Combining Rolling Circle Amplification Research Articles

Fluorogenic RNA Aptamer-Based Amplification and Transcription Strategy for Label-free Sensing of Methyltransferase Activity in Complex Matrixes.

DNA methyltransferase is significant in cellular activities and gene expression, and its aberrant expression is closely linked to various cancers during initiation and progression. Currently, there is a great demand for reliable and label-free techniques for DNA methyltransferase evaluation in tumor diagnosis and cancer therapy. Herein, a low-background fluorescent RNA aptamer-based sensing approach for label-free quantification of cytosine-guanine (CpG) dinucleotides methyltransferase (M.SssI) is reported. The fluorogenic light-up RNA aptamers-based strategy exhibits high selectivity via restriction endonuclease, padlock-based recognition, and RNA transcription. By combining rolling circle amplification (RCA), and RNA transcription with fluorescence response of RNA aptamers of Spinach-dye compound, the proposed platform exhibited efficiently ultrahigh sensitivity toward M.SssI. Eventually, the detection can be achieved in a linear range of 0.02-100 U mL-1 with a detection limit of 1.6 × 10-3 U mL-1. Owing to these superior features, the method is further applied in serum samples spiked M.SssI, which delivers a recovery ranging from 92.0 to 107.0% and a relative standard deviation <7.0%, providing a promising and practical tool for determining M.SssI in complex biological matrices.

Localized surface plasmon resonance biosensor chip surface modification and signal amplifications toward rapid and sensitive detection of COVID-19 infections

We developed a multi-pronged approach to enhance the detection sensitivity of localized surface plasmon resonance (LSPR) sensor chips to detect SARS-CoV-2. To this end, poly(amidoamine) dendrimers were immobilized onto the surface of LSPR sensor chips to serve as templates to further conjugate aptamers specific for SARS-CoV-2. The immobilized dendrimers were shown to reduce surface nonspecific adsorptions and increase capturing ligand density on the sensor chips, thereby improving detection sensitivity. To characterize the detection sensitivity of the surface-modified sensor chips, SARS-CoV-2 spike protein receptor-binding domain was detected using LSPR sensor chips with different surface modifications. The results showed that the dendrimer-aptamer modified LSPR sensor chip exhibited a limit of detection (LOD) of 21.9 pM, a sensitivity that was 9 times and 152 times more sensitive than the traditional aptamer- or antibody-based LSPR sensor chips, respectively. In addition, detection sensitivity was further improved by combining rolling circle amplification product and gold nanoparticles to further amplify the detection signals by increasing both the target mass and plasmonic coupling effects. Using pseudo SARS-CoV-2 viral particles as detection targets, we demonstrated that this combined signal intensification approach further enhanced the detection sensitivity by 10 folds with a remarkable LOD of 148 vp/mL, making it one of the most sensitive SARS-CoV-2 detection assays reported to date. These results highlight the potential of a novel LSPR-based detection platform for sensitive and rapid detection of COVID-19 infections, as well as other viral infections and point-of-care applications.

First report of Cotton leaf curl Multan virus infecting Millettia pinnata in Pakistan

