Rolling Circle Amplification Products Research Articles

Self-Assembled mRNA-Responsive DNA Nanosphere for Bioimaging and Cancer Therapy in Drug-Resistant Cells

DNA assembly has provided new opportunities for the development of a novel drug delivery system (DDS) for real-time monitoring and precision treatment of cancer lesions. Herein, we propose mRNA-responsive DNA nanospheres (DNA-NS), whose self-assembly can be triggered by products of rolling circle amplification and functional hairpins and deliver anticancer drug doxorubicin (DOX) for bioimaging and cancer therapy. It has been demonstrated that DNA-NS exhibited good stability in biological environments. Hence, DNA-NS can serve as a universal platform of detections of mRNA related to various tumor cells. DNA-NS can also be applied in the mRNA-dependent DDS. For drug-resistant cells, which are widely present in actual cancer models, DNA-NS can effectively overcome the efflux action of drug-resistant cells to improve the therapeutic efficacy of DOX. In summary, this study provides a potential strategy for constructing the endogenous mRNA-responsive DDS for cancer diagnosis and chemotherapy in vivo.

Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography

Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies.Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of ~1 μm-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes.The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.

A multiplex platform for digital measurement of circular DNA reaction products.

Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold ∼100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold ∼500 ng/l), using the same antibody pair.

In Situ Formation of Multifunctional DNA Nanospheres for a Sensitive and Accurate Dual-Mode Biosensor for Photoelectrochemical and Electrochemical Assay.

Herein a photoelectrochemical (PEC) and electrochemical (EC) dual-mode biosensor with cationic N,N-bis(2-(trimethylammonium iodide)propylene)perylene-3,4,9,10-tetracarboxydiimide (PDA+)-decorated multifunctional DNA spheres in situ generated on an electrode was proposed for sensitive and accurate detection of miRNA-141. By employing a target-related ternary "Y" structure cleavage cycling reaction, the target DNA was converted into massive output DNA anchored on a TiO2 substrate, and hence triggering the rolling circle amplification (RCA) reaction. Upon addition of magnesium ions and PDA+, the long DNA tails of the RCA product were condensed in situ to form multifunctional DNA spheres. Notably, the distance between DNA spheres and TiO2 substrate was short, thus forming an effective PDA+-TiO2 sensitization structure with fast electron transfer for acquiring an extremely enhanced PEC signal with assistance of ascorbic acid (AA). Meanwhile, cationic PDA+ with a large planar π-π skeleton enabled favorable redox-activity and substantial loading on DNA spheres, directly producing an obviously well-defined cathodic peak for implementing EC biodetection on the same sensing platform. This approach not only avoided difficult assembly of diverse signal indicators but also significantly improved the sensitivity by utilizing cleavage cycling amplification and RCA strategies. Moreover, the distinct dual-response signals from two different transduction mechanisms and independent signal transduction can mutually support accuracy improvement. As a result, detection ranges of 0.1 fM to 1 nM for PEC and 2 fM to 500 pM for EC were obtained for miRNA-141, providing a universal and efficient biosensing method with promising applications in bioanalysis and early disease diagnosis.

Real‐time analysis of switchable nanocomposites of magnesium pyrophosphates and rolling circle amplification products

AbstractRolling circle amplification (RCA) is a robust isothermal nucleic acid amplification method producing nanocomposites of DNA and inorganic magnesium pyrophosphate precipitates. Although the conformation and structure of such nanocomposites impact applications, most studies of them have been performed at the end‐point after exposure to a treatment. Here, we use real‐time optomagnetic measurements of the hydrodynamic size of magnetic nanoparticles (MNPs) to study the growth of RCA products grafted onto MNPs as well as the effect of post‐RCA temperature annealing and chemical treatment. As a key result, we show that secondary structures in the RCA products trap and partially protect magnesium pyrophosphate precipitates and that these are reversibly released upon heating above a characteristic temperature defined by the sequence of the RCA product. These findings provide a deeper mechanistic understanding of the synthesis and structure of DNA nanocomposites, which impacts applications of DNA nanocomposites in sensing and drug delivery.

