Rolling circle amplification (RCA)

Vadim V. Demidov Editor

Rolling Circle
Amplification
(RCA)
Toward New Clinical Diagnostics and
Therapeutics


Rolling Circle Amplification (RCA)


Vadim V. Demidov
Editor

Rolling Circle
Amplification (RCA)
Toward New Clinical Diagnostics
and Therapeutics


Editor
Vadim V. Demidov
Global Prior Art Inc.
Boston, MA, USA

ISBN 978-3-319-42224-4
ISBN 978-3-319-42226-8
DOI 10.1007/978-3-319-42226-8

(eBook)

Library of Congress Control Number: 2016955094
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To Andrew Fire, Eric Kool, Paul Lizardi,
David Zhang, Ulf Landegren, Jerry Ruth,
David Driver and Jeff Auerbach, who were
the first few to realize the great potential of
rolling replication of small DNA circles, and
who introduced this innovative methodology
to bioresearchers in the 1990s, therefore
laying the foundations of current RCA
technology

Advance Praise for Rolling Circle
Amplification (RCA)

“The ability to amplify nucleic acids in the test tube has revolutionized nearly every aspect
of research and diagnostics based on genetic material. For the past few decades the polymerase chain reaction (PCR) has been the dominant amplification method. However
because PCR requires thermal cycling it is difficult to adapt to some important experimental applications. A few different approaches to circumvent the need for thermal cycling have
been developed but most of these have failed to gain widespread adoption. The exception
is rolling circle amplification (RCA) which has matured into a widely used powerful alternative to PCR for many applications. This book provides a badly needed compendium of
innovative RCA methods and applications. It should help further increase the community
of scientists that have employed RCA in research and diagnostic programs.”
—Charles Cantor
Professor Emeritus of Biomedical Engineering, Boston University
Executive Director, Retrotope Inc. (USA)
“When I came in early 1990s upon the publications of Margarita Salas and coworkers on
the mechanism of circular DNA replication by bacteriophage phi29 DNA polymerase I
became immediately enamored with the idea that one could build DNA nanomachines to
perform interesting and useful tasks. Twenty years have elapsed, and many other colleagues
in the field have come to share my love of biomolecular complexes that run on DNA circles
and can stand on surfaces to do their work, or perhaps generate particles able to penetrate
into cells to perform more ambitious tasks. In this new book Vadim Demidov has assembled an enticing menu of articles that illustrate the evolution of the RCA field, including
improved protein parts for building superior DNA nanomachines, enhanced modalities of
amplification and detection, diagnostic applications, and even a sampling of potential therapeutic applications. The reader will appreciate that while RCA has come of age, there is no
lack of exciting surprises, turns, and twists in the continuing evolution of the technology.”
—Paul Lizardi
Professor of Pathology, Yale University School of Medicine (retired)
Investigator, University of Granada, Spain
President, PetaOmics, Inc., San Marcos, Texas

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Advance Praise for Rolling Circle Amplification (RCA)
“The rolling circle concept for isothermal amplification is appealingly simple and has been
adopted widely. This timely book details some of the promising applications under development that the technology makes possible.”
—Eric Kool
George and Hilda Daubert Professor of Chemistry, Stanford University, USA
“Rolling circle amplification offers some unique advantages in molecular biotechnology
and molecular medicine. This book is a valuable occasion to illustrate the range of opportunities this creates before a wide readership, hopefully stimulating more to take advantage
of this exciting technology.”
—Ulf Landegren
Professor of Molecular Medicine, Uppsala University, Sweden
“RCA is one of the excellent signal amplification technologies. Several chapters of this
opportune book demonstrate that when other biochemical tools are combined with RCA, it
transforms into even a more effective method—it’s worth reading and learning this.”
—Tsugunori Notomi, Ph.D.
Executive Officer and General Manager
R&D Division, Eiken Chemical Co., Ltd., Japan
“Since its discovery in the 1990s, RCA has become a highly versatile, widely used DNA
amplification tool in many fields where limitations of sensitivity, specificity, or laborious
sample preparation and/or signal amplification procedures had previously hindered applications using other tools. These applications now include assays and procedures in fields such
as immunohistochemistry, nanotechnology, genomics, proteomics, biosensing, drug discovery, flow cytometry, etc. RCA greatly extends the utility of these methods and Vadim
Demidov has introduced a very valuable resource with this book which is the first book
entirely devoted to RCA technology although RCA has been around for over 20 years!”
—Filiz M. Aslan, Ph.D.
Technology and Innovation Development Office (TIDO)
Boston Children’s Hospital, Harvard Medical School