Millettia pinnata (Pongame oiltree, Sukh chain; family Fabaceae) is an important leguminous tree widely grown and utilised as a herb in Southeast Asia and the Indian subcontinent. In October 2020, symptoms of stunted growth, leaf curling (upward and downward) and vein thickening (Figures 1 and 2), which are associated with infection by Begomovirus species in other hosts, were observed on M. pinnata, growing near cotton fields (30.3°N, 73.0°E), in Faisalabad, Punjab Province, Pakistan. DNA was isolated from ten leaves (S1 to S10), one each from ten diseased plants, and one leaf from two asymptomatic plants (AS11 and AS12) using a cetyl trimethyl ammonium bromide protocol (Doyle & Doyle, 1990). Rolling circle amplification in combination with PCR using begomovirus universal primers (BegomoF/BegomoR) (Briddon & Markham, 1994) amplified a c. 2.8 kb product from all the diseased samples. PCR assays for the Begomovirus alphasatellite (primer pair DNA101F/DNA101R) (Bull et al., 2003) and betasatellite (primer pair Beta01F/Beta02F) (Briddon et al., 2002) each amplified a c. 1.4 kb product from all the diseased samples. Neither begomovirus genome nor satellite DNA was detected in the asymptomatic samples. Amplicons from the begomovirus genome (AV-2 and AV-3 from S1 and S2; MZ268122-MZ268123), alphasatellite (AV-36 from S1; MZ057253) and betasatellite (AV-8 and AV-10 from S1, AV-11 and AV-12 from S2, and AV-13 from S3; MZ057231- MZ057235), were cloned in a general purpose PTZ57R/T cloning vector (Thermo Fisher Scientific, USA), Sanger sequenced, and the sequences deposited in GenBank. The genome sequences shared >99% nucleotide identity with Cotton leaf curl Multan virus (CLCuMuV) and were most closely related to CLCuMuV strain NAS-14 (MK357257), which was found in cotton in the same region of Faisalabad (Ahmed et al., 2021). They also had 94% and 87% nt similarity with the CLCuMuV-Raj (AF363011) and CLCuMuV-Dar strains (EU365613) respectively. Moreover, the alphasatellite and betasatellite sequences shared 98% similarity with Cotton leaf curl Multan alphasatellite (CLCuMuA; MK357286.1) and betasatellite (CLCuMuB; MT920327.1), respectively. The five CLCuMuB betasatellite sequences (MZ057231-MZ057235) shared 99% identity and the two Begomovirus genomic sequences (MZ268122-MZ268123) from the M. pinnata samples had 99% identity with each other. Southern hybridization (Zubair et al., 2017) confirmed the detection of Begomovirus genomic DNA and quantitative real-time PCR (Shafiq et al., 2017) assays confirmed the detection of CLCuMuV in three samples, S1, S2 and S3, selected for further analysis. To the best of our knowledge, this is the first report of CLCuMuV in Millettia pinnata which may serve as an alternative host of the virus in the off season when cotton is not grown. Routine monitoring and detection of CLCuMuV is essential to prevent the devastating leaf curl disease it causes from spreading worldwide. The authors acknowledge support in Sanger sequencing from Dr. Brian Scheffler and his group from USDA-ARS.

Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue

Highly multiplexed spatial mapping of transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing chemistry. We benchmark a High Sensitivity Library Preparation Kit from CARTANA that circumvents the reverse transcription needed for cDNA-based in situ sequencing (ISS) via direct RNA detection. We found a fivefold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell clustering. With this increased efficiency, we also found to maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap to other fluorescent in situ hybridization-based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS.

Identification and Characterization of Circular Single-Stranded DNA Genomes in Sheep and Goat Milk.

In recent years, a variety of circular replicase-encoding single-stranded (CRESS) DNA viruses and unclassified virus-like DNA elements have been discovered in a broad range of animal species and environmental samples. Key questions to be answered concern their presence in the human diet and their potential impact on disease emergence. Especially DNA elements termed bovine meat and milk factors (BMMF) are suspected to act as co-factors in the development of colon and breast cancer. To expand our knowledge on the occurrence of these potential pathogens in human nutrition, a total of 73 sheep and 40 goat milk samples were assayed by combining rolling circle amplification (RCA), PCR and Sanger sequencing. The present study further includes retail milk from the aforementioned species. We recovered 15 single stranded (ss) circular genomes. Of those, nine belong to the family Genomoviridae and six are members of the unclassified group of BMMF. Thus, dairy sheep and goats add to dispersal of CRESS viruses and circular ssDNA elements, which enter the food chain via milk. The presence of these entities is therefore more widespread in Bovidae than initially assumed and seems to be part of the common human nutrition.

RNase H-dependent amplification improves the accuracy of rolling circle amplification combined with loop-mediated isothermal amplification (RCA-LAMP).

The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.

Immunodetection and counting of circulating tumor cells (HepG2) by combining gold nanoparticle labeling, rolling circle amplification and ICP-MS detection of gold.

A method is described for counting circulating tumor cells (CTCs). It is making use of inductively coupled plasma mass spectrometry (ICP-MS) along with a dual amplification strategy by combining rolling circle amplification (RCA) and gold nanoparticle (Au NP) labeling. HepG2 cells, as a representative CTC line, were captured by anti-epithelial cellular adhesion molecule (EpCAM) immobilized on a microplate, then specifically labeled with biotinylated anti-asialoglycoprotein receptor (ASGPR). Taking streptavidin (SA) as the bridge, the biotinylated RCA primer was conjugated to HepG2 cells. When the RCA reaction was triggered, long ssDNA with tandem repeats generated on the cell surface. Then, Au NP functionalized detection DNA (signal probes) was added to hybridize with the ssDNA. After removing the redundant signal probes, Au NPs conjugated on target HepG2 cells were subjected to ICP-MS detection. By adopting such a dual amplification strategy, a 756-fold improvement in sensitivity is accomplished compared to the method involving only Au NP labeling without RCA. The limit of detection is as low as 3 HepG2 cells (15 cell mL-1) which is the lowest LOD in ICP-MS based methods for cell counting. Besides, the method provides good selectivity, a wide linear range of 10-1000 HepG2 cells (50-5000 cells mL-1), and relative standard deviations of 6.3% (n = 7; 50 HepG2 cells (250 cells mL-1)). The method was successfully applied to HepG2 cell counting in spiked human blood samples and gave good recoveries. Graphical abstract Schematic presentation of an ICP-MS based immunoassay for the sensitive circulating tumor cells counting by combining rolling circle amplification (RCA) with gold nanoparticle (Au NP) labeling. ICP-MS: inductively coupled plasma mass spectrometry; ASGPR: asialoglycoprotein receptor; EpCAM: epithelial cellular adhesion molecule.

