turned doubtfully. She lacked her usual faith in her sharp tongue in
dealing with a calloused fellow like Minardi. She had taken but a step when
the draperies parted and Minardi, wearing the same clothes, expression, and
carnation as on the previous evening, bulked before them. He had heard
Rodrigo's voice talking with Maria, and he was taking no chances. His fat,
weak face was trying its best to assume hard, menacing lines. His ill-kept,
corpulent body was drawn up as straight as possible with unrighteous
indignation. He relaxed for an instant to turn around and drag by the wrist
from the other side of the curtain his daughter, Rosa.
 Rosa had been brought to the scene with some difficulty. She flashed
indignation at her father through swollen eyes. Actually propelled now into
the presence of Rodrigo, she glanced half defiantly, half shamefacedly at
him, then stood regarding the floor.

Victor Minardi started at once toward Dorning, taking up again with

undiminished vigor the torrent of abuse and threat which he had hurled at the
American at the Café Del Mare. He was persisting in his belief that Dorning
was Count Torriani, the man who was to pay.

Rodrigo stepped between the gesticulating Italian and the uncertain

Dorning. "I am Count Torriani. Now, what is it you want?"

Minardi wheeled upon Rodrigo. "So—it was you! Ah. Why did you not
say so before, eh?" And he launched into a fresh flood of indignation.

Rodrigo raised a hand to stop him. He perceived that this fellow could
not be easily overawed. Minardi wanted money and would probably
continue to be a howling nuisance until he got it. Rosa, Rodrigo suspected
shrewdly, was in the plot with her father. Certainly she would not otherwise
have revealed her love affair with Rodrigo to Minardi and, instead of
keeping her rendezvous at the Café Del Mare, allowed the noisy old man to
come on a blackmailing expedition in her place. Any tenderness Rodrigo had
previously felt for Rose Minardi disappeared. His lips curled as he looked at
her dark head, cast down in assumed modesty.

When Minardi had calmed down, Rodrigo snapped, "How much do you
want?"

Minardi's anger faded. His eyes lighted up with greed. "Five thousand
lira," he replied in a business-like tone.

"You come high," said Rodrigo.

Minardi's hand went to his greasy inside coat pocket, "I have here letters
that are worth more than that," he said. "Letters you have written to my
Rosa. There are such things as breach of promise suits. The newspapers
would like them, eh? The Torrianis are not popular at Naples, eh?"
 In spite of himself, Rodrigo winced a little. This fat, futile old reprobate
began to assume the proportions of a real danger. Rodrigo essayed frankness.
"You know so much about the Torrianis," suggested he, "you perhaps know
that I have not five thousand liras at the moment."

Minardi shrugged his stooped shoulders. "Even if that is true, you can
get them," he said. And he looked significantly at John Dorning, an
interested and somewhat disgusted spectator at the scene.

Rodrigo's slim fingers were drumming nervously upon the Beniti cabinet
which he had just been displaying to his guest. In their nervous course over
the top of the cabinet the finger points met the smooth surface of an
elaborately wrought silver vase standing there. Rodrigo looked down. He
hesitated an instant, then caught up the vase in his hand.

"This was made by the great Cellini himself," he remarked to Minardi.

"It is worth at least twice the amount you are blackmailing me for. You can
easily dispose of it in Naples. I do not, of course, admit any of your silly
accusations. However, take this vase—and go at once."

He held the exquisitely formed metal toward Minardi. John Dorning's

eyes made a hasty appraisal of it. He half opened his lips to protest against
this careless disposal of the little silver masterpiece. But Minardi, hardly
looking at it, snarled, "No. I want money."

Dorning said at once to Rodrigo, "Give him money then. I will buy the
vase. I'll give you twice what he wants—ten thousand liras—and make a
handsome profit if I ever want to dispose of it." He took out his purse.

Rodrigo regarded his guest with puzzled surprise. "I don't want you to do
this for me, Dorning. I——"

"Please believe me, it is merely a matter of business," Dorning cut in

quietly. "I am in Italy for the purpose of picking up just such bargains." He
counted out the money and offered it to Rodrigo. The young Italian hesitated
an instant, then took the proffered notes, counted them and started to hand
half to Minardi.
 "You want something for your money, don't you?" Dorning interjected.
"Your letters?"