Preface

In 2005, I wrote the editorial article entitled “10 years of rolling the minicircles:
RCA assays in DNA diagnostics” [Expert Rev Mol Diagn 5(4):477–478], where I
have summarized significant achievements of the first decade of rolling circle
amplification (RCA) technology in identification of pathogens, oncogenes, hot spot
mutations, and SNPs, as well as in multiplexed genomics and proteomics profiling
with microarrays. A year before, I have compiled and edited the book published by
Horizon Bioscience that covered existing DNA amplification techniques, with several chapters being devoted to various innovative diagnostic methods involving
RCA. Since then, another decade has passed, and continuing developments in the
RCA-based diagnostics become more and more capable and more close to real-life
applications.
I have worked with RCA for several years, which resulted in a number of related
research and review publications, some of which are referenced in the aforementioned editorial. And though I left the research bench a while ago by switching to the
intellectual property consulting business, I have kept an eye on the RCA innovations, and I am pleased to see a great progress in the RCA field toward the development of new clinical diagnostics. I am also excited about the recently discovered
RCA pharmaceutical capabilities. All this prompted me to compile the book presenting these new RCA achievements.
To my knowledge, this is the first book devoted entirely to the RCA technology,
and it intends to present the current state of the art of this technology related to
nucleic acid diagnostics with the major focus on clinically relevant applications.
Notably, the RCA technology is now extending beyond the field of molecular diagnostics (where new robust RCA methods and sensors have been developed that are
presented in the corresponding “diagnostic” section of this book) into the area of
drug delivery vehicles and nucleic acid drugs. In accord with that, a section of this
book is devoted to prospective RCA-based therapeutics. Two other sections cover
new enzymes useful in RCA and RCA-involving techniques with enhanced signal
amplification.
Note that with exception of the first chapter, all other chapters deal with RCA of
small, ≤50-nt-long DNA circles since only short circularized DNAs serve as
ix

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Preface

convenient probes in the RCA-based diagnostics and/or as templates in the RCAbased therapeutics. The stand-alone chapter describes the engineered DNA polymerases with enhanced abilities for RCA of long circular DNAs in genomics and
sequencing protocols. And I decided to present these innovations just to illustrate
the possible ways of RCA improvements and also to encourage testing these new
polymerases in diagnostic RCA.
To those readers who may consider the contents of this book as somewhat patchy,
I would say that according to PubMed database, nearly 800 articles related to RCA
have been presently published, with more than 100 articles being published last
year. Hence, it was inevitable for me to focus the first book on RCA to a certain area,
and I chose the area of clinical diagnostics and prospective therapeutics as the one
more close to my expertise.
Therefore, although exciting, certain RCA-related topics are out of the scope of
this book, such as the RCA applications in DNA nanotechnology, RCA-derived
sequencing templates and cell-free RCA cloning, or the use of RCA to introduce
random mutations. But even so, with several dozen interesting studies performed
and published in recent years on RCA-based diagnostics and emerging therapeutics,
it was a tough task for me to choose among them for a reasonable, and rather limited, number of chapters.
In my opinion, the choice of topics covered in this book is sufficient to show the
wealth of ideas in this particular area of RCA research. Still, I would like to express
my sincerest apologies to those researchers developing innovative RCA diagnostics
and therapeutics, who are not presented in this book.
Given the great progress achieved in the clinically related diagnostic RCA applications and in the RCA sensors field, as it is presented in this book, I anticipate that
commercial RCA-based diagnostic kits and sensors will soon be on the market
[to the best of my knowledge, no commercial RCA diagnostics are so far available].
I also believe that some RCA-based drugs will enter preclinical trials in not so distant future.

