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 © Springer International Publishing Switzerland 2016 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland 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 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 vii viii 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 x 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 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 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 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 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 xi 162 L.E Ruff et al Fig 14.1 DeNAno selection and target binding assessment (a) A 100-nt oligo, made up of two 20-nt primer sites and a 60-nt random region, is ligated via a 40-nt “linker” oligo (aka “splint”) This splint is also used as the initiating primer for RCA to make DeNAno particles A pool of 1010–1011 unique DeNAno particles is incubated with the target of interest in the selection process, and unbound particles are washed away Bound particles are amplified by PCR, followed by asymmetric PCR to enrich for the template strand The template strands are then re-ligated and the entire selection process is repeated several times, until binding clones dominate the pool (b) An example of a successful selection is shown DeNAno pools from each round of selection (and library) were labeled with a fluorescently labeled oligo complementary to one of the primer sites, incubated with target-coated beads, washed, and analyzed on a fluorescent plate reader The increase in fluorescence detected corresponds to the increased binding of fluorescently labeled DeNAno to the beads after every round of selection The dotted line is the background fluorescence (beads only) (c) A selected, fluorescently labeled DeNAno clone is shown binding to its target by flow cytometry Initial, non-enriched library DeNAno and target only are used as negative controls Positive clone binding is indicated by an increase in fluorescence of the target population polymerase (Fig 14.1a) Importantly, the RCA step is easily tunable, and DNA nanoparticles of different sizes can be made by altering the concentration of dNTPs in the reaction or the reaction time The resulting DeNAno particles are concatemers with complementary sequence to the circularized oligo Due to the randomized nucleotide sequence, each nanoparticle features unique 2D and 3D structures, allowing DeNAno libraries to be treated as pools of diverse binding reagents, each with the potential to bind a different target A DeNAno particle can be labeled postproduction by hybridization with complementary oligos carrying a label Examples of this include fluorescently labeled oligos and biotin-labeled oligos Direct conjugation of DeNAno particles with molecular cargo, drug, or dye is also possible DeNAno particles have the intrinsic advantage that they can be sequenced, cloned, and amplified using conventional or high-throughput DNA-related methods, and they are thus almost infinitely multiplexable 14 DeNAno: A Novel Multivalent Affinity Reagent Produced by Selection… DeNAno Selection 163 The selection method for DeNAno is similar to that used for aptamers (systemic evolution of ligands by exponential enrichment—SELEX), in which a diverse library of DeNAno is incubated with a target (cell, protein, small molecule), the target is washed to remove nonbinding particles, and the bound particles are re-amplified via PCR using the 5′ and 3′ primer sites For DeNAno, similarly selected template strands are then amplified by symmetric PCR followed by asymmetric PCR, and new DeNAno particles are generated via ligation/circularization of the enriched template strand with subsequent RCA amplification The selection process is repeated until only high-binding particles enrich and