"Naturally," replied Rodrigo, flushing a little. He was not used to being

prompted. As he took the packet of note-paper from Minardi's greasy hands
he now made an over-elaborate show of checking them up. "They are all
here," he decided, speaking curtly and more to Dorning than to Rosa's papa.
To the latter he continued even more curtly, "Now get out. If I see you about
here again I will turn you over to the police."

Minardi bowed impudently. He made a move to seize the silent Rosa's

hand, but she eluded him. Suddenly she opened shrill soprano abuse of her
father. "I hope you're satisfied now!" she cried. "You have humiliated me,
your daughter. You've sold my honest love for money, made me appear a
low, scheming woman. I hate you." With a swift movement she slipped over
to Rodrigo, who stood with arms folded, regarding her with a wry smile.

"Please tell me you do not think I plotted this with him," she pleaded, her
dark, warm face quite near to his. "It is not for money I love you. I did not
come to the café last night, because I was angry with you for telling me I am
bad tempered. I cried all last night over that, Rodrigo. But I am not angry at
you now. I am angry only at Papa." Her soft arms attempted to steal around
Rodrigo's neck. "Tell me that you still love me," she begged in a low, husky
voice.

Still he stood rigid. He shot an apologetic smile at Dorning. Even now he

felt the attraction of this creature of primitive emotions, though he suspected
she was acting.

"But you are bad tempered, Rosa," he jibed, disengaging her arms. "And
I think you are somewhat of a liar besides."

She fairly flung herself away from him at that, standing with heaving
bosom and flashing eyes. She was still cursing him when her father laid
violent hands upon her and led her out of the house.

Rodrigo shrugged his shoulders and lit a cigarette. "A charming

creature," he remarked flippantly to Dorning. Nevertheless Rodrigo was
rather ashamed of the scene the two Minardis had made in front of the
American. Somehow Dorning had already assumed an importance to him
much more than that of a casual and congenial guest. It was not that Dorning
had stepped into an embarrassing situation with ten thousand liras. It was the
spirit that had prompted the American's action. Rodrigo sensed a quiet
strength in the man that he himself somehow lacked, a strength that in the
troublous future confronting him he would like to have near him.

"The trouble with women," Rodrigo remarked, "is that they cannot keep
love in its proper place. It soon ceases to be a game with them and becomes
a mad scramble to possess a man. Then comes jealousy, bad temper,
remorse, and complications such as you have just seen."

"'Love is to man a thing apart; to woman their whole being,'" Dorning

quoted. He did not think his host had acquitted himself with especial credit
in the "complications." There was a tawdriness about the Minardis and the
scene they had created unbecoming to a man who owned original Cellinis
and other treasures. Art, to Dorning, was about all there was in life. The
Rosas were superficial annoyances that had never yet entered into his own
career, though he was quite aware that they existed in the careers of most
other men. He had been immediately attracted to his host by their mutual
interest in art. The charm of the man, his good looks, his facile tongue, his
wit and deftness in conversation had added to the attraction. Why should
such a man love such a common creature as Rosa Minardi and consent to be
blackmailed by her father? Dorning resolved to forget Rosa and turned the
conversation to tapestries.

But Rodrigo's thoughts were not entirely diverted from "complications."

"There is an amusing tradition about those tapestries," he said. "You will
observe that the ones near the window seat are identical with those at the
door leading into the outer hall. Well, my worthy ancestor whose portrait
you have praised, Francesca Torriani, once found their similarity his
undoing. It seems that he was entertaining a very lovely married lady in this
room, a Countess. Her husband, the Count, followed her to the rendezvous.
Suddenly in the middle of my ancestor's love-making, the Countess caught
sight of her husband outside. 'Quick,' she cried, 'where can I hide?' Francesca
thrust her behind the tapestry by the door.
 "The Count entered, very angry and his hand upon his sword hilt. 'Where
is my wife? I saw her come here,' he bellowed. Francesca swore like a
gentleman that the lady was not present. The Count insisted and started
searching. His eye caught the outline of a lady's foot showing beneath the
tapestry. With a loud cry of rage he tore the tapestry to one side and revealed
not his Countess but quite another lady! Another of Francesca's lady friends
had sought shelter when the Countess entered, behind the tapestries by the
window seat. All might have been well had not the Countess, hearing from
her hiding-place a woman's voice, been assailed by jealousy and, casting
discretion to the winds, come forth breathing fire and brimstone."