My other dream is that the contents of this book would stimulate novel promising developments in the RCA field. So, I am glad with the opportunity given to me
to compile and edit this book. And I am very grateful to all contributors and to the
Springer editors and production managers who made this book possible.
Boston, MA

Vadim V. Demidov


Contents

1

Introduction: 20+ Years of Rolling the DNA
Minicircles—State of the Art in the RCA-Based Nucleic
Acid Diagnostics and Therapeutics .......................................................
Vadim V. Demidov

Part I
2

3

4

5

6

Improved DNA Polymerases and New DNA
and DNA/RNA Ligases Useful in RCA

Improvement of ϕ29 DNA Polymerase Amplification
Performance by Fusion of DNA Binding Motifs ..................................
Miguel de Vega, José M. Lázaro, and Margarita Salas
Preparation of Circular Templates by T4 RNA Ligase 2
for Rolling Circle Amplification of Target microRNAs
with High Specificity and Sensitivity.....................................................
Yifu Guan, Bin Zhao, Guojie Zhao, Chidong Xu, and Hong Shang
Use of DNA CircLigase for Direct Isothermal Detection
of Microbial mRNAs by RNA-Primed Rolling Circle
Amplification and Preparation of ø29 DNA Polymerase
Not Contaminated by Amplifiable DNA ...............................................
Hirokazu Takahashi, Yoshiko Okamura, and Toshiro Kobori

Part II

1

11

25

37

RCA-Involving Techniques with Enhanced Signal Amplification

Nicking Enzyme-Assisted Branched-Chain RCA Reaction
for Cascade DNA Amplification.............................................................
Xiaoli Zhu, Chang Feng, and Genxi Li

49

Combining Isothermal Amplification Techniques: Coupled
RCA-LAMP .............................................................................................
Laura E. Ruff, Jessie-Farah Fecteau, Dina Uzri, and Bradley T. Messmer

57

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xii

Contents

Part III
7

8

9

Emerging RCA Diagnostics

Detection of Vascular Disease-Related Single Nucleotide
Polymorphisms in Clinical Samples Using Ramified Rolling
Circle Amplification ................................................................................
James H. Smith and Thomas P. Beals

67

Ultrasensitive Isothermal Detection of Protein Analytes
Using Rolling Circle Amplification in Microscale Platforms ..............
Saheli Sarkar, Pooja Sabhachandani, and Tania Konry

85

Rolling Circle Amplification with Padlock Probes
for In Situ Detection of RNA Analytes ..................................................
Anja Mezger, Malte Kühnemund, and Mats Nilsson

99

10

PNA-Assisted Rolling Circle Amplification for Detection
of DNA Marker Sequences in Human Cells ......................................... 107
Anastasia I. Gomez and Irina V. Smolina

11

Sensor Systems with Magnetic and Optomagnetic Readout
of Rolling Circle Amplification Products .............................................. 123
Mikkel F. Hansen, Marco Donolato, Jeppe Fock, Mattias Strömberg,
Maria Strømme, and Peter Svedlindh

Part IV Prospective RCA-Based Therapeutics
12

DNA Nanoclews for Stimuli-Responsive Anticancer

Drug Delivery .......................................................................................... 141
Wujin Sun and Zhen Gu

13

RCA-Assisted Self-assembled DNA Origami Nano-constructs
as Vehicles for Cellular Delivery of Diagnostic Probes
and Therapeutic Drugs ........................................................................... 151
Shiping Song and Chunhai Fan

14

DeNAno: A Novel Multivalent Affinity Reagent Produced
by Selection of RCA-Generated DNA Nanoparticle Libraries ........... 161
Laura E. Ruff, Dina Uzri, Jessie-F. Fecteau, Mehmet Hikmet Ucisik,
and Bradley T. Messmer

Index ................................................................................................................. 169


About the Editor

Vadim V. Demidov, Ph.D. is a Senior Analyst in the
Biotechnology & Pharmaceuticals Group at Global
Prior Art, Inc. (Boston, USA), an intellectual property
research and analysis firm.
Demidov received his M.S. degree in Physical/
Chemical Engineering from Moscow Institute of Physics
and Technology (MIPT, a leading Russian technical university, aka “the Russian MIT”) and his Ph.D. degree in
Biophysics from the Institute of Molecular Genetics of

the Russian Academy of Sciences (IMGRAS) and MIPT. Before joining the Global
Prior Art company in 2008, he has worked for almost 30 years in academia and
industry worldwide, serving lately as a research professor and senior scientist at
prestigious institutions, such as Moscow Institute of Biotechnology and Institute of
Molecular Genetics (Russia), Copenhagen University (Denmark), and George
Mason University and Boston University (USA).
Demidov is well known in the molecular biology and biotechnology field for his
innovative studies related to peptide nucleic acid (PNA). He has introduced the use
of PNA openers for the detection of specific sequences within DNA duplexes under
non-denaturing conditions, with some of these applications involving rolling circle
amplification (RCA).
During his research career, Demidov has published over 50 peer-reviewed
research papers, of which 5 are related to RCA technology. In addition, he holds
several US and international patents on nucleic acids biotechnology and environmental monitoring. He has presented at many international conferences, wrote several review articles and book chapters, including those on RCA technology, and
published the book entitled DNA Amplification: Current Technologies and
Applications (Horizon Bioscience, 2004).
Demidov also served as the Editorial Board member for Trends in Biotechnology,
Expert Review of Molecular Diagnostics, Expert Opinion on Medical Diagnostics,

