key: cord-0810975-yir5dia8
authors: Goo, Nam-In; Kim, Dong-Eun
title: Rolling circle amplification as isothermal gene amplification in molecular diagnostics
date: 2016-07-29
journal: Biochip J
DOI: 10.1007/s13206-016-0402-6
sha: cb2ac105735bcf360963ae001284f4dd3ed8eff9
doc_id: 810975
cord_uid: yir5dia8

Rolling circle amplification (RCA) developed in the mid-1990s has been widely used as an efficient isothermal DNA amplification process for molecular diagnosis. This enzymatic process amplifies target DNA sequences with high fidelity and specificity by using the strand displacing DNA polymerases. The product of RCA is long single-stranded DNA that contains tandem repeat of target sequence. Isothermal reaction amplification condition of RCA has an advantage over conventional polymerase chain reaction, because no temperature cycling devices are needed for RCA. Thus, RCA is suitable tool for point-of-care detection of target nucleic acids as well as facile detection of target genes. Combined with various detection methods, RCA could amplify and detect femtomolar scale of target nucleic acids with a specificity of one or two base discrimination. Herein, RCA technology is reviewed with an emphasis on molecular diagnosis of microRNAs, infectious pathogens, and point mutations.

Rolling circle amplification (RCA) was introduced in the middle of 1990s as a new isothermal DNA amplification method 1, 2 . As shown in Figure 1A , the am-plification of DNA is based on circular template and strand-displacing DNA polymerases, such as phi 29 DNA polymerase 3, 4 . Especially, phi 29 DNA polymerase harboring 3′ to 5′ exonuclease activity for proofreading contributes to high fidelity of DNA polymerization. As product of RCA, long single-stranded DNA (ssDNA) products containing tandemly repeating sequences complementary to the circular target DNA are generated 5 , which can be often observed as long stretch of ssDNA under atomic force microscopy (AFM, Figure 1A ).

Ligation-RCA (L-RCA) is one of RCA variations, which is based on padlock probe 6, 7 , a linear singlestranded template DNA for RCA reaction ( Figure 1B) . The 5′-and 3′-end of padlock probes are designed to be hybridized with target nucleotides such as genomic DNA or microRNA (miRNA). The only padlock probe that is hybridized with its target nucleotides is eligible for ligation reaction to form circular DNA templates for subsequent amplification process. Addition of deoxynucleotide triphosphates (dNTPs), primer DNA, and phi 29 DNA polymerase to the circular template DNA can initiate polymerization of target DNA sequence. In addition, branched RCA (BRCA), or hyperbranched RCA (HRCA) has been developed as expanded variations of RCA ( Figure 1C ), which amplify DNA exponentially with forward and reverse primers 8 . HRCA products are double-stranded DNAs (dsDNA), whereas the ordinary RCA products are long ssDNAs. Thus, dsDNA-specific intercalating fluorescent dyes such as SYBR Green (SG) has been often used for the detection of the HRCA products 9 .

RCA has an obvious advantage over polymerase chain reaction (PCR); unlike PCR, RCA is performed at an isothermal reaction condition at room tempera-ture or at 30℃. Since RCA does not need thermal cycling for amplification of DNA, no additional devices such as thermal cyclers, are needed for amplification of DNA through RCA. Thus, RCA as isothermal amplification of target gene is regarded as a suitable tool for point-of-care detection of target genes. In addition, RCA can amplify target DNA sequences with high specificity and sensitivity; 1 copy of target DNA in 100,000 copies of non-target DNAs can be amplified by RCA 10 . High sensitivity and specificity makes RCA as a feasible tool for detection of single nucleotide polymorphisms (SNPs) 8, [11] [12] [13] [14] [15] [16] [17] , microRNAs (miRNA) 9,18-29 , bacterial [30] [31] [32] [33] [34] , and viral nucleic acids [35] [36] [37] [38] [39] . Amplified DNA products can be detected with diverse methods including fluorescence measurement 8 , colorimetric assay 32, 37 , enzymatic luminescence assay 20, 21 , or elec-tric signals 33 . Herein, we reviewed recent advances in molecular diagnosis methods based on RCA for specific detection of target nucleotides such as miRNAs and infectious pathogens, and point mutations causing SNP.