Specific and simultaneous detection of micro RNA 21 and let-7a by rolling circle amplification combined with lateral flow strip

Owing to the pivotal function in post transcriptional gene modification, miRNA biomarkers are playing crucial role in tracking and diagnosing various forms of tumors in a short time period. Hence, the need to develop simple, sensitive and specific detection of miRNAs for precise diagnosis arises. This current study is aimed to develop a detecting platform by combining rolling circle amplification with AuNps-based lateral flow strip (LFS-RCA) for simultaneous detection of miRNA 21 and miRNA let-7a. The current methodology is simple, sensitive, specific and selective for miRNA let-7a and miRNA 21 with the limits of detection (LOD) as low as 20 pM and 40 pM, respectively. In this technique, rolling circle amplification is playing an essential role in increasing sensitivity and reducing experimental cost. Moreover, the padlock probe with high specificity can immediately identify the simultaneous amplification of multiple miRNAs targets, which may contribute in saving sample volume and detection time. Hopefully, in future with further development, this developed technique can be chosen as a potential tool for detection of miRNAs in clinical diagnosis.

A dual signal amplification-assisted DNAzyme biosensor for ultrasensitive detection of Argonaute 2 activity.

We develop a new DNAzyme biosensor for Argonaute 2 (Ago2) assay based on target-initiated rolling circle amplification in combination with an 8-17 DNAzyme-mediated dual signal amplification strategy. This biosensor can achieve ultrahigh sensitivity with a detection limit of 0.35 pM, and it can be further used for the screening of Ago2 inhibitors and the accurate measurement of endogenous Ago2 from HeLa cell extracts.

Aptamer and rolling circle amplification-involved sandwich assay for platelet-derived growth factor-BB with absorbance analysis

An aptamer-based absorbance assay for PDGF-BB was developed by combining rolling circle amplification reaction and catalysis with horseradish peroxidase.

An aptamer-based chemiluminescence method for ultrasensitive detection of platelet-derived growth factor by cascade amplification combining rolling circle amplification with hydroxylamine-enlarged gold nanoparticles

An ultrasensitive and selective chemiluminescence assay for PDGF-BB detection using the powerful signal amplification capability of RCA and hydroxylamine-amplified AuNPs.

Light-dependent segregation of begomoviruses in Asystasia gangetica leaves

Asystasia gangetica (Acanthaceae) from tropical Africa and Asia is used as source of food and for medical applications. Plants collected in West Africa in the 1980s with typical geminivirus symptoms showed an unusual symptom segregation that included vein yellowing, curling and mosaic, which were present simultaneously or separately on different leaves of the same plant or on different plants propagated as cuttings from a single plant. Rolling-circle amplification in combination with restriction fragment length polymorphism analysis followed by deep sequencing of the RCA products identified two geminiviruses in these plants. One with a bipartite genome, Asystasia begomovirus 1, and the other with a monopartite genome together with its defective DNA, Asystasia begomovirus 2. The relationship between leaf symptoms and virus distribution under different light regimes was investigated, and showed for the first time an unusual segregation of symptoms and viruses, either within a single plant, or even within a leaf.

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

Molecular beacon structure mediated rolling circle amplification for ultrasensitive electrochemical detection of microRNA based on quantum dots tagging

MicroRNA-16 (miR-16) frequently deleted or downregulated in patients with B cell chronic lymphocytic leukemia and thus can be used as promising biomarkers in B-CLL diagnosis and prognosis. Herein, an ultrasensitive miR-16 detection was carried out by combining rolling circle amplification (RCA), quantum dots tagging and anodic stripping voltammetric detection (ASV). The method could specifically quantify miR-16 over a 6-decade dynamic range and reach an ultra-low detection limit of 0.32 aM.