"What happened then?" asked Dorning smiling, amused in spite of

himself.

"There was a terrific four-handed clash. Poor Francesca was half mad
with anxiety. The Count challenged him to a duel. In the fight, Francesca,
who, unlike the rest of the Torrianis, was no swordsman, was killed."

"And quite a proper climax to the adventure it was," John Dorning

declared soberly.

"Proper—why!" Rodrigo asked. "Because Francesca had been too stupid

to learn swordsmanship?"

"No—because of his interest in a lady who belonged to another."

"The lady should not have taken Francesca's love so seriously as to have
become jealous. When will women understand that when they take our
admiration seriously they kill it?"

"Not at all," Dorning returned stoutly. "That is exactly the wrong

attitude. I do not understand it in you—you who are so intelligent and
sensible about other things. There are so many other things for you to
interest yourself in than in these petty love affairs."

Rodrigo straightened. He did not relish criticism. In the next instant,

realizing that Dorning was honest in his questioning and rather pleased that
he had aroused his quiet guest to such a pitch, he relaxed and asked calmly,
"What other interests do you recommend for a reckless and extravagant
gentleman, like myself, who now finds himself penniless and equipped for
nothing in the world but for amusing the ladies and for being amused by
them?"

"If you will pardon me—are you really in straightened circumstances?"

"Yes. I am in debt. Economy was not one of my father's virtues, nor did
he take the trouble to develop it in me." Rodrigo, fearing to be
misunderstood, added, "Not that I am in need of a loan, you understand. You
have done quite enough for me, and I am grateful."

"What are you thinking of doing then?"

"I can either marry the first single rich lady or widow who will have me,
or I can sell or rent this place and its contents."

"You would do neither of those two foolish things."

"Why not?" Rodrigo was curious. He was secretly rather pleased at the
personal turn the conversation had taken, for, with all his worldliness and
experience along romantic lines, it seemed that Dorning's common sense
might be valuable in considering the rather dismaying future.

"Have you ever considered entering trade?" Dorning asked tentatively.

"My father was in trade. There is nothing unpleasant about it to me.

What sort of trade?"

Thus encouraged, John Dorning revealed what was in his mind. "We—
Dorning and Son," he explained, "have gone in recently, to a very extensive
degree, for Italian antiques. My mission over here is for the purpose of
adding to our stock. Also, if possible, to acquire a man to manage that
department of our business, someone who is an expert in that line and who at
the same time is fitted to deal with our rather exclusive clientele. It occurs to
me that you might be that man, if you would care to consider it."

Rodrigo did not reply at once. He took three or four steps in silence,
thoughtfully, away from Dorning. Go to America! Enter business! He
recalled the deprecatory manner in which his father had always talked about
business and the great relief it had been for the elder Torriani to leave the
Indian trade and settle down at last to be a gentleman again. And he was very
much like his father in so many ways. The business of John Dorning, to be
sure, was art, something he, Rodrigo, loved. It was not like the mad
commercial scramble of ordinary trade. There was nothing commercial
about Dorning. Something within Rodrigo said "Go." Something in
Dorning's offer was lifting off his mind the almost physical weight that
oppressed him every time he considered the future.

"I will accept your offer and return with you to America," Rodrigo said
with quiet suddenness.

John Dorning started. He had not suspected such a quick and decisive
answer. "Fine," he said. "Can you arrange your affairs to sail with me next
week on the Italia?"

