Circular Template Research Articles

A Multi-RNAi Microsponge Platform for Simultaneous Controlled Delivery of Multiple Small Interfering RNAs.

Packaging multiple small interfering RNA (siRNA) molecules into nanostructures at precisely defined ratios is a powerful delivery strategy for effective RNA interference (RNAi) therapy. We present a novel RNA nanotechnology based approach to produce multiple components of polymerized siRNA molecules that are simultaneously self‐assembled and densely packaged into composite sponge‐like porous microstructures (Multi‐RNAi‐MSs) by rolling circle transcription. The Multi‐RNAi‐MSs were designed to contain a combination of multiple polymeric siRNA molecules with precisely controlled stoichiometry within a singular microstructure by manipulating the types and ratios of the circular DNA templates. The Multi‐RNAi‐MSs were converted into nanosized complexes by polyelectrolyte condensation to manipulate their physicochemical properties (size, shape, and surface charge) for favorable delivery, while maintaining the multifunctional properties of the siRNAs for combined therapeutic effects. These Multi‐RNAi‐MS systems have great potential in RNAi‐mediated biomedical applications, for example, for the treatment of cancer, genetic disorders, and viral infections.

A Multi‐RNAi Microsponge Platform for Simultaneous Controlled Delivery of Multiple Small Interfering RNAs

AbstractPackaging multiple small interfering RNA (siRNA) molecules into nanostructures at precisely defined ratios is a powerful delivery strategy for effective RNA interference (RNAi) therapy. We present a novel RNA nanotechnology based approach to produce multiple components of polymerized siRNA molecules that are simultaneously self‐assembled and densely packaged into composite sponge‐like porous microstructures (Multi‐RNAi‐MSs) by rolling circle transcription. The Multi‐RNAi‐MSs were designed to contain a combination of multiple polymeric siRNA molecules with precisely controlled stoichiometry