Novel animal papillomavirus sequences and accurate phylogenetic placement

All amniotes are probably infected by specific papillomaviruses (PVs), but knowledge about PV diversity remains sparse. An insufficient taxon sampling, and a focus on humans as hosts, may perturb phylogenetic analyses leading to wrong conclusions about PV evolution. We performed a systematic approach to explore the diversity of PVs combining rolling circle amplification with the use of “universal” primers to search for the presence of novel PV sequences in animal samples. We communicate 12 sequences putatively corresponding to novel PVs gained from 10 host species in eight mammal families: Bovidae, Canidae, Cervidae, Equidae, Hominidae, Phocoenidae, Procyonidae and Pteropodidae. The phylogenetic position of the new sequences was inferred with an evolutionary placement algorithm under a Maximum Likelihood framework using a pre-computed, well-resolved tree constructed with the E1–E2–L1 gene sequences as a backbone. The new sequences were phylogenetically diverse and could be respectively placed with confidence within all four PV crown groups. The prevailing presence of sequences from the crown groups Alpha+Omikron-PVs and Beta+Xi-PVs may correspond to an increased viral diversity in these taxa, or rather reflect a combination of anthropocentric bias and preferential amplification from commonly used “universal” primers. Our results combined with literature data support the view that the number and diversity of animal PVs is overwhelmingly large.

Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR

Background The study aimed to develop an effective method to quantitate HBV covalently closed circular DNA (cccDNA) using small section of formalin fixed paraffin-embedded (FFPE) liver biopsy. Methods Plasmid-safe ATP-dependent DNase (PSAD)-treated samples were subjected to rolling circle amplification (RCA) prior to real-time PCR mediated by cccDNA-selective primers. Human beta-actin gene was used as a reference control. Results Compared to the classical method, i.e., PSAD digestion + real-time PCR, introduction of RCA increased the lower limit of detection for about 2 logs with good inter- and intra-assay reproducibility. HBV cccDNA was detected in 91.5% (119/130) of the FFPE samples. The cccDNA levels (copy/cell) between FFPE liver tissues and fresh frozen counterpart tissues were comparable. The median of cccDNA level in HBeAg-positive patients was higher than that in HBeAg-negative ones (52.60 vs. 31.25 copies/cell, P < 0.01). Intrahepatic cccDNA level was positively correlated with intrahepatic HBV total DNA level, but not obviously correlated with serum HBV DNA or alanine aminotransferase levels. Conclusions The method could sensitively and specifically quantitate intrahepatic HBV cccDNA in micro FFPE liver biopsy tissue for evaluation of HBV replication status in the liver.

Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA

To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA). HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA. The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases. The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.

Genomic diversity of sweet potato geminiviruses in a Brazilian germplasm bank

Begomoviruses cause major diseases of sweet potato worldwide impairing considerably the yields of this important food staple. Since sweet potato plants are vegetatively propagated and globally transported, they are prone to accumulate and disseminate geminiviruses. Effective diagnostic tools are, therefore, desirable. We studied the genomic diversity of geminiviruses present in naturally-infected sweet potato accessions belonging to a Brazilian germplasm bank collection. Fifty-five samples from different sweet potato accessions displaying geminivirus-like symptoms were analyzed by combining rolling circle amplification (RCA) with restriction fragment length polymorphism (RFLP) and sequencing. The restriction enzyme MspI ( HpaII) revealed diverse band patterns in 55 samples and digestion with BamHI, SstI or PstI resulted in full-length sweet potato geminivirus DNAs of about 3 kb in 46 samples. In addition, smaller fragments were identified as either viral “Defective DNAs” (D-DNAs) or mitochondrial plasmid DNAs. The diversity of sweet potato-associated geminiviruses was found to be very high under Brazilian conditions. Representative viral full-length DNAs have been cloned and sequenced yielding two new tentative species, three strains and several variants of previously described sweet potato geminiviruses. Sequence comparisons identified footprints of recombination in their genomes underscoring the risk of generating new geminiviruses in vegetatively propagated germplasm bank material. The sites of recombination were found in conjunction with predicted hairpin structures. We propose diagnostic routines to screen germplasm bank material for geminiviruses by the rapid and reliable RCA/RFLP as the technique of choice.

Classification of scleroderma

Asystasia gangetica (Acanthaceae) from tropical Africa and Asia is used as source of food and for medical applications. Plants collected in West Africa in the 1980s with typical geminivirus symptoms showed an unusual symptom segregation that included vein yellowing, curling and mosaic, which were present simultaneously or separately on different leaves of the same plant or on different plants propagated as cuttings from a single plant. Rolling-circle amplification in combination with restriction fragment length polymorphism analysis followed by deep sequencing of the RCA products identified two geminiviruses in these plants. One with a bipartite genome, Asystasia begomovirus 1, and the other with a monopartite genome together with its defective DNA, Asystasia begomovirus 2. The relationship between leaf symptoms and virus distribution under different light regimes was investigated, and showed for the first time an unusual segregation of symptoms and viruses, either within a single plant, or even within a leaf.