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About the Editor

Current Medicinal Chemistry, and Open Medicinal Chemistry. Dr. Demidov was
awarded Silver Medal from All-Union National Exhibition of Economic
Achievements (Moscow, USSR, 1988) and Medal of Honor from International
Biographical Centre (Cambridge, UK, 2007). He is listed in several international

biographical directories, including Who’s Who in Science and Engineering, Who’s
Who in Medicine and Healthcare, Who’s Who in America, Who’s Who in the World,
and Dictionary of International Biography.


Contributors

Thomas P. Beals Research and Development, Thorne Diagnostics, Beverly,
MA, USA
Vadim V. Demidov, Ph.D. Global Prior Art, Inc., Boston, MA, USA
Marco Donolato, Ph.D. BluSense Diagnostics ApS, Copenhagen Ø, Denmark
Chunhai Fan Division of Physical Biology, Shanghai Institute of Applied Physics,
Chinese Academy of Science, Jiading, Shanghai, China
Jessie-Farah Fecteau, Ph.D. Research and Development, Abreos Biosciences,
San Diego, CA, USA
Chang Feng State Key Laboratory of Pharmaceutical Biotechnology, Department
of Biochemistry, Nanjing University, Nanjing, China
Jeppe Fock, Ph.D. Department of Micro- and Nanotechnology, Technical
University of Denmark, DTU Nanotech, Kongens Lyngby, Denmark
Anastasia I. Gomez Department of Biomedical Engineering, Boston University,
Boston, MA, USA
Zhen Gu, Ph.D. Joint Department of Biomedical Engineering, University of North
Carolina at Chapel Hill and North Carolina State University, Raleigh, NC, USA
Division of Molecular Pharmaceutics and Center for Nanotechnology in Drug
Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel
Hill, Chapel Hill, NC, USA
Department of Medicine, University of North Carolina School of Medicine, Chapel
Hill, NC, USA
Yifu Guan, Ph.D. Department of Biochemistry and Molecular Biology, China
Medical University, Shenyang, Liaoning, China

xv


xvi

Contributors

Mikkel F. Hansen, Ph.D. Department of Micro- and Nanotechnology, Technical
University of Denmark, DTU Nanotech, Kongens Lyngby, Denmark
Toshiro Kobori, Ph.D. (Life Science) NanoBiotetchnology Laboratory, Food
Engineering Division, National Food Research Institute, National Agriculture and
Food Research Organization, Tsukuba, Ibaraki, Japan
Tania Konry, Ph.D. Department of Pharmaceutical Sciences, Northeastern
University, Boston, MA, USA
Malte Kühnemund Science for Life Laboratory, Department of Biochemistry and
Biophysics, Stockholm University, Solna, Sweden
José M. Lázaro Instituto de Biología Molecular “Eladio Viñuela” (CSIC), Centro
de Biología Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autónoma,
Madrid, Spain
Genxi Li School of Life Sciences, Shanghai University, Shanghai, China
Bradley T. Messmer, Ph.D. Moores Cancer Center, University of California, San
Diego, La Jolla, CA, USA
Abreos Biosciences, San Diego, CA, USA
Anja Mezger Science for Life Laboratory, Department of Biochemistry and
Biophysics, Stockholm University, Solna, Sweden
Mats Nilsson Science for Life Laboratory, Department of Biochemistry and
Biophysics, Stockholm University, Solna, Sweden
Yoshiko Okamura, Ph.D. (Engineering) CREST, Japan Science and Technology
Agency, Higashihiroshima, Hiroshima, Japan