dominate the pool (selection scheme is shown in Fig 14.1a) In published papers, this occurred in 6–9 rounds of selection (Ruff et al 2014; Steiner et al 2010), but with enhancements to the process, faster selection in 3–5 rounds is now possible (L.E.R unpublished observation) A single DeNAno particle likely binds to multiple proteins or small-molecule targets on the surface of one cell While DeNAno can be incubated with cells directly, because of their low affinity for individual targets, such as proteins, these types of targets must be loaded onto a bead and presented to the DeNAno library as an aggregate Upon successful selection, there are increased amounts of DNA bound to the bead/cell, leading to increased DNA amplification during the PCR step (observed on qPCR machine by a shift in Ct) To achieve high specificity of DeNAno to its target, counterselections may also be required, involving exposure of the particles to cells or proteins that closely resemble the intended target, but which are missing the key component to which DeNAno binding is desired To assess the capacity of DeNAno to bind to its target, fluorescently labeled nanoparticles could be used: in a successful selection, the number of particle bound to the target increases in every round, or until binding sites are saturated An example of a successful selection, round-by-round, is followed via fluorescent readout on a fluorescent plate reader (see Fig 14.1b) The binding of fluorescently labeled selected clone to its target can be compared to nonselected nanoparticles (library) by flow cytometry to prove the enrichment of the cell-binding nanoparticles (see Fig 14.1c) A pool of selected DNA nanoparticles can be cloned into a plasmid vector to allow for testing of individual clones to choose the most optimal ones This is done by ligating the 100-bp PCR product (PCR-amplified sequence comprising 20 + 20-nt primer sites + 60-nt random insert) from the last round of selection into the plasmid vector This plasmid can be sequenced, and the 100-bp insert can be amplified by PCR and asymmetric PCR to generate the template strand required for ligation/RCA Alternately, for high-throughput analysis, adapter primers can be added to the 100-bp PCR product instead, and next-generation sequencing can be performed to produce >100,000 sequences In this case, it is beneficial to sequence PCR products from all rounds of selection to follow the enrichment of clones throughout the selection process As sequence motifs are often identified in binding clones, this process provides more thorough clone analysis when trying to determine which clones are optimal It also allows for analysis of selected clones when no dominant clone emerges from a small-scale analysis by plasmid cloning approach 164 L.E Ruff et al DeNAno Comparison to Aptamers Aptamers, one inspiration for DeNAno, have been used as DNA affinity reagents for over 25 years (Ellington and Szostak 1990; Tuerk and Gold 1990) In general, oligonucleotide aptamers are A) transitions, 78 F FA See Folic acid (FA) FA-PEG-NHS See Folic acid-polyethylene glycol 2000-N-hydroxysuccinimidyl ester (FA-PEG-NHS) Factor II (Prothrombin/FII) gene, 68 H Hexamer primers, 14 High Pure PCR cleanup Micro kit, 42 172 HIV-1 DNA, 50, 52 fluorescent detection, 55 NRCA-based detection, 54–55 Hologic’s Invader Assay, 73 HRCA See Hyperbranched rolling circle amplification (HRCA) Human breast cancer cell line MCF-7, 143 Human tick-borne pathogen, 101 Hybridization C-probe concentrations in, 71 DNA, 69 Hydrodynamic diameters, 125, 126 Hyperbranched rolling circle amplification (HRCA), 50–54, 59–62 I iCycler real-time fluorescence reader, 74 Immuno-RCA, 86 amplicons, 92 amplification protocol, 92–93 cytokines, 95 immobilization, 87–90 In situ RCA-based bi-plex mutation, 103 In situ RNA detection, 101–102 Incubation