A novel fluorescent aptasensor for aflatoxin M1 detection using rolling circle amplification and g-C3N4 as fluorescence quencher

Aflatoxin M1 is a prevailing natural carcinogenic mycotoxin, found in milk. Herein, we reported a sensitive fluorescent aptasensor for AFM1, based on rolling circle amplification (RCA) technology to improve the sensitivity of the assay. The detection of AFM1 was achieved using KYF4: Eu3+ time-resolved fluorescent nanoparticles (KYF4: Eu3+ TRFNPs) as signal probe and graphitic carbon nitride (g-C3N4) nanosheet as a quencher. The AFM1 aptamer act as a recognition element and as a primer for DNA synthesis. TRFNPs attached to complementary DNA (TRFNPs-cDNA) acts as a signal probe. In the absence of target, a rolling circle template (RCT) ligated to aptamer and RCA reaction initiated. The TRFNPs-cDNA was strengthened to amplified RCA product (RCAP) to make RCAP/TRFNPs-cDNA duplex. This duplex cannot be adsorbed onto g-C3N4, and ultimately no fluorescence quenched. In the presence of AFM1, aptamer recognized the target and aptamer-target complex was formed. Hence, no aptamer hybridized with RCT, therefore, no RCA and duplex formation. Consequently, free TRFNPs-cDNA adsorbed onto g-C3N4 and resulted in quenched fluorescence. The assay provides a lower detection limit (0.0194 pg/mL) than the previously reported assays and has good recoveries between 92–99.8 %. Hence, the present method would be a suitable method for the quantification of small molecules.

Tomato Leaf Curl New Delhi Virus Found Associated with Eggplant Yellowing Disease in Italy

HomePlant DiseaseVol. 104, No. 7Tomato Leaf Curl New Delhi Virus Found Associated with Eggplant Yellowing Disease in Italy PreviousNext DISEASE NOTES OPENOpen Access licenseTomato Leaf Curl New Delhi Virus Found Associated with Eggplant Yellowing Disease in ItalyGiuseppe Parrella, Elisa Troiano, Sukchan Lee, and Eui-Joon KilGiuseppe Parrella†Corresponding author: G. Parrella; E-mail Address: giuseppe.parrella@ipsp.cnr.ithttp://orcid.org/0000-0002-0412-4014Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), 80055 Portici (NA), ItalySearch for more papers by this author, Elisa TroianoInstitute for Sustainable Plant Protection, National Research Council (IPSP-CNR), 80055 Portici (NA), ItalySearch for more papers by this author, Sukchan LeeCollege of Biotechnology and Bioengineering, Sungkyunkwan University, 16419 Suwon, KoreaSearch for more papers by this author, and Eui-Joon KilCollege of Biotechnology and Bioengineering, Sungkyunkwan University, 16419 Suwon, KoreaSearch for more papers by this author AffiliationsAuthors and Affiliations Giuseppe Parrella1 † Elisa Troiano1 Sukchan Lee2 Eui-Joon Kil2 1Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), 80055 Portici (NA), Italy 2College of Biotechnology and Bioengineering, Sungkyunkwan University, 16419 Suwon, Korea Published Online:12 May 2020https://doi.org/10.1094/PDIS-12-19-2635-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus (genus Begomovirus, family Geminiviridae), was originally described in tomato in India in 1995 (Padidam et al. 1995). The virus apparently remained confined to the Asian continent for about 20 years and has only recently been discovered in Europe. In Italy, ToLCNDV was detected for the first time in 2015 in zucchini squash in Sicily (Panno et al. 2016) and later on in other regions of continental Italy (Panno et al. 2019). Recently, epiphytotics of ToLCNDV were reported in central and south Italy, associated with yellowing and leaf curling symptoms in pepper crops (Luigi et al. 2019). In 2016, a large-scale survey was conducted to assess the distribution and the genetic diversity of the viral isolates spreading in Italy. Samples collected at that time included cucurbits and solanaceous plants, including five leaf samples from five distinct eggplant (Solanum melongena L.) plants of cultivar Violetta di Napoli that showed yellowing and light curling of the apical leaves and noticed in a cultivation located in Campania region (Castel Volturno municipality). A few Bemisia tabaci individuals were noticed associated to the cultivation. All five leaf samples tested positive for ToLCNDV with ImmunoStrip (Agdia, Elkhart, IN). These results were confirmed by PCR using ToLCNDV-specific primers TLCNDVCP1/TLCNDVCP2 (Parrella et al. 2018) on the total DNA extracted from each sample using an E.Z.N.A. Plant DNA kit (Omega Bio-tek, Norcross, GA). Amplicons of the expected size (∼1.0 kb) were obtained only from the five symptomatic plants, and the nucleotide sequences of these isolates were identical. One representative sample (Som-166/16) was selected for full-length amplification of the genome (DNA-A and DNA-B-like sequences) using the rolling circle amplification (RCA) method with an Illustra TempliPhi amplification kit (GE Healthcare, Piscataway, NJ), in accordance with the manufacturer’s instructions. RCA products were digested with different restriction endonucleases to obtain a 2.8-kb linear DNA fragment. Among the different enzymes tested, BamHI resulted in a maximum DNA fragment length of 2.8 kb, which was cloned into a BamHI-linearized pUC19 plasmid. The ligated products were transformed into a competent DH5α strain of Escherichia coli, and the positive clones were sequenced in both orientations at Microsynth Seqlab (Göttingen, Germany). The obtained full-length DNA-A (2,738 nt; GenBank MN782303) and DNA-B (2,686 nt; GenBank MN782304) sequences showed the highest percentage of nucleotide identity with the ToLCNDV Italian isolates Caa-164/16 (GenBank MK732932) for the DNA-A (99.82%) and Cum-45/16 (GenBank MF688671) for the DNA-B (99.48%). Eggplant leaf yellowing caused by a ToLCNDV variant has been previously described in India (HQ264185 for DNA-A and HQ264186 for DNA-B) (Pratap et al. 2011), but the Italian and the Indian isolates exhibited only 90.20 and 79.00% nucleotide identity with respect to the nucleotide sequences of DNA-A and DNA-B. The Som-166/16 isolate was very similar at the molecular level to previously reported isolates from Mediterranean countries and belonged to the European strain ToLCNDV-ES, because the percentage of nucleotide identity was 98.83% for DNA-A with the ToLCNDV-ES strain A-MU.13.ME/4.3 (GenBank MH577751) and 97.88% for DNA-B with the ToLCNDV-ES strain B-AL.15.ZU/2.1 (GenBank MH577658) (Moriones et al. 2017; Panno et al. 2019). The ToLCNDV-ES strain evolved from ToLCNDV isolates of Asian origin and adapted to infect cucurbits (Moriones et al. 2017). Nevertheless, this report presents further evidence, in addition to a previous report (Luigi et al. 2019), that ToLCNDV-ES isolates can potentially pose a threat not only to cucurbits but also to solanaceous crops as well, because this is the first finding of ToLCNDV in eggplant in Italy. Control measures against ToLCNDV are limited and mainly based on vector control, cultivation in protected environments, and the prompt elimination of infected materials.The author(s) declare no conflict of interest.