Graduate School of Advanced Sciences of Matter, Hiroshima University,
Higashihiroshima, Hiroshima, Japan
Laura E. Ruff, Ph.D. Moores Cancer Center, University of California, San Diego,
La Jolla, CA, USA
Pooja Sabhachandani Department of Pharmaceutical Sciences, Northeastern
University, Boston, MA, USA
Margarita Salas Instituto de Biología Molecular “Eladio Viñuela” (CSIC), Centro
de Biología Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autónoma,
Madrid, Spain
Saheli Sarkar, Ph.D. Department of Pharmaceutical Sciences, Northeastern
University, Boston, MA, USA
Hong Shang Key Laboratory of AIDS Immunology of National Health and Family
Planning Commission, Department of Laboratory Medicine, The First Affiliated
Hospital, China Medical University, Shenyang, Liaoning, China
James H. Smith, B.Sc. (Hons.), Ph.D. Research and Development, Thorne
Diagnostics Inc., Beverly, MA, USA


Contributors

xvii

Irina V. Smolina Department of Biomedical Engineering, Boston University,
Boston, MA, USA
Shiping Song Division of Physical Biology, Shanghai Institute of Applied Physics,
Chinese Academy of Science, Jiading, Shanghai, China
Mattias Strömberg, Ph.D. Division of Solid State Physics, Department of
Engineering Sciences, Uppsala University, The Ångström Laboratory, Uppsala,
Sweden
Maria Strømme, Ph.D. Division of Nanotechnology and Functional Materials,

Department of Engineering Sciences, Uppsala University, The Ångström Laboratory,
Uppsala, Sweden
Wujin Sun, M.S. Joint Department of Biomedical Engineering, University of
North Carolina at Chapel Hill and North Carolina State University, Raleigh,
NC, USA
Division of Molecular Pharmaceutics and Center for Nanotechnology in Drug
Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel
Hill, Chapel Hill, NC, USA
Peter Svedlindh, Ph.D. Division of Solid State Physics, Department of Engineering
Sciences, Uppsala University, The Ångström Laboratory, Uppsala, Sweden
Hirokazu Takahashi, Ph.D. NanoBiotetchnology Laboratory, Food Engineering
Division, National Food Research Institute, National Agriculture and Food Research
Organization, Tsukuba, Ibaraki, Japan
CREST, Japan Science and Technology Agency, Higashihiroshima, Hiroshima, Japan
Department of Molecular Biotechnology, Graduate School of Advanced Sciences
of Matter, Hiroshima University, Higashihiroshima, Hiroshima, Japan
Mehmet Hikmet Ucisik, Ph.D. Department of Biomedical Engineering, School of
Engineering and Natural Sciences, Istanbul Medipol University, Istanbul, Turkey
Dina Uzri, Ph.D. Research and Development, Abreos Biosciences, San Diego,
CA, USA
Miguel de Vega, Ph.D. in Biology Genome Dynamics and Function, Centro de
Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain
Chidong Xu Center of Medical Physics and Technology, Hefei Institutes of
Physical Science, CAS, Hefei, Anhui, China
Bin Zhao Key Laboratory of National Sport Bureau, Department of Human
Movement Sciences, Shenyang Sport University, Shenyang, China
Guojie Zhao Department of Biochemistry and Molecular Biology, China Medical
University, Shenyang, Liaoning, China
Xiaoli Zhu Laboratory of Biosensing Technology, School of Life Sciences,
Shanghai University, Shanghai, China

Chapter 1

Introduction: 20+ Years of Rolling the DNA
Minicircles—State of the Art in the RCABased Nucleic Acid Diagnostics
and Therapeutics
Vadim V. Demidov

1

The RCA Concept and Plausible Mechanism

The idea of using DNA minicircles for isothermal amplification of specific nucleic
acid sequences originated in the pioneering investigations of several researchers
who filed independent patent applications in the early and mid-1990s (Ruth and
Driver 1992; Auerbach 1994; Kool 1998; Lizardi 1998; Zhang et al. 1999). Although
these researchers focused on different aspects of the technology (for example, its
ability to mediate target vs. signal amplification, or its exquisite sensitivity), their
cumulative efforts resulted in the development of the seminal concept of RCA,
which was thoroughly validated and further advanced in the subsequent proof-ofprinciple studies (Fire and Xu 1995; Liu et al. 1996; Lizardi et al. 1998; Zhang et al.
1998; Banér et al. 1998).
The RCA principle is based on the fundamental property of a circle, i.e., the endlessness of a circular line. For a ring of DNA copied by DNA polymerase, this feature of circles means that the polymerase enzyme, if initiated by a primer at some
point on circular DNA, will generate a single-stranded DNA concatemer by moving
around DNA minicircle and repeatedly synthesizing its linear replicas (see Fig. 1.1a)
until the process is terminated by some reasons. In fact, this principle is successfully
used by living cells in replication of circular plasmids and viral genomes (Gilbert
and Dressier 1968; Baker and Kornberg 1992), but its workability is not so obvious
in case of small DNA circles. Indeed, the intuitive representation of rolling circle
mechanism shown in Fig. 1.1a and adopted in essentially all publications dealing