MicroRNAs (miRNAs) are non-coding single-stranded RNAs (ssRNA) that have ~25 nucleotides in length. MiRNAs anneal to 3′-untranslated region (3′-UTR) of target mRNA and induce degradation of the mRNA, leading to inhibition of the target gene translation, as a post-transcriptional regulation of target gene 40 . It has been known that miRNAs are closely associated The DNA polymerase unwinds dsDNA (strand displacement) and synthesizes long ssDNA with high processivity and fidelity. The AFM image shows that the length of RCA product varies at least hundred nanometers to few micrometers. AFM image was reproduced from Ref. 5 with a permission. (B) Schematic illustration of L-RCA. In the presence of target nucleotide, the padlock probe hybridizes with the target and both ends of the padlock probe are ligated to form circular template DNA by DNA ligase. DNA polymerization is then initiated from 3′-end of RCA primer by DNA polymerase. (C) Schematic representation of HRCA. During the extension of the first primer (primer 1), addition of the second primer (primer 2), which is complementary to the first RCA product, initiates synthesis of complementary RCA product, resulting in formation of double-stranded RCA product.

with various cell metabolisms such as cell differentiation, proliferation, fat metabolism, and cell death 41 . Of importance, some miRNAs are significantly overexpressed in cancerous tissues compared with normal tissues 42, 43 . Therefore, detection and quantification of miRNAs in clinical samples are important for diagnosis of certain cancer.

The sensitivity and target specificity of RCA made it suitable for detection and quantification of miRNA. In 2006, Jonstrup et al. reported padlock probe and L-RCA based miRNA detection method 18 . They used padlock probes targeting several miRNAs and the miRNAs are used as both target molecule and primer of DNA polymerization. The resulting RCA products are detected by autoradiography. Compared with Northern blot, a commonly-used RNA detection method which require micrograms of RNA samples for detection, RCA detected target miRNAs in nanograms of total RNA sample. In addition to L-RCA, BRCA was also utilized for detection of miRNAs; Cheng et al. reported a BRCA-based fluorescence detection of let-7a miRNA 9 . For fluorescence detection of BRCA product, SYBR Green I (SG I) dye, which intercalates into double-stranded DNA (dsDNA) and shows green fluorescence, was utilized in this method ( Figure 2A ). The BRCA-based miRNA detection could detect 10 fM of miRNA and discriminate let-7a from let-7b and let-7c, which differ in one or two bases in sequence.

In addition to fluorescence detection of miRNA using fluorescent dye, indirect detection of RCA product using luminescence assay coupled with other enzymatic reactions was adopted for detection of miRNAs. Mashimo et al. reported miRNA detection based on BRCA coupled with bioluminescent (BL) pyrophosphate assay ( Figure 2B ) 20 . The BL assay utilizes target miRNA molecule as primer of the DNA polymerization reaction. During the synthesis of RCA product, inorganic pyrophosphates (PPi) are released when dNTPs are incorporated. Adenylyl transferase then converts released PPi to ATP, which provides chemical energy for firefly luciferase to generate light. Target RNA could be detected up to 0.1 fM through this method, and the luminescence showed linearity as a function of target miRNA amount. Sun et al. also developed miRNA quantification system using RCA coupled with enzymatic luminescence assay ( Figure 2C ) 21 . In this assay, 2′-deoxyadenosine-5′-O-(1-thiotriphosphate) (dATPαS) was used as a replacement of dATP for generation of adenosine 5′-phosphosulfate (APS) and PPi during the DNA polymerization. The released APS reacts with the PPi to form ATP by the catalytic activity of ATP-sulfurylase, and the generated ATP provides energy for subsequent reaction of firefly luciferase to generate luminescence. This method claims to detect 0.01 pg of target miRNA and discriminate let-7d miRNA from other let-7 miRNA families (let-7a, let-7b, let-7c, and let-7e).