Rodrigo was sure that he could. Now that he was committed to the
plunge, he was positively gay about it. The two young men spent the rest of
the day talking the arrangements over. In the afternoon they journeyed in to
Naples in Rodrigo's car and entered an agreement with the fussy Italian real
estate agent to rent the palace of the Torrianis to the family of a young
American author who had just made a fortune out of a best-selling novel and
wished to write its sequel along the romantic shore of the Bay of Naples.

CHAPTER IV

The great floating hotel glided steadily ahead over the smooth, black
waters of the Mediterranean. Somewhere within her hull, boiler fires were
roaring and a labyrinth of machinery was driving furiously, but only a slight,
muffled throb reached the ears of the lone passenger standing at the rail
directly under the bridge. Over his head he could hear the regular tread of
the watch officer as he paced his monotonous round. In front of him was the
dark immensity of the night, broken only when he lowered his eyes to take
in the lights from the port-holes and the jagged streaks of phosphorescence
streaming back from the bow as it cut the water.

Rodrigo was quite happy. His ripening friendship with Dorning, the new
clean life into which every minute of the ship's progress was carrying him,
the cool, damp darkness that surrounded him, added to his content. He
snapped his cigarette into the Mediterranean and with a peaceful sigh walked
into the crowded, brilliantly lighted saloon in search of his friend.

The waiter was standing expectantly at Dorning's table, while Dorning,

menu card in hand, was looking about for Rodrigo. Another man sat at the
table with the American, a small, nervous, middle-aged man, who was also
fingering a menu.

"I feared you had changed your mind and leaped overboard or
something," Dorning smiled as Rodrigo approached. "I want you to meet Mr.
Mark Rosner, Rodrigo. Mr. Rosner—Count Torriani." Rodrigo bowed and
slid into his place at the table.

"Mr. Rosner is an old friend of my father's," Dorning explained. "We met

by chance at the door of the saloon."

Rosner elaborated upon the explanation in a rapid, clipped voice. "I

worked for Dorning and Son for a long time, Count Torriani. I left them five
years ago to open a shop of my own in London. I did rather well, but you
know how it is—once an American, always an American. There is no town
in the world like New York. I sold out my place in London six months ago.
Since then I have been traveling in Italy acquiring a stock, and I am on my
way back to New York to go into business there."

He directed his conversation toward Rodrigo, evidently awed a bit by the

young Italian's title and reserved manner and anxious to make an impression.
Mark Rosner was a rare Jewish type, an impractical æsthete who disliked
business life intensely but who nevertheless was consumed by the urge to
make money. The struggle had whitened his mop of unruly hair prematurely,
stooped his fat shoulders, and worn his nerves to ragged edges. The truth
was that his London venture had been a failure and his new stock had been
bought in Italy on borrowed capital. His delight at meeting John Dorning
again had been partly caused by genuine pleasure at coming upon the son of
a man he had always liked and admired and partly by the thought that he
might derive aid later from the Dornings in getting started in New York.

"Count Torriani is to become associated with us in New York," Dorning

remarked when the waiter had departed with the three orders. Dorning, now
that Rodrigo had arrived, would rather the third party were not present. He
remembered Rosner as a valuable employee, but as one who was always
timid in taking responsibility and evasive and whining when things went
badly. However, he was too kind-hearted to snub the fellow.

Rosner replied in his jerky voice, "Really? You couldn't join a concern
with a finer reputation, Count Torriani. Dorning and Son are the leaders in
their line in New York, as you probably know. Sometimes I wish I had never
left your father, John." Dorning secretly smiled at Rosner's sudden
familiarity. "But you know how it is—there is a certain satisfaction in being
on your own, in spite of the risk involved."

He went on to relate in considerable detail the difficulties that had beset

his venture in London. In the midst of his recital the food arrived. Rodrigo
and John Dorning, who were hungry and bored, fell to at once and heard
only snatches of the remainder of Rosner's querulous discourse. Englishmen
of the art world, according to Rosner, were prejudiced against Americans in
the same line of business, particularly Americans of Semitic extraction. He
gave instances of alleged discriminations against him.