with RCA reactions (including the chapters of this book) cannot actually be real,
though it suitably depicts the RCA principle and the outcome of RCA reaction. This
is due to the known fact that it is very difficult to form circles of double-stranded

V.V. Demidov (*)
Global Prior Art, Inc., 21 Milk St., Boston, MA 02109, USA
e-mail:
© Springer International Publishing Switzerland 2016
V.V. Demidov (ed.), Rolling Circle Amplification (RCA),
DOI 10.1007/978-3-319-42226-8_1

1


2

V.V. Demidov

Fig. 1.1 Schematics of the RCA process. (a) The commonly accepted view of RCA reaction that
is going on a free DNA minicircle with the use of a single primer. Strand-displacement ability of a
DNA polymerase is assumed in this case. (b) More realistic representation of the RCA-generating
complex where N represents nucleotides in the DNA minicircle (could be A,T,C,G) and C represents complementary nucleotides in the RCA-generated DNA strands (could be T,A,G,C). Given a
small, ≤100-nt size of DNA minicircles used in RCA and the strong rigidity of duplex DNA fragments with these lengths, only part of the circular probe can be base paired at any given time
(shown as :). Symbols ▬►and >> represent the DNA polymerase and unraveling of DNA
duplex, respectively. The right curved arrow indicates rotation of circular DNA template in forward direction to allow extension of nascent DNA strand, whereas left curved arrow illustrates
turning motion of the template, which serves to relax twisting that arises at the site of DNA
synthesis. Bold letters at the end of nascent DNA strand represent sequence of a primer, which is
used to initiate the RCA process

1

Introduction: 20+ Years of Rolling the DNA Minicircles…

3

DNAs shorter than 150–200 bp because of enormous rigidity of such short DNA
duplexes (Ulanovsky et al. 1986; Hagerman 1988; Koo et al. 1990). Therefore, it is
highly unlikely to form a stable duplex all the way around a ≤100-nt-long DNA
circle (which is a typical size used in RCA) because of the tight radius of curvature.
The more plausible RCA mechanism is shown in Fig. 1.1b: the newly synthesized DNA strand is not hybridized completely around the circle during its enlongation since only about a half of DNA minicircle can be involved in a linear duplex
formed with the product DNA strand. The replication thus proceeds on a remaining single-stranded part of a circle, with DNA polymerase working to extend a
short straight double-stranded region, and polymerase can continue this process
obviously only if there is unwinding behind the polymerase (Fire and Xu 1995;
Liu et al. 1996).
Such an unwinding could be spontaneous as the result of thermal fluctuations
leading to fast transient unzippering at the end of DNA helix (Lazurkin et al. 1970;
Porschke 1974; Lukashin et al. 1976). Alternatively, the RCA-capable DNA polymerases could have a kind of associated helicase activity (Sakurai et al. 1993) and
the energy for both polymerization and helicase activity of these enzymes would be
derived from utilization by DNA polymerase of nucleoside triphosphates during the
RCA reaction (Fire and Xu 1995). Accordingly, most likely the actual shape and
performance of a replicating complex during the RCA processes resembles the
movement of a caterpillar track on the road which is synchronously created in front
of the track, as it moves ahead (Fig. 1.1b).
One more unusual feature of the RCA process comes from the fact that the
nascent DNA strand forms the right-handed helix with the replicated strand of a
DNA minicircle. This means that twist in the growing DNA helix at the site of DNA
synthesis requires relative rotation between DNA polymerase and replicated DNA
strand. In case of linear DNAs and large DNA circles, there is no obstacle for such
rotations, but the ≤100-nt-long DNA circles could not encircle any RCA-active