In place of using padlock probe based RCA for detection of miRNA, some researchers used hybridiza tion of miRNA to dumbbell 19 or hairpin-shaped pro be 22 19 . The dumbbell probe is composed of 3 domains: miRNA-binding domain (MBD), SYBR green I binding domain (SGBD), and loop domain. Once the targeting sequences of MBD hybridize with target miRNA, closed template for RCA forms and DNA polymerization occurs in the presence of phi 29 polymerase and miRNA as primer. The RCA product forms repeating SGBDs and the SG I dye intercalates into double-stranded region of the SGBD, which generates detectable green fluorescence. Detection limit of this method was calculated as low as 1 fM. Rather than the conventional padlock probe DNA for L-RCA, Li et al. adopted hairpin-shaped probe mediated RCA (HP-RCA) for detection of miRNA ( Figure 3B ) 22 . The hairpin probe contains miRNA binding domain, which targets miR-486-5p, and circular template binding domain, which also acts as primer for DNA polymerization. In the presence of target miRNA, the hairpin structure of the hairpin probe melts and the target miRNA binds to the probe, which exposes recognition site of circular template. The exposed probe sequence then binds with circular template to initiate polymerization of RCA product. The amplified DNA products are detected by SYBR Green II dye, which can stain ssDNA to emit green fluorescence. Detection limit of this method was calculated as low as 10 fM of target miRNA and could discriminate closely related miRNA families.

Very recently, our group also reported a fluorometric system for the detection of miRNA using L-RCA, graphene oxide (GO), and fluorescent peptide nucleic acid (F-PNA) probe 29 . This assay method is featured by unique properties of GO such as high affinity to single-stranded nucleic acids and quenching of nearby fluorescence via long-range energy transfer. As shown in Figure 3C , the padlock probe DNA complementary to a target miRNA was specifically ligated to form circular DNA and then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. High sen-sitivity (i.e. LOD of pM range) and selectivity of the assay for miRNAs allows the efficient detection of multiple miRNAs in a mixture, using a simple 96-well format that can be completed within an hour.

Infectious diseases are still major cause of death in developing countries and causing millions of deaths every year. Thus, detection of infectious pathogens such as bacteria and viruses is important for early diagnosis and prevention of disease spreading. RCA has been de-veloped for detection of pathogens due to its sensitivity, simplicity, and specificity. More importantly, RCA is advantageous in point-of-care diagnosis because RCA is an isothermal DNA amplification method and no sophisticated device is needed. For detection of bacteria, research groups targeted genomic DNAs of target bacterium.

Gomez et al. developed a colorimetric detection method of bacterial pathogens using HRCA combined with nicking endonuclease, which they called exponential linear RCA (ELRCA) (Figure 4) 32 . They used bis-PNA openers to expose target genomic DNA sequence for padlock probe binding. The exponential amplifica- tion of DNA is proceeded by HRCA. By the activity of nicking endonuclease, multiple gaps are generated on the forward strand. Polymerization can be initiated from the gap; thus displacement of nicked pieces can occur because of the strand-displacing activity of the DNA polymerase. The ssDNA pieces fold to form G-quadruplex structures, which show catalytic activity of oxidization of 2,2′-azino-bis (3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) to ABTS-in the presence of Hemin and H2O2. The reaction progress was monitored by measuring absorbance at 412 nm. This method was able to detect bacterial genomic DNA as low as Figure 5B) 33 . SPR biosensor monitors the change of refractive index, which is caused by the hybridization of RCA products and capture probes immobilized on the surface of AuNPs. The limit of detection was as low as 10 pM of target DNA, and this method could specifically detect 42,000 cfu/ mL (5 pg/μL) of M. tuberculosis genomic DNA and 37,000 cfu/mL (2 pg/μL) of Mycobacterium avium genomic DNA.

