Rolling Circle Amplification Products Research Articles

Special RCA based sensitive point‐of‐care detection of HPV mRNA for cervical cancer screening

AbstractDeveloping the sensitive point‐of‐care testing (POCT) of oncogenic nucleic acids from human papillomavirus (HPV) infection is essential in preventing cervical cancer, especially in resource‐limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT via nucleic acid‐based aggregation under isothermal conditions. However, the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity. Here, a minimum secondary structured RCA technique (MSS‐RCA) is developed by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions. The amplification efficiency of MSS‐RCA could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS‐RCA, HPV16 E6/E7 nucleic acid target was detected with a personal glucose meter (PGM) with a sensitivity of 5 fm (50 zmol in 10 µL). This integrated MSS‐RCA‐PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00% and negative predictive values of 96.00% (77.77% to 99.40%, 95% CI). MSS‐RCA‐PGM provides a sensitive POCT platform for the detection of nucleic acid biomarkers for screening of cervical cancer or other diseases.

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer.

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL-1, 0.77 ng∙μL-1, and 1.1 ng∙μL-1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL-1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.

Nonribosomal Peptide Synthetase Specific Genome Amplification Using Rolling Circle Amplification for Targeted Gene Sequencing.

Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using Pseudomonas fluorescens ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and Escherichia coli (non-target), and a specific primer designed for the NRPS was used for the RCA reaction. The resulting RCA product (RCP) amplified only the Pseudomonas genome. The NRPS was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and NRPS sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of NRPS in unknown bacteria, including viable but non-culturable bacteria.

First report of sweepoviruses infecting Ipomeas batatas L. cultivars and landraces in Trinidad.

Root crops, referred to as ground provisions in the Caribbean, are traditional staples in Trinidad. One widely consumed example is sweet potato (Ipomeas batatas L.). The crop is mainly produced by subsistence farming which together with imports from neighboring Caribbean countries meet domestic demand (Singh et al. 2008). The Central Experiment Station, situated in the eastern part of Trinidad, maintains a sweet potato germplasm collection comprising both imported and locally-sourced landraces for cultivar development and distribution of propagules. In May 2017 chlorosis and leaf curling symptoms, typically associated with sweepoviruses, were observed on imported cultivars, Centennial, Jewel, 86 BM 31, TIB 313, TIB 8 21 1, and S128, and the landraces, Kick Up Jenny, John, and Carrot. Leaf samples from these nine symptomatic plants were collected for analysis, along with samples from the asymptomatic landrace, Chickenfoot. Total nucleic acids were extracted (Sharma et al. 2008) and the samples were assayed by PCR using degenerate primers SPG1 and SPG2 (Li et al. 2004) that target the replication associated protein gene (ORF C1), a highly conserved region of sweepoviruses. Amplicons of 912-bp were obtained from two of the nine symptomatic plants (TIB 8 21 1, Kick Up Jenny), but not from the asymptomatic Chickenfoot. The same samples were assayed by PCR amplification using primers SpvF and SpvR (Avelar 2015) which are specific to a highly conserved 632-bp region of the coat protein gene (ORF V1) of sweet potato leaf curl virus (SPLCV). All 10 samples tested positive for SPLCV, including the asymptomatic landrace, Chickenfoot. The ORF V1 PCR products from three of the 10 samples, namely Chickenfoot, TIB 8 21 1, and Kick Up Jenny, were cloned and sequenced (two clones per sample). Comparison of the sequences (GenBank accession nos. OR882007 [Chickenfoot], OR913125 [TIB 8 21 1] and OR913126 [Kick Up Jenny]) identified up to 4% nt sequence variability between samples. In BLASTn analysis, they were most closely related to the SPLCV isolate China:Sichuan (GenBank accession no. KJ013557), sharing 94 to 98% nt identity. Total nucleic extracts from one representative sample (TIB 8 21 1) was used as template for rolling circle amplification (RCA, TempliPhi Amplification Kit, GE Healthcare Life Sciences, Piscataway, NJ, USA). Digestion of the RCA product with StuI (Thermo Scientific, MA, USA) yielded ~2.8 kb DNA fragments indicative of monomeric full length genomes. Digested fragments were cloned, completely sequenced and deposited in GenBank under the accession nos. OR866202 (2,821 nts) and OR866203 (2,828 nts). Two species of sweepoviruses were detected. In BLASTn analysis, OR866202 showed 95% nt identity with sweet potato golden vein associated virus (SPGVaV) US:MS:1B-3 (GenBank accession no. HQ333143.1) which is a recombinant virus comprised of SPLCV and sweet potato leaf curl Georgia virus (SPLCGV) (Zhang and Ling 2011) and in BLASTx analysis OR866202 was most similar (92-99%) to SPLCV isolates from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ACY79479.1), Peru (GenBank accession no. ACY79466.1) and China (GenBank accession nos. ACY79439.1). OR866203 shared 96% nt identity with SPLCV China:Henan25(8):2012 (GenBank accession no. KF040465.1) in BLASTn analysis and BLASTx analysis revealed ≥ 94% aa sequence identity with SPLCV from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ADZ96559.1), Peru (GenBank accession no. ACY79479.1), China (GenBank accession no. ACY79466.1). and Spain (GenBank accession no. QWQ56365.1). Both Trinidad isolates also showed 90-96% nt identity with SPLCV from Korea (GenBank accession no.s KT992061.1, KT992064.1, unpublished). This is the first detection of sweepoviruses in Trinidad. SPGVaV has been reported in Brazil, the United States and Korea (Kil et al. 2014), while SPLCV has been described in other Caribbean islands, including Cuba, Jamaica, Puerto Rico, St. Vincent (Cuellar et al. 2015), and Barbados (Alleyne et al. 2019), as well as several countries in South America. Although Koch's postulates were not completed, our findings suggest that sweet potato crops in Trinidad harbor sweepoviruses, notwithstanding efforts to distribute pathogen-free materials and, in some instances, the apparent absence of visible symptoms on infected plants. Further studies on the management and impact of these viruses are necessary, including their prevalence in the sweet potato production regions of Trinidad.