DNA polymerase which all have diameters larger than that of DNA circles. This
evidently precludes circling of the DNA polymerase around the DNA strand (as
well as circling of the DNA minicircle around the DNA polymerase) since the bulky
enzyme cannot physically pass through the small circular DNA template.
Due to such a topological constraint, the polymerase enzyme should act essentially as a fixed surface while the DNA template must continually twists inward on
itself (Fire and Xu 1995). This is possible since about a half of DNA minicircle
remains in a single-stranded form (see Fig. 1.1b) so it can turn itself “inside out” by
freely rotating around phosphodiester bonds, thus relaxing any arising twisting.
Accordingly, the combined forward and twisting motion of the template would provide the necessary constant interface between polymerase and template (Fire and
Xu 1995; Liu et al. 1996). It is also expected that the duplex unwinding behind the
polymerase complex counteracts the emerging DNA winding (Liu et al. 1996).
As a result of all these restraints, DNA polymerization in the course of RCA reaction proceeds ~10 times slower relative to that on linear templates (Liu et al. 1996).
Still, this rate is high enough to generate 102–103 repeats of the complementary to
minicircle sequence in less than an hour (Liu et al. 1996; Lizardi et al. 1998). Besides,


4

V.V. Demidov

RCA amplicons can be readily involved into other isothermal DNA amplification
schemes to devise assays with branched cascade of reactions, thus enabling to amplify
the probe DNA sequences ≥109-fold at a single temperature (Lizardi et al. 1998;
Kühnemund et al. 2014 and chapters in Part II of this book). Real-time detection of
branched RCA reactions is also possible (Smolina et al. 2004).

2

Unique Advantages Offered by RCA to Molecular
Diagnostics and Molecular Medicine

RCA reactions have several distinctive features that offer unique advantages to the
RCA-based molecular diagnostics (see Table 1.1 and chapters in Part III of this
book). Most important is that RCA has no need in costly instrumentation to cycle
the temperature, as it is required with the widely used PCR-based DNA diagnostics.
This is especially beneficial during epidemics in low-income regions, when thousands of samples must be rapidly tested (Rodrigues et al. 2015). Furthermore, by
some simple methods RCA amplicons can be visualized by naked eye and/or
recorded by a camera phone without involvement of any detector probes (Xie et al.
2014), which is also beneficial for design of low-cost diagnostics and allows expedient remote analysis of diagnostic data.
RCA amplicons also have certain features that are favorable for the development
of RCA-based drugs. For instance, the concatemeric nature of RCA amplicons
makes it possible to generate the drug-loaded polyvalent reagents that specifically
bind the intended surface-exposed targets on cancer cells and kill them (Zhang et al.
2013). Moreover, special selection process produces RCA-based multivalent
reagents with extremely high avidity to unknown targets exposed on surface of
specific cells (see chapter by Ruff et al. in Part IV of this book). In addition, RCA
amplicons can be readily converted into compact nanoparticles loaded with therapeutic and sensing substances, which are able to penetrate into live cells and to
Table 1.1 Distinctive RCA features beneficial for molecular diagnostics
•

•

•
•
•

Isothermal nature of RCA reaction and its appealingly simple mechanism entails a low cost
of RCA diagnostics and makes them well amenable for miniaturization and automation in
high-throughput analyses
RCA reaction generates ultra-long single-stranded concatemers of DNA repeats, which is

favorable for detection of RCA amplicons with a variety of labeled hybridization probes, as
well as for their non-instrumental and label-free detection
RCA capacity to directly yield the surface-bound amplification products offers significant
advantages to in situ- or microarray-based diagnostic assays
RCA reactions with padlock probes exhibit an excellent sequence specificity that is favorable
for genotyping or mutation detection
RCA reactions are well resistant to many contaminants, which allows detection of target
DNA and RNA molecules in crude complex mixtures also containing large excess of host
nucleic acids


1

Introduction: 20+ Years of Rolling the DNA Minicircles…

5

deliver these materials inside target cells (Hamblin et al. 2012; Chen et al. 2015a
and the first two chapters in Part IV of this book). In this connection, it is worth to
note that the RCA-like process can be performed with DNA minicircles by RNA
polymerases to generate long concatemeric RNAs (Daubendiek et al. 1995), which
could serve both as drug carriers and as a therapeutic cargo (Seyhan et al. 2006; Lee
et al. 2012). Also, it was recently reported that protein-encoding circular RNAs can
be efficiently translated by the RCA-like process to produce abundant protein products both in vitro and in vivo (Abe et al. 2013, 2015).