"I don't suppose, though, that it's much different in New York," Rosner
admitted. "I remember many of the old-line concerns were against foreigners
there too, and I don't suppose it has changed much. I recall how Henry
Madison opposed your father's taking on that Italian sculptor, Rinaldi, and
how pleased he was when the chap fell down and had to be let out. You were
there then, weren't you, John?"

John did not look over-pleased. "Rinaldi was not the man for the job," he
said with a frown. "My father was carried away with his enthusiasm for the
man's work in clay. Rinaldi was no good out of his studio, and Madison
quickly recognized it. The fact that Rinaldi was a foreigner had nothing to do
with the matter."
 Rodrigo now listened with interest for the first time since he had sat
down at the table. He foresaw that his career with Dorning and Son might
not prove as unruffled as he had anticipated. This did not greatly annoy him.
He had little of the eccentric artistic temperament, and there was enough of
the merchant blood in him to enable him to adapt himself to office work. At
least, he hoped so. If obstacles arose, he would overcome them.

"Who is Mr. Madison?" Rodrigo asked politely.

"He is the manager of our establishment," John explained. "There is no

cause for alarm, Rodrigo. He is the most honest, fairest person alive."

Rosner, glancing furtively from one of his tablemates to the other, sensed
that he had rather put his foot into it. Why had he not remembered that
Count Torriani was a foreigner? He flushed with embarrassment and, to
change the subject, asked John, "Is your father still active in the business?"

Dorning's sensitive face clouded. He answered, "No, my father has not

been in very good health for the past year or so. He is staying at our place at
Greenwich and only gets down to the office once or twice a month."

"Then you have charge?"

"Yes—with the able assistance of Madison and the rest of our staff. It
isn't a very difficult job, as you can imagine. The long-standing reputation of
Dorning and Son and the organization my father built up don't leave a very
great deal for the head of the concern to do."

"All the same, it's quite a responsibility for a young fellow only a few
years out of college, John, and I congratulate you." What there was of
shrewdness in Mark Rosner now showed in his dark, ineffective eyes. Young
Dorning was evidently kind-hearted, and, of necessity, inexperienced. An
appeal to him for assistance by an old employee of his father's would
probably meet with a favorable response.

After dinner the two younger men contrived to rid themselves of

Rosner's company temporarily on the plea that they wished to unpack their
bags. Having accomplished this task, they drifted into the smoking-room,
where the card players were already hard at it. Waiters were running here
and there with tinkling glasses. The air was hazy with the smoke of many
cigarettes and cigars.

A corpulent gentleman with the wine-ruddy face and expansive clothes

and manners of a London theatrical producer, as indeed he was, approached
the two friends as they stood surveying the scene. "Would you two
gentlemen care to make up a table at bridge?" he asked.

Bridge was John's favorite diversion. He played a careful, serious-

minded game for pleasure rather than for profit. He looked suggestively at
Rodrigo, who shrugged affirmatively. The Italian would have been happier
at baccarat or some other continental game which moved more quickly than
bridge. But he was willing to please, and it occurred to him that his funds
would not permit his participation in baccarat as played in this smoking-
room, for a few moments' observation had shown him that the stakes were
very high.

The red-faced Englishman guided them over to a table near the stairway.
A gaunt, pale, long-haired man was already seated there, surrounded by
three tipped-up chairs. He was idly shuffling the cards and dropped them to
rise as his companion reappeared. The introductions revealed that the stout
Englishman was Gilbert Christy, producer of the Christy Revues, which
Rodrigo was familiar with as elaborate girl-and-music shows relying upon
well-drilled choruses and trick stage effects rather than cleverness for their
success. The lean Englishman was Clive Derrick, leading man in Christy's
current show. The Christy Revue was transporting itself overseas, after a
brief and rather unremunerative engagement at Rome and Naples, to try its
luck on Broadway.

"André Chariot has been filling his pockets in America," boomed

Christy, whose voice was as loud as his vest. "Why not I?"

Rodrigo agreed that the chances were excellent, being too polite to
explain that Charlot's divertissements were clever, while Christy was about
to offer America something which Ziegfeld and other native New York
producers were already doing better than anybody else in the world.