Simultaneously responsive microfluidic chip aptasensor for determination of kanamycin, aflatoxin M1, and 17β-estradiol based on magnetic tripartite DNA assembly nanostructure probes.

The authors describe a microfluidic chip-based aptasensor platform combined with magnetic tripartite DNA structure-functionalized nanocomposites to achieve simultaneous determination of kanamycin (KANA), aflatoxin M1 (AFM1), and 17β-estradiol (E2) in milk. The two-duplex tripartite DNA nanostructure was first assembled on the surface of magnetic beads. When the aptamer on the probes recognized the specific target, the aptamer-target would be released into the supernatant. The pre-primer@circular DNA template structure initiates rolling circle amplification (RCA) by phi29 polymerase. After magnetic separation, the magnetic nanocomposites were added into a solution containing three different lengths of complementary strands to the RCA products. The number of complementary strands significantly decrease, and this can be quantitated by the microfluidic chip. Further, the employment of magnetic nanocomposites and microfluidic chip not only resolve the complex matrix interference, but also dramatically enhances the determination selectivity and sensitivity. This aptasensor allows for determination of KANA, AFM1, and E2 with limits of detection as low as 0.32pgmL-1, 0.95pgmL-1, and 6.8pgmL-1, respectively. This novel method exhibits the advantages of excellent stability and fast response time (< 3min on microfluidic chip platform) for simultaneous determination of KANA, AFM1, and E2 in milk samples and ensures food safety. Graphical abstract.

Strand displacement-triggered G-quadruplex/rolling circle amplification strategy for the ultra-sensitive electrochemical sensing of exosomal microRNAs.

Emerging evidence suggests that exosomal microRNAs are potential biomarkers for the early diagnosis and prognostic assessment of tumor. Here, we design a strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy for highly specific and sensitive electrochemical sensing of exosomal microRNAs. In the presence of exosomal miRNA-21, a locked nucleic acid (LNA)-labeled toehold mediated strand displacement reaction (TMSDR) is initiated, releasing output P2 to trigger the subsequent RCA reaction by hybridizing with the C-rich circular template. Then the obtained G-rich RCA products can bind to the probe anchored on the surface of gold electrode and generate G-quadruplex conformations. Based on the TMSDR-triggered G-quadruplex/RCA strategy, the detection limit of this electrochemical biosensor is down to 2.75 fM. Moreover, our biosensor exhibits excellent repeatability, stability, and high consistency compared to RT-PCR for clinical detection. In conclusion, this assay is expected to provide a hopeful strategy for the early non-invasive diagnosis and prognostic estimation of cancer. Graphical abstract Schematic illustration of electrochemical sensing of exosomal microRNAs based on strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy.