3

Optimistic View of the Long-Term RCA Prospects
in Biomedical Fields

Until recently, PCR was the primary technique of DNA amplification in the area of
DNA diagnostics and it still remains one of the most useful methods for DNA analysis. But PCR requirement for high-precision temperature cycling makes it difficult
to adapt PCR to a number of important diagnostic applications, therefore strongly
justifying a quest for robust isothermal alternatives.
RCA methodology stands alone among a few other isothermal amplification
techniques because it was able to mature, after a large number of basic and applied
studies, into a widely used powerful alternative to PCR for many applications
whereas other such techniques have failed to gain widespread adoption. Figure 1.2
shows the dynamics of publications dealing with various RCA-based techniques

Fig. 1.2 Dynamics of RCA publications presented as the number of articles published in the corresponding 5-year period (data from PubMed database)


6

V.V. Demidov

and revealing quasi-exponential growth in the number of such publications since
RCA inception till present time.
Note that this graph corresponds to publications related not only to biomedical
RCA applications since they are now extending to DNA sequencing, cell-based and
cell-free cloning, molecular nanotechnology, and even to chemical sensing (see for
example Ohmichi et al. 2002; Takahashi et al. 2009; Lou et al. 2013; Wang et al.
2013; Chen et al. 2015b). Still, this burst of activity correctly represents the trend in
biomedical RCA applications, too, as the majority of current RCA-based studies are
concentrated in this area.
In view of this significant progress, I therefore expect that RCA will soon become
the method of choice for scoring SNPs and to identify biomarkers associated with
certain somatic disorders. Besides, it may be of practical use for reliable pathogen
detection at the molecular level thus helping to combat the germ-related diseases

and preventing biothreat. I also believe that some RCA-based drugs and drug delivery vehicles will enter preclinical trials in the near future, and the contents of this
book support my expectations.
After all, I would like to conclude my introductory chapter with the words of
Paul Lizardi, which he wrote in his comments on this book: “…while RCA has
come of age, there is no lack of exciting surprises, turns, and twists in the continuing
evolution of the technology.”

References
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Introduction: 20+ Years of Rolling the DNA Minicircles…

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Part I

Improved DNA Polymerases
and New DNA and DNA/RNA
Ligases Useful in RCA


Chapter 2

Improvement of ϕ29 DNA Polymerase
Amplification Performance by Fusion of DNA

Binding Motifs
Miguel de Vega, José M. Lázaro, and Margarita Salas

1

Introduction

Most of the modern genomic, phylogenetic, and epidemiological studies rely on the
amplification of tiny amounts of DNA (Demidov and Broude 2004). The DNA amplification techniques rest on the DNA synthetic properties of the DNA polymerases
from both thermophilic and mesophilic organisms that have led to the development of
a large variety of isothermal and temperature-cycling amplification protocols.
Although the polymerase chain reaction (PCR; Mullis and Faloona 1987) is still
the most widely used methodology for DNA amplification, it has two limitations:
dependence on at least a limited knowledge of the sequence to be amplified and
relatively short amplicons production. Among the alternative amplification technologies developed to yield large amounts of high quality DNA for genomic studies
were those based in the unique features of bacteriophage ϕ29 DNA polymerase,
such as the isothermal Multiple Displacement Amplification (MDA; Dean et al.
2001, 2002).
The ϕ29 DNA polymerase is the only enzyme involved in the replication of the
phage ϕ29 genome. Based on amino acid sequence similarities and its sensitivity to
specific inhibitors, ϕ29 DNA polymerase was included in the eukaryotic-type family B of DNA-dependent DNA polymerases (Bernad et al. 1987). As any other DNA
polymerase, it accomplishes sequential template-directed addition of dNMP units
onto the 3′-OH group of a growing DNA chain, showing discrimination for mismatched dNMP insertion by a factor from 104 to 106 (Esteban et al. 1993). In addition, ϕ29 DNA polymerase catalyzes 3′–5′ exonucleolysis, i.e., the release of dNMP

M. de Vega, Ph.D. in Biology (*) • J.M. Lázaro • M. Salas, Ph.D. in Chemistry (*)
Instituto de Biología Molecular “Eladio Viñuela” (CSIC), Centro de Biología
Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autónoma,
C/Nicolás Cabrera 1, Cantoblanco, Madrid 28049, Spain
e-mail: ;
© Springer International Publishing Switzerland 2016

V.V. Demidov (ed.), Rolling Circle Amplification (RCA),
DOI 10.1007/978-3-319-42226-8_2

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