RCA has been also used for detection of viruses, which is based on the detection of genomic DNA or RNA of target viruses. Wang et al. designed a detection method using HRCA targeting severe acute respiratory syndrome coronavirus (SARS-CoV) RNA 35 . They tested liquid-phase RCA and solid-phase RCA, which proceeds in a reaction buffer and on surface of magnetic bead coated with oligo (dT), respectively. Gel electrophoresis was performed for analysis of the RCA products, and both methods could detect single-copy of SARS-CoV RNA. However, accurate quantification of the target RNA was not attained because the detection of the RCA products was based on gel electrophoretic signal, which is insufficient for quantification of the samples. To overcome the lack of quantification, Hamidi et al. developed real-time monitoring methods of H5N1 influenza virus RNA using HRCA combined with colorimetric 37 or fluorometric 38 assay. For fluorescence detection of H5N1 RNA, cDNA synthesis was performed and subsequent HRCA was proceeded by the phi 29 polymerase. SG I was adopted to generate fluorescence signal in the presence of HRCA product. The limit of detection was calculated as low as 9 fM of target, and the signal could be obtained within 3 h. The colorimetric assay was based on characteristics of Hydroxy Naphthol Blue (HNB), which is known as metal chelator. HNB has sky blue color at pH 8.8 with absorption peak at 650 nm. In the presence of metal ions such as Mg 2+ , HNB captures Mg 2+ to form HNB-Mg complex and the absorption peak shifts to 530 nm, which appears in dark blue color. During the polymerization of DNA by Bst DNA polymerase, PPi is released and chelates Mg 2+ in reaction buffer to form PPi-Mg complex. As RCA proceeds, more PPi-Mg complexes are generated and Mg 2+ ions are released from HNB-Mg complexes. Thus, the absorption peak shifts from 530 nm to 650 nm and the color change can be observed via naked eye. By measuring the absorption at 650 nm, the presence of H5N1 influenza virus in sample can be detected. The limit of detection was calculated as low as 28 fM of target and could detect H5N1 virus in real samples.

Single nucleotide polymorphisms (SNPs) are alteration of single base in specific position of a gene sequence, which make individual difference of disease susceptibility 44, 45 and drug response 46 . In addition, SNPs are known to be cause of cancers 47 and genetic diseases 48, 49 . Thus, sensitive detection of SNPs is crucial for diagnosis of multiple diseases. RCA has been widely adopted in detection of SNPs because of its high sensitivity and specificity. The first SNP analysis using RCA was reported by Lizardi et al. in 1998 8 . Their RCA strategy was designed for detection of CFTR G542X gene causing cystic fibrosis. Oligonucleotide probe was immobilized on glass surface and the probe was ligated with RCA primer depending on the sequence of the target DNA. Amplification of DNA sequence occurred selectively in the presence of circularized template DNA, and the amplified DNA was monitored by fluorophore-labeled probe. Using a similar RCA method but different report system, Zhang et al. designed electrochemical label-free SNP detection system using RCA 17 . They immobilized capture probe, which captures RCA products, on the gold electrode and used methylene blue as signal molecule or detection of RCA products. This method can detect 40 amol of mutant strand and distinguish 1 target mutant from 5,000 non-target wild type DNAs. The SNP detection methods described above are using immobilized oligonucleotides as amplification and signal detection platform. Those methods are expected to be utilized for microarray-type SNP assay system. Molecular beacons are oligonucleotide probes containing both fluorophore and quencher. Without target nucleic acids, the fluorescence of molecular beacons is quenched because hairpin structure of the probes places fluorophore and quencher at near distance. Fluorescence of molecular beacons is recovered when the probes specifically hybridized with their target nucleic acids. Based on the specificity of the molecular beacons, they are widely used as fluorescent probe for SNP genotyping using RCA. Faruqi 

Success of molecular diagnostics are heavily dependent upon the target specificity and sensitivity of the detection method as well as target gene amplification.