Mechanistic investigation of bead-based padlock rolling circle amplification under molecular crowding conditions

We present a mechanistic investigation of bead-based padlock rolling circle amplification (RCA) under molecular crowding conditions, a sensitive and selective DNA detection method we have developed. Several important points to optimize the method were clarified: (i) the increase in the number of RCA products is proportional to the excluded volume of poly(ethylene glycol) (PEG), (ii) PEG facilitates ligation of padlock probe to form circular concatemers and monomers, both of which may act as a template for RCA, and (iii) hybridization of detection probe to the products may be facilitated at higher PEG concentrations.

CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification.

False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.

Rolling circle amplification based colorimetric determination of Staphylococcus aureus.

A colorimetric microplate assay for determination of Staphylococcus aureus DNAis described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.μL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination ofStaphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increasesensitivity.

On-Chip DNA Analysis of Tuberculosis Based on Magnetic Nanoparticle Clustering Induced by Rolling Circle Amplification Products

We present on-chip implementation of an isothermal nucleic acid amplification assay with optomagnetic detection. Isothermal rolling circle amplification (RCA) was designed for the highly specific detection of a single point mutation in the subsequence of the katG catalase peroxidase gene responsible for resistance against the antibiotic isoniazid in Mycobacterium tuberculosis . The assay was integrated using streptavidin-coated magnetic microbeads (MMBs) as a movable substrate to transport the synthetic katG target and products between the sequential processing steps. RCA products were detected via optomagnetic measurements of the binding of functionalized magnetic nanoparticles (MNPs). Binding of MNPs to RCA products leads to an increase of their hydrodynamic size and consequently a shift of the optomagnetic signal to lower frequencies. We first analyzed the affinity of DNA probes used for target capture on MMBs and binding of MNPs to RCA products, respectively. This enabled us to choose conditions where amplification products were released from the MMBs and where their binding to MNPs was optimal. The optimized assay was transferred to a polymer chip with three connected chambers filled with reaction buffers for capture, RCA, and detection. The chip was operated in a setup integrating temperature control, transport of the DNA on magnetic carriers, and dose-dependent detection of MNP clustering. For a total assay time of about 2 h, we demonstrate specific detection of the antibiotic resistance in a synthetic tuberculosis DNA target with a detection limit of 4 pM.

The sweet detection of rolling circle amplification: Glucose-based electrochemical genosensor for the detection of viral nucleic acid

Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 μg mL−1 of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.

Digital Rolling Circle Amplification–Based Detection of Ebola and Other Tropical Viruses

Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.

Label-free polygonal-plate fluorescent-hydrogel biosensor for ultrasensitive microRNA detection

Traditional methods for microRNA detection are always dependent on large-scale instruments, labelled DNAs or cumbersome processes, which cannot meet the requirements of on-site detection. To overcome above limitations, we proposed a label-free polygonal-plate fluorescent-hydrogel (PPFH) biosensor for on-site detection of microRNA. In our study, all experimental procedures were easy to handle and performed at a constant temperature without any precision instruments or labelled probes. The target miR-21 initiated the rolling circle amplification (RCA) reaction to produce extensive elongate DNA chains, which offered a facile, economical and efficient strategy to construct a new type of DNA hydrogel called polygonal-plate hydrogel. The RCA reaction endpoint can be judged by naked eye in unconstrained environment. Moreover, this turn-on fluorescent biosensor worked spontaneously by integrating thioflavin T (ThT) with the whole G-quadruplex which was obtained by hybridizing RCA products with a pair of split G-quadruplex probes. The fluorescence intensity of the system was in good linear correlation with the miR-21 concentration in the dynamic range of 0.5–50 nM and the limit of detection was calculated to be 5 pM. The feasibility was also confirmed by detecting spiked serum samples. These results exhibited high stability and dependable detection capability of the PPFH biosensor and provided sufficient support for the practical on-site detection. Moreover, a surprising finding showed the morphological structure of polygonal plates can be controlled by adjusting RCA reaction time and miR-21 concentration. Overall, the PPFH biosensor is friendlier and more affordable compared with traditional methods and makes much progress in on-site microRNA detection.