mixture, 21, 22 Infectious agents, detection of, 62 In-house-made readout unit, 129 In-phase susceptibility, 130 Instrumentation, MNPs chip-based AC susceptometer, 128–129 optomagnetic reader, 129–130 tabletop AC susceptometer, 127–128 Integrated DNA Technologies Inc., 143 Intensity of transmitted laser light, 126 Intracellular delivery of diagnostic probes, 155–157 Isothermal amplification, 50, 57–59 Isothermal detection, protein analytes, 85 Isothermal dsDNA amplification, 14 Isothermal RAM assay, 80 Isothermal signal amplification, 99 ISMMS See Icahn School of Medicine at Mount Sinai (ISMMS) K Kinetics ligation reaction of, 29 Kits FDA-cleared, for SNP detection, 67 Index L Laboratory reagents, 41 LAMP See Loop-mediated isothermal amplification (LAMP) Laser light, intensity of transmitted, 126 Let-7 miRNA, 30, 32 Ligases, 26–28, 31, 33, 39, 45, 49 Ligation-dependent probes, 68 Linear RCA (LRCA), 49–52, 54 Long ssDNA strands, 152 Loop-mediated isothermal amplification (LAMP), 57 M M13mp18 ssDNA, 21 M13mp8 ssDNA, 21 Magnapure LC Instrument, 71 Magnetic beads, 73, 78, 80 Magnetic dynamics, 125, 127, 129 Magnetic nanoparticles (MNPs) detection of digested amplicons, 133–135 instrumentation chip-based AC susceptometer, 128–129 optomagnetic reader, 129–130 tabletop AC susceptometer, 127–128 multiplexing, 133 phase-based detection, 132–133 rotational response, 124–127 turn-off detection, 130 turn-on detection, 131–132 Magnetic readout, 131 Magnetic sensors, 137 Magnetic susceptibility, 125–127 Magnetoresistive chip system, 129 MB See Molecular beacon (MB)-based assay MDA See Multiple Displacement Amplification (MDA) Melting curve analysis, 73 Messenger RNAs (mRNAs), 37 Methylenetetrahydrofolate reductase (MTHFR) enzyme A1298C mutation, 68, 73 C677T mutation, 68, 73 isoforms of, 68 Microarray method, 25 Microbial detection methods, 37 Microbial RNAs, 37–39 Microplate Reader, 27, 148 Micro RNAs (miRNAs), 37 bodily fluids, 25 detection, 26 discovery of, 25 let-7, 30 173 Index MB assay, ligation efficiency analysis, 27–28 methods for analysis, 25, 26 as novel biomarkers, 25 RCA, miRNA analysis, 28 reagents, 27 sequence-specific detection, RCA, 30–31 Microspheres cytokines, 94 fiber-optic microarray, 91 functionalization, 90–91 imaging system, 92 optical encoding, 90 MNP See Magnetic nanoparticles (MNPs) MNP-based detection, 135–136 Molecular beacon (MB)-based assay, 26 assessing ligation reactions, 28–29 FRET based, 27 ligation efficiency analysis, 27–28 nucleotide validation, 27 oligonucleotides validation, 27 Molecular inversion probes, 68 MRCA See Multiply primed RCA (MRCA) mRNAs See Messenger RNAs (mRNAs) MTHFR See Methylenetetrahydrofolate reductase (MTHFR) enzyme Multiple Displacement Amplification (MDA), 11 Multiplexing, 133 cytokines, 94 protein detection assays, 85 Multiplex RAM assay, 81 Multiply primed RCA (MRCA), 14, 18, 22 assays, 18 efficiency, 18–19 Multivalent aptamers, 164 Mutations A1298C, 68, 73 C677T, 68, 73 G1691A, 68 G20210A, 68 N Nanocapsule (NCa), 143, 146–147 Nanocs Inc., 143 Nanoparticles, 99 DNA, 142 lipid and polymeric, 142 Nanotechnology approach, 151 Netlike rolling circle amplification (NRCA) technique, 50, 52 agarose gel-electrophoresis, 56 fluorescent signals, 54 HIV-1 target, detection of, 54–55 mechanism and validation, 52–54 principle, 53 reaction, 51 system, 52 Nicking endonucleases (enzymes) circularization of padlock probe, 51 fluorescent detection, 52 gel electrophoresis analysis, 51, 52 materials, 50 NRCA-based detection, HIV-1 target, 54–55 NRCA mechanism and validation, 52–54 RCA amplicons, visualization of, 52 Non-coding RNAs, 25 Non-hybridizing probe backbone, 100 Non-small cell lung cancer tissues, 102 Novel biomarkers, miRNAs, 25 NRCA See Netlike rolling circle amplification (NRCA) technique N-terminal exonuclease, 12 Nucleic acid hybridization, 142 