Rolling circle amplification (RCA) is an isothermal DNA amplification method that is based on circular template and high fidelity of strand-displacing DNA polymerases, such as phi 29 or Bst DNA polymerase. RCA and its variations were combined with fluorescence detection, colorimetric assay, enzymatic luminescence assay, or electric signal for the detection of target nucleic acids, such as human or bacterial genomic DNAs, miRNAs, and viral RNAs. The detection sensitivity of the RCA based methods are as low as femtomolar scale of target nucleic acids. Furthermore, those methods could discriminate the sequence of the target nucleic acids, even one or two bases could be discriminated by RCA. Therefore, RCA is expected to be an excellent point-of-care diagnostic tool for SNPs, miRNAs, and viral pathogens. 

Rolling replication of short DNA circles

Rolling Circle DNA Synthesis: Small Circular Oligonucleotides as Efficient Templates for DNA Polymerases

Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication

Highly efficient DNA synthesis by the *: Bacterial, viral nucleic acids, **: Single Nucleotide Polymorphism (point mutation), ***: Not determined

phage phi 29 DNA polymerase. Symmetrical mode of DNA replication

Periodic DNA nanotemplates synthesized by rolling circle amplification

Padlock probes: circularizing oligonucleotides for localized DNA detection

Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification

Mutation detection and single-molecule counting using isothermal rolling-circle amplification

Highly sensitive determination of mi-croRNA using target-primed and branched rolling-circle amplification

Combining nucleic acid amplification and detection

A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification

Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms

Genotyping of multiple single nucleotide polymorphisms with hyperbranched rolling circle amplification and microarray

Integration of DNA ligation and rolling circle amplification for the homogeneous, endpoint detection of single nucleotide polymorphisms

A label-free strategy for SNP detection with high fidelity and sensitivity based on ligation-rolling circle amplification and intercalating of methylene blue

A microRNA detection system based on padlock probes and rolling circle amplification

A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microR-NA detection

Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay

Rapid and direct microRNA quantification by an enzymatic luminescence assay

Isothermally sensitive detection of serum circulating miRNAs for lung cancer diagnosis

High specific and ultrasensitive isothermal detection of microRNA by padlock probe-based exponential rolling circle amplification

Homogeneous and label-free detection of microRNAs using bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification

A rolling circle amplification-based DNA machine for miRNA screening coupling catalytic hairpin assembly with DNAzyme formation

Ultrasensitive detection of microRNA through rolling circle amplification on a DNA tetrahedron decorated electrode

Chemiluminescence detection of DNA/ microRNA based on cation-exchange of CuS nanoparticles and rolling circle amplification

A DNA logic gate based on strand displacement reaction and rolling circle amplification, responding to multiple low-abundance DNA fragment input signals, and its application in detecting miRNAs

Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide

Mycobacterium tuberculosis detection via rolling circle amplification

Sensitive colorimetric detection of Listeria monocytogenes based on isothermal gene amplification and unmodified gold nanoparticles

Visual detection of bacterial pathogens via PNA-based padlock probe assembly and isothermal amplification of DNAzymes

Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor

Label-free and highly sensitive electrochemical detection of E. coli based on rolling circle amplifications coupled peroxidase-mimicking DNAzyme amplification

Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification

Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing

Colorimetric monitoring of rolling circle amplification for detection of H5N1 influenza virus using metal indicator

Real-time detection of H5N1 influenza virus through hyperbranched rolling circle amplification

Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV

Oncomirs -micro RNAs with a role in cancer

MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing

Unique microRNA molecular profiles in lung cancer diagnosis and prognosis

Exosomal microRNA: a diagnostic marker for lung cancer

IFNL3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance

Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease

Pharmacogenetics and the practice of medicine

Association studies for finding cancer-susceptibility genetic variants

The genetics of autistic disorders and its relevance: a review of the literature

Detecting shared pathogenesis from the shared genetics of immune-related diseases

Copyright for the published papers belongs to the Korean BioChip Society