Hyperbranched RCA-mediated construction of a dendritic DNA nanostructure for ratiometric fluorescence biosensing

As a robust isothermal strand amplification technique, rolling circle amplification (RCA) is popularly adopted for constructing various homogeneous fluorescence biosensing strategies. However, the limited functional unit decoration on linear RCA products and the slow homogeneous reaction kinetics usually restrict their performance. Herein, by designing a target biorecognition-triggered hyperbranched RCA reaction to construct an Mg2+-dependent DNAzyme (MNAzyme)-decorated dendritic DNA nanostructure, a novel ratiometric fluorescence biosensing method was proposed. The catalyzed cleavage of MNAzymes toward a short stand labeled with the FAM fluorophore and its quencher realized a “signal-on” fluorescence output. Meanwhile, a “signal-off” fluorescence output of 2-aminopurine (2-AP) labeled at a DNA hairpin was realized through the DNA hybridization with the nanostructure. Besides efficient acceleration of homogeneous reaction kinetics, these fluorescence responses were dramatically amplified by the high decoration of MNAzymes on the dendritic nanostructure and its strong signal suppression toward 2-AP. Together with the exonuclease III-amplified target biorecognition reaction, a very low detection limit of 0.53 fg mL–1 and a six-order magnitude linear range were endowed for kanamycin antibiotic assay. This excellent analytical performance and its high selectivity, and good operability determine promising prospect of the method for practical applications.

High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

High‐Throughput Single‐Cell, Single‐Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics

AbstractThere is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single‐cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single‐cell genomic, RNA, and protein analysis, their application to sub‐cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose‐based droplet microfluidic approach for single‐cell, single‐mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi‐step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single‐mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design.