First Report of Tomato Leaf Curl New Delhi Virus Infecting Crossandra infundibuliformis in India

Crossandra infundibuliformis (L.), commonly known as firecracker flower, is a common flowering plant belonging to the family Acanthaceae. It is commonly cultivated in southern India for its attractive flowers. Symptoms of leaf yellowing, leaf mottling, leaf curling, and distortion as well as stunted growth and flower abortion were observed in C. infundibuliformis plants grown in Dharmapuri district (Tamil Nadu, India). In 2011, we observed the first symptom of leaf curl disease, and a partial sequence of tomato leaf curl New Delhi virus (ToLCNDV) was deposited in GenBank (JN410650). Further, in a survey conducted from 2013 to 2015, a total of 30 symptomatic samples suspected of begomovirus infection were collected along with a nonsymptomatic sample. In particular, intact twin icosahedral geminivirus particles (∼18 × 30 nm) were observed by high-resolution transmission electron microscopy in a partially purified extract from symptomatic leaves. Total DNA was extracted and subjected to PCR using coat protein gene-specific degenerate primers (FP/RP) (Riyaz et al. 2013) and replication-associated protein gene-specific degenerate primers ToLCNDVRepFP (5′-ATGAAGTAWGAACAGCCRCAC-3′) and ToLCNDVRepRP (5′-CCATCCGAACATTCAGGGAG-3′). PCR products of the expected sizes of ∼500 and ∼850 bp were obtained from 25 out of 30 plants, and respective PCR products were eluted from gels and sequenced. In sequence analysis, the isolate from C. infundibuliformis (CI1) showed 89 to 92% nucleotide identity with the ToLCNDV isolates reported earlier from India (KM269361, KM269355, and KP868764). Rolling circle amplification (RCA) was performed using a TempliPhi kit (GE Healthcare). RCA products were subjected to PCR amplification employing abutting primers AKKCI1F (5′-ACCTAATGGCAGAATTGTAATTTTG-3′) and AKKCI1R (5′-AATATTATACGAATGGCCGCTTTAG-3′) for DNA-A and BKKCI1F (5′-ACCGAAAGGCCGCGAAAATTTATCCTG-3′) and BKKCI1R (5′-AATATTAGACGAAAGGCCGCTATGA-3′) for DNA-B. The amplified PCR products were ligated into pJET1.2/blunt vector and cloned using a CloneJet PCR cloning kit (Thermo Scientific, U.S.A.). The clones were sequenced by primer walking, and the corresponding sequences assembled to DNA-A and DNA-B fragments were deposited under GenBank accessions MK395548 and MK395549, respectively. DNA-A (2,742 bp) showed 96% sequence identity with the DNA-A component of a ToLCNDV isolate infecting the bitter gourd crop in India (KP868764), whereas DNA-B (2,694 bp) showed 87% identity with the DNA-B component of a ToLCNDV isolate from India infecting chrysanthemum (MG758147). In addition, association of a betasatellite was also confirmed through PCR assay using universal primer pair Beta01/Beta02 (Briddon et al. 2002), followed by sequencing. The nucleotide sequence of the associated betasatellite (1,352 bp) shared 93.7% identity with the betasatellite component of tomato leaf curl New Delhi betasatellite (ToLCuNDB) reported from New Delhi (JX679002) on tomato. The betasatellite sequence was deposited in GenBank (MK040727). This study records the coexistence of bipartite ToLCNDV and ToLCuNDB in C. infundibuliformis with the evidence of their full-length genome for the first time from India.

Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman

BackgroundIn Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection.MethodsCircular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation.ResultsThe full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites.ConclusionsThe leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

Optomagnetic Detection of Rolling Circle Amplification Products.

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Enzymatically Synthesized DNA Polymer as Co-carrier for Enhanced RNA Interference.

Polymerization of small interfering RNA (siRNA) has been demonstrated as a promising strategy to improve siRNA delivery, which will change the low-charge and rigid properties of single siRNA and enhance its electrostatic interactions with cationic polymers. For such polymerization strategy, a major breakthrough is still needed to fully eliminate chemical processes and further improve the nanocomplex-forming ability of polymerized siRNAs. Herein, the extremely strong interaction between the DNA product of rolling circle amplification (RCA) and linear poly(ether imide) (PEI) has been disclosed; accordingly, a stable nanocomplex is formed just at its charge neutralization point, which benefits from the high molecular weight of the RCA product (>3 000 000 Da). In addition, as the sequence of the RCA product is determined by the cyclic template, the programmable nature of DNA can simplify the optimization process and maximize the hybridization efficiency between RCA and sticky siRNAs, realizing a superior siRNA polymerization efficiency. Depending on these two effects, the RCA DNA is utilized as a cocarrier material to organize siRNA polymerization and substantially reduce the usage amount of PEI, which greatly improves RNAi efficiency of PEI/RCA-siRNA polyplex both in vitro and in vivo, providing evidence that RCA DNA is a promising material to promote the RNAi-based therapeutics.