Nucleic acid-assisted detection technologies, 85 Nucleic acid target detection, 80 Nucleocapsid protein (NP), 102 O Oligonucleotides, 20, 27, 38, 135 circular probe preparation, 39 evaluation of ligation efficiency, 28 MB assay, valuation of, 27 phosphorylated, 42 uncircularized, 42 Oncoviral SNPs typing, 114–116 On-demand drug delivery, 142 One-pot annealing, 153 Optomagnetic reader, 129–130 readout system, 128 sensors, 137 signal, 126 technique, 132 P Padlock gap probes, 102 Padlock probe-RCA, 59 Padlock probes, 58–60, 68, 80, 99, 123 advantages, 99 application of, 26 backbone sequence, 123 circularization of, 28, 29, 51, 61, 124 let-7 members, 31 174 Padlock probes (cont.) ligation, 100 RCA diagnostics with, 99–101 RCA with, 101–102 Patient DNA, 71 PCR See Polymerase chain reaction (PCR) PCR-based kits, 80 Peptide nucleic acid (PNA) openers binding sites, 108 hybridization, 107 PD-loop, 109 Pfu DNA polymerase, 14 PG-RCA See Primer generation-rolling circle amplification (PG-RCA) method Phase-based detection, 132–133 Phase lag, 126, 132, 135 phi29 polymerases, 27 Phosphorilation, 39 Phosphorylated oligonucleotides, 42 pJLW2 plasmid, 20 Plasmid amplification, 14 PNA See Peptide nucleic acid (PNA) openers PNA-assisted RCA advantage, 109 DNA targeting, 108, 114 EBV, 115 FISH microscopy, 111 fluorescent in situ detection, 112 human DNA, 112 target site MT-ND3, 112 Point of care (POC) testing, 59 Point-of-care testing (POCT), 38, 40 Polyacrylamide gel electrophoresis, 52 Polymerase chain reaction (PCR), 11, 26, 59 Primer generation-rolling circle amplification (PG-RCA) method, 50 Polymeric nanocapsule (NCa), 143 Polymerization, 13, 16–18, 21–22 PPi See Pyrophosphate (PPi) Primer/template junctions, 17 Primers, 14, 51, 57, 60, 68, 70, 71 candidate, 71 design, 59 pair concentrations, 71 re-annealing of, 57 Processive polymerization, 21–22 Proteins fusion, 19, 20 Pyrophosphate (PPi), 40 Q QIAamp DNA blood mini kit, 71 Qiagen Gel-Extraction Kit Columns, 22 Quality-control algorithm, 75 Index Quant-iT™ ssDNA Assay Kit, 42 Quantum dots (QDs), 155 Quasi-exponential amplification, 57, 59, 62 Quencher DABCYL, 27 QuikChange® (Stratagene), 20 R RAM-assay-based process, 69 Ramification amplification, 62 Ramified RCA assay automated sample-processing, 74 clinical samples, DNA preparation/control, 71–73 detection, 68 elution plate, 74 magnetic beads, 73 methodology overview, 69–70 nucleic acids detection reagents design and quality control, 70–71 RAM amplification, 74 reagents, 70 replicate, 75 response time post-assay interpretation, 74–75 sample processing for RAM assays, 73 targets, 67 RAW264.7 cells, 155, 157 RCA See Rolling circle amplification (RCA) RCR See Rolling circle replication (RCR) RCA-based DNA origami, 151, 152 RCA-based nucleic acid advantages, amplicons, drug delivery vehicles, RCA-driven synthesis, NCl, 143 RCA-LAMP, coupled amplicons, 62 amplification, 60 coupled, 58, 59 design, 59–60 experimental validation, 60, 61 RCP monomerization, 99 RCA products (RCPs), 99 Real-time detection, 38, 43 Real-time fluorescent signals, 52 Real-time RP-RCA reaction, 42–43 Real-time signals, RAM, 69 Recombinant E coli, 41 Response time quality control, RMA assay, 74, 75 Restriction site, 62 Reverse transcription (RT), 101 Reverse transcription PCR (RT-PCR), 37 Index Reverse transcription-quantitative PCR (RT-qPCR) method, 25 Ribo-oligonucleotides, 28 RNA in situ detection, 101–102 primer, 38 sample from recombinant E coli, 41 RNA-primed RCA (RP-RCA) additional laboratory reagents, 41 circular probe for, 38 ø29 DNA polymerase, 40–41 DNA probe circularization, 42 exonuclease for circular DNA purification, 39–40 oligonucleotides for circular probe preparation, 39 real-time RP-RCA reaction, 42–43 RNA sample from recombinant E coli, 41, 42 Robust isothermal method, 26 Rolling circle amplification (RCA) amplicons, 87, 124, 133–136, 142 assays, biomedical fields, 5–6 diagnostic applications, 54 diagnostics with padlock probes, 99–101 DNA polymerization, EpCAM detection, 89 glass slides, 87 LRCA, 49 for miRNA analysis, 28 molecular diagnostics, 4–5 molecular medicine, 4–5 nucleic acid sequences, posttranslational modifications, 85 products detection, 124 proteomic data, 85 replication, right-handed helix, sensors, 137 sequence-specific detection, miRNAs, 30–31 single-molecule detection, 86 on slides, 88 target vs signal amplification, thermal fluctuations, topological constraint, visualization of, amplicons, 52 with padlock probes, 101–102 Rolling circle replication (RCR), 16, 18, 21 RP-RCA See RNA-primed RCA (RP-RCA) RT See Reverse transcription (RT) 175 RT-PCR See Reverse transcription PCR (RT-PCR) RT-qPCR See Reverse transcriptionquantitative PCR(RT-qPCR) method S SanPrep Column DNA Gel Extraction Kit, 50, 52 Scaffolds ssDNA, 151, 152 Scaffold/staples, 153, 154 Self-priming DNA stem-loop, 58 Self-replication, 60 Sephadex G-50 columns, 21 Shapiro-Wilks test, 73 Signal amplification technique, 49 Single-base discrimination, 50 Single-cell analysis cellular and molecular dynamics, 86 encapsulation, 87 resolution, 95 Single-cell single-copy DNA imaging cells-in-flow DNA detection, 114–116 cytogenetic aberrations, 109 FISH, 109 interphase nuclei, 112–113 mitochondrial DNA, 112 multi-target detection, 110–111 PNA-assisted RCA, 109 Single-molecule manipulation methods, 13 Single nucleotide polymorphisms (SNPs), 14, 107 A>C transversion, 78 allele determinations, 76 configurations, 79 C>T transition, 78 detection, 67 G>A transitions, 78 locus, 71 RAM assay platform, 67 Single nucleotide variations (SNVs), 102 Single-stranded DNA (ssDNA), 14, 50, 68, 141, 142 Single-stranded RNA (ssRNA), 101 SNPs See Single nucleotide polymorphisms (SNPs) SNVs See Single nucleotide variations (SNVs) SoftMax Pro software, 51 Solarbio Technology Co., Ltd., 50 SpectraMax M3 Multi-Mode Microplate Reader, 51, 52 Splint oligonucleotide, 38 ssDNA See Single-stranded DNA (ssDNA) 176 Staples, 151 Strand-displacing activity, 57 Streptavidin particles, 70 Superconducting quantum interference device (SQUID), 127, 128 SYBR Green I dye, 50 SYBR Green II dye, 27 SYBR-Green fluorescence, 68, 69 Synthetic oligonucleotides, 135 T T4 DNA ligase, 26, 27, 38 ligation efficiencies of, 30 L-ON and R-ON by, 28 T4 RNA ligase 1, 27, 30 T4 RNA ligase 2, 26, 27, 29–30 Tabletop AC susceptometer, 127–128 TAMRA-labeled 2D DNA nanoribbons, 155 TAMRA-labeled oligodeoxynucleotides, 155 Tapping mode, 145 Taq DNA polymerases, 14 Target detection, SNP, 80 Target-specific capture probe, 71, 78 Target-specific padlock probes, 101 Target-specific RCA, 101–102 Terminal Protein Region-1 (TPR1), 12 Terminal Protein Region-2 (TPR2), 12 Therapeutic drugs, 151, 155–157 Thermocycling equipment, 37 Three-dimensional (3D) DNA nanoribbons, 152–155 Index Thrombosis-related polymorphisms, 68 Time-dependent magnetic response, 125 Topoisomerase V, 19 TPR1 See Terminal Protein Region-1 (TPR1) TPR2 See Terminal Protein Region-2 (TPR2) Transfection reagents, 151 Tumor-associated point mutations, 102 Tumor necrosis factor (TNF)-α secretion, 156, 157 Turn-off detection, 130 Turn-on detection, 131–132 Two-dimensional (2D) DNA nanoribbons, 152–155 U UltraPower DNA/RNA Safedye, 42 Uncircularized oligonucleotides, 42 Universal primer, 21 V Vascular disease, 67, 68 Vero monkey cells, 101 Viral nucleocapsid protein (NP), 102 Viral RNA (vRNA), 102 W Wide Mini-Sub Cell GT Cell, 50 Wild-type ϕ29 DNA polymerase, 16, 17 .. .Rolling Circle Amplification (RCA) Vadim V Demidov Editor Rolling Circle Amplification (RCA) Toward New Clinical Diagnostics and Therapeutics... 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... 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