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

The construction of COFs functionalized CRISPR electrochemical sensor for ultrasensitive detection of bacteria by hyper-branched rolling circle amplification

Rapid, sensitive, and low-cost foodborne pathogens detection is crucial for dealing with foodborne diseases caused by bacteria. In this work, we designed a new CRISPR/Cas9-mediated electrochemical biosensor for ultrasensitive detection of Escherichia coli (E. coli) based on multifunctional covalent organic framework-based nanocomposite (GOx-AuNPs-COF-H2) and hyperbranched rolling circle amplification (HB-RCA) reaction. When specific DNA targets were present in the system, the targets could be recognized and cleaved by CRISPR-Cas9 system, which triggered strand displacement reaction (SDR) and HB-RCA reaction. By introducing GOx-AuNPs-COF-H2 nanocomposite, H2 probe could hybridize with RCA products to bind to the electrode surface. After added glucose solution, GOx could catalyze it in the detection system and thus offered significant current signals for bacteria detection. This approach showed high analytical performance for assay of E. coli with the detection limit (LOD) of 10 CFU/mL. Furthermore, real samples were detected by this approach, demonstrating its promising potential for foodborne pathogens detection in food monitoring and clinical diagnosis.

Sensitive, Low-Background-Signal miRNA Analysis via the Self-Priming-Initiated Color Reaction Loaded on a Rolling Circle Amplification Product.

MicroRNAs (miRNAs) have been regarded as potential biomarkers in evaluating various diseases, such as pregnancy-induced hypertension and cancers. However, sensitive and reliable miRNA detection is still a challenge due to the low amplification efficiency and high background signal. Herein, we developed a colorimetric method for miRNA detection utilizing the self-priming-initiated color reaction loaded on a rolling circle amplification (RCA) product. In this method, a biotin-labeled RCA product is fixed on the surface of the streptavidin-coated wells, and the interfering components in samples are removed to avoid false reactions, thus reducing the background signals. Two signal amplification processes, including RCA and self-priming-initiated chain extension, endow the method with high sensitivity and a low limit of detection at the 10 fM level. In conclusion, our approach offers a promising perspective on sensitive and reliable miRNA detection and has the potential to be further utilized in biomedical research and early cancer detection.

Template-controllable rolling circle amplification for dual protein sensitive analysis.

Conjoint analysis of multiple protein biomarkers can improve the accuracy of disease analysis. Rolling circle amplification (RCA) generates different products by designing circular templates, which can subsequently bind with specific probes to generate various fluorescence signals; thus, it has potential for application in the analysis of various protein biomarkers. Current RCA approaches to detect proteins commonly follow an indirect primer-controlled RCA mode. And the molecular beacon probe combines with RCA products through free collision to generate signals, resulting in lower reaction efficiency. Herein, we propose a direct template-controlled RCA mode using nanosheets as carriers and quenchers for fluorescent probes to simultaneously detect two protein biomarkers. A dual functional magnetic bead was first designed to recognize and capture two proteins while releasing two templates for subsequent RCA. RCA products compete with probes loaded on two-dimensional metal-organic framework nanosheets for hybridization, completing the transition from single-stranded to double-stranded DNA. Double-stranded DNA is far from the nanosheets, and the recovered fluorescence signal can be used to evaluate the concentration of target proteins. This method exhibits excellent analytical performance and can successfully achieve the analysis of Tau and AβO in Alzheimer's disease clinical cerebrospinal fluid samples.

The single strand template shortening strategy improves the sensitivity and specificity of solid-state nanopore detection.

Through controlling the ssDNA product length of rolling circle amplification with AcyNTP, here we develop a nanopore signal enhancement strategy (STSS), which can successfully transfer the short oligonucleotide targets into long ssDNAs with appropriate lengths that can generate significant translocation currents. By labelling the RCA product with tags such as tetrahedral structures and isothermal amplicons, the resolution, signal specificity, and target range of the STSS can be further extended.

Cas-Rainbow: Cas12a-driven single-reaction multiplex detection system

Recently, CRISPR/Cas system has garnered considerable attention owing to its high cleavage efficiency. However, it lacks the ability to differentiate between signal sources. To address this, we have developed a multiplex detection system called Cas-Rainbow. This system combines CRISPR/Cas with rolling circle amplification (RCA) and hybridization chain reaction (HCR) to enable the detection of multiple miRNAs using reporter probes labeled with different fluorescent groups. The RCA product can trigger the formation of HCR and activate the Cas12a/crRNA complex, achieving the simultaneous detection of multiple miRNAs. Detection limits of three miRNAs (miR-155, miR-10b, and miR-21) were 33 fM, 41 fM, and 79 fM, respectively. Cas-Rainbow system could effectively identify these three miRNAs in serum, distinguishing breast cancer patients from healthy donors with accuracy of 100 %. Moreover, this system exhibited exceptional performance in logic gate circuits, showcasing its remarkable versatility and robustness. In summary, our proposed method offers a new approach for multiplex detection based on the CRISPR/Cas system.

Label-free fluorescence detection of human 8-oxoguanine DNA glycosylase activity amplified by target-induced rolling circle amplification

BackgroundHuman 8-oxoG DNA glycosylase 1 (hOGG1) is one of the important members of DNA glycosylase for Base excision repair (BER), the abnormal activity of which can lead to the failure of BER and the appearance of various diseases, such as breast cancer, bladder cancer, Parkinson's disease and lung cancer. Therefore, it is important to detect the activity of hOGG1. However, traditional detection methods suffer from time consuming, complicated operation, high false positive results and low sensitivity. Thus, it remains a challenge to develop simple and sensitive hOGG1 analysis strategies to facilitate early diagnosis and treatment of the relative disease. ResultsA target-induced rolling circle amplification (TIRCA) strategy for label-free fluorescence detection of hOGG1 activity was proposed with high sensitivity and specificity. The TIRCA strategy was constructed by a hairpin probe (HP) containing 8-oxoG site and a primer probe (PP). In the presence of hOGG1, the HP transformed into dumbbell DNA probe (DDP) after the 8-oxoG site of which was removed. Then the DDP formed closed circular dumbbell probe (CCDP) by ligase. CCDP could be used as amplification template of RCA to trigger RCA. The RCA products containing repeated G4 sequences could combine with ThT to produce enhanced fluorescence, achieving label-free fluorescence sensing of hOGG1. Given the high amplification efficiency of RCA and the high fluorescence quantum yield of the G4/ThT, the proposed TIRCA achieved highly sensitive measurement of hOGG1 activity with a detection limit of 0.00143 U/mL. The TIRCA strategy also exhibited excellent specificity for hOGG1 analysis over other interference enzymes. SignificanceThis novel TIRCA strategy demonstrates high sensitivity and high specificity for the detection of hOGG1, which has also been successfully used for the screening of inhibitors and the analysis of hOGG1 in real samples. We believe that this TIRCA strategy provides new insight into the use of the isothermal nucleic acid amplification as a useful tool for hOGG1 detection and will play an important role in disease early diagnosis and treatment.

Ratiometric Fluorescence Biosensing of Tandem Biemissive Ag Clusters Boosted by Confined Catalytic DNA Assembly.

The reaction kinetics and yield of traditional DNA assembly with a low local concentration in homogeneous solution remain challenging. Exploring confined catalytic DNA assembly (CCDA) is intriguing to boost the reaction rate and efficacy for creating rapid and sensitive biosensing platforms. A rolling circle amplification (RCA) product containing multiple tandem repeats is a natural scaffold capable of guiding the periodic assembly of customized functional probes at precise sites. Here, we present a RCA-confined CCDA strategy to speed up amplifiable conversion for ratiometric fluorescent sensing of a sequence-specific inducer (I*) by using string green-/red-Ag clusters (sgAgCs and srAgCs) as two counterbalance emitters. Upon recognition of I*, CCDA events are operated by two toehold-mediated strand displacements and localized in repetitive units, thereby releasing I* for recycled signal amplification in the as-grown RCA concatemer. The local concentration of reactive species is increased to facilitate rapider dsDNA complex assembly and more efficient input-output conversion, on which the clustering template sequences of sgAgCs and srAgCs are blocked and opened, enabling srAgCs synthesis but opposite to sgAgCs. Thus, the fluorescence emission of srAgCs goes up, while sgAgCs go down. With the resultant ratio featuring inherent built-in correction, rapid, sensitive, and accurate quantification of I* at the picomolar level is achieved. Benefiting from efficient RCA confinement to enhance reaction kinetics and conversion yield, this CCDA-based strategy provides a new paradigm for developing simple and diverse biosensing methodologies.

A Colorimetric Biosensing Platform with Aptamers, Rolling Circle Amplification and Urease-Mediated Litmus Test.

Here we report on an ultra-sensitive colorimetric sensing platform that takes advantage of both the strong amplification power of rolling circle amplification (RCA) and the high efficiency of asimple urease-mediated litmus test.The presence of atarget triggers the RCA reaction, and urease-labelled DNA can hybridize to the biotinylated RCA products and beimmobilized onto streptavidin-coated magnetic beads. The urease-laden beads are then used to hydrolyze urea, leading to an increase in pH that can be detected by a simple litmus test. We show this sensing platform can be easily integrated with aptamers for sensing diverse targets via the detection of human thrombin and platelet-derived growth factor (PDGF) utilizing structure-switching aptamers as well as SARS-CoV-2 in human saliva using a spike-binding trimeric DNA aptamer. Furthermore, we demonstrate that this colorimetric sensing platform can be integrated into a simple paper-based device for sensing applications.

A Colorimetric Biosensing Platform with Aptamers, Rolling Circle Amplification and Urease‐Mediated Litmus Test

AbstractHere we report on an ultra‐sensitive colorimetric sensing platform that takes advantage of both the strong amplification power of rolling circle amplification (RCA) and the high efficiency of a simple urease‐mediated litmus test. The presence of a target triggers the RCA reaction, and urease‐labelled DNA can hybridize to the biotinylated RCA products and be immobilized onto streptavidin‐coated magnetic beads. The urease‐laden beads are then used to hydrolyze urea, leading to an increase in pH that can be detected by a simple litmus test. We show this sensing platform can be easily integrated with aptamers for sensing diverse targets via the detection of human thrombin and platelet‐derived growth factor (PDGF) utilizing structure‐switching aptamers as well as SARS‐CoV‐2 in human saliva using a spike‐binding trimeric DNA aptamer. Furthermore, we demonstrate that this colorimetric sensing platform can be integrated into a simple paper‐based device for sensing applications.

Programmable all-DNA hydrogels based on rolling circle and multiprimed chain amplification products.

Soft, biocompatible, and tunable materials offer biomedical engineers and material scientists programmable matrices for a variety of biomedical applications. In this regard, DNA hydrogels have emerged as highly promising biomaterials that offer programmable self-assembly, superior biocompatibility, and the presence of specific molecular identifiable structures. Many types of DNA hydrogels have been developed, yet the programmability of the DNA building blocks has not been fully exploited, and further efforts must be directed toward understanding how to finely tune their properties in a predictable manner. Herein, we develop physically crosslinked all-DNA hydrogels with tunable morphology and controllable biodegradation, based on rolling circle amplification and multiprimed chain amplification products. Through molecular engineering of the DNA sequences and their nano-/microscale architectures, the precursors self-assemble in a controlled manner to produce soft hydrogels in an efficient, cost-effective, and highly tunable manner. Notably, we develop a novel DNA microladder architecture that serves as a framework for modulating the hydrogel properties, including over an order of magnitude change in pore size and up to 50% change in biodegradation rate. Overall, we demonstrate how the properties of this DNA-based biomaterial can be tuned by modulating the amounts of rigid double-stranded DNA chains compared to flexible single-stranded DNA chains, as well as through the precursor architecture. Ultimately, this work opens new avenues for the development of programmable and biodegradable soft materials in which DNA functions not only as a store of genetic information but also as a versatile polymeric biomaterial and molecularly engineered macroscale scaffold.

Simplified rolling circle amplification driven in situ generation of electrochemiluminescent silver nanoclusters for APE1 activity monitoring

Apurinic/apyrimidinic endonuclease 1 (APE1) plays an essential part in the base excision repair whose abnormal expression is closely correlated to the occurrence of tumors. Therefore, a sensitive assay of APE1 is crucial for the early diagnosis of cancer. Herein, we developed a simplified rolling circle amplification (RCA) strategy to drive in situ production of electrochemiluminescent (ECL) silver nanoclusters (Ag NCs) for APE1 activity monitoring. Unlike conventional RCA, which required locker DNA and padlock to form the circular template and hybridize with primer to initiate amplification, we proposed a simplified RCA that employed hairpin (HP) and padlock to directly form the circular template to initiate the amplification. Through reasonable design of the amplification templates, the RCA products were cytosine-rich (C-rich) sequences for silver ions adsorption, which were electrochemically reduced to Ag NCs. Combined with Cu-based metal-organic frameworks (Cu-MOFs) as co-reaction accelerator to facilitate the reduction of S2O82-, the ternary ECL system (Ag NCs/S2O82-/Cu-MOFs) demonstrated strong ECL intensity. The simplified RCA strategy combined with the ternary ECL sensing platform achieved the sensitive analysis of APE1 with a linear range of 1 × 10−8 to 1 × 10−2 U/μL and a detection limit of 2.29 × 10−9 U/μL, which presented a novel approach for sensitive monitoring of APE1 activity.

First Report of Tomato Leaf Curl Cebu Virus Associated with Stachytarpheta jamaicensis Golden Yellow Mosaic Disease in Taiwan.

Stachytarpheta jamaicensis (L.) Vahl, also known as snack weed, is an exotic plant in Taiwan. In April 2021, severe golden yellow mosaic leaves (Fig. S1) were observed on S. jamaicensis plants in Taichung City, Taiwan. Samples from eight symptomatic and two asymptomatic plants were collected from the public flowerbed. Total DNA was extracted from each of the collected samples by using a modified CTAB method (Echevarría-Machado et al. 2005). PCR with Begomovirus degenerate primers (PAL1v1978/PAR1c715; Rojas et al. 1993) was conducted. The expected 1.5-kb fragment was amplified only from the 8 symptomatic samples. Two randomly selected amplicons were cloned on pCRII-TOPO TA vector (Invitrogen Co., San Diego, CA, USA) and sequenced with the ABI3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) at National Chung Hsing University (NCHU). After NCBI BLASTn analysis, the sequences were shown to be most closely related to tomato leaf curl Cebu virus (ToLCCeV) isolates (EU487042, EU487025, KU946997), with 92.4-92.5% nucleotide sequence identity by using the CLUSTAL W method of MegAlign program (DNASTAR, Inc., Madison, WI, USA). A ToLCCeV specific primer pair (FJJ2021-165 /166 5'-ACTTACAGGCCCATGTATCG-3' / 5'-GAATGGGTATCCGAGCACG-3') was designed to amplify and sequence the remaining half of viral DNA. The expected 1.6-kb amplicon was amplified only from the symptomatic samples. The full-length of DNA-A consisted of 2.7-kb nucleotides (ToLCCeV isolate stachy, ON525110 and ON525111) and contained six open reading frames (two in viral sense, V1 to V2 and four in the viral complementary sense, C1 to C4) and the conserved nonanucleotide motif (TAATATTAC). The full-length DNA-A of ToLCCeV stachy isolates shared 99.9% nucleotide identity to each other and 91.2-92.4% and 91.3-92.5% nucleotide identities to other ToLCCeV isolates (EU487042, EU487025, KU946997) available in NCBI GenBank. Besides, ToLCCeV is a monopartite begomovirus that harbors no DNA-B. Thus, there were no bands amplified from the degenerate primer pair for DNA-B (DNABLC2 / DNABLV2; Green et al. 2001). Furthermore, the infectious clone was constructed by using phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) for rolling circle amplification (RCA). The RCA product was partially digested with ApaI (NEB) and ligated into the binary vector pCambia0380 (AF234290). The resulting recombinant vector was transformed into Agrobacterium tumefaciens C58. A. tumefaciens C58, containing the infectious ToLCCeV-Stachy DNA-A vector, was grown overnight in LB broth containing kanamycine (50 μg/ml) at 28°C. S. jamaicensis and Nicotiana benthamiana (Nb, four to six leaf stage) plants were agroinoculated to confirm the infectivity of the ToLCCeV clone. The leaf curling and blister symptoms were observed on the Nb systemic leaves 17-day post inoculation (dpi) and the golden yellow mosaic symptom noticed on S. jamaicensis systemic leaves 30-dpi. The presence of the viral DNA in the inoculated plants was confirmed by PCR using the specific primer pair of ToLCCeV. To the best of our knowledge, this is the first report of the monopartite begomovirus, ToLCCeV, associated with golden yellow mosaic disease in S. jamaicensis in Taiwan. The existence of ToLCCeV might severely impact the tomato and pepper industry because they are the natural hosts of ToLCCeV (Tsai et al. 2011) and ToLCCeV may be transmitted by the whitefly, Bemisia tabaci, in Taiwan (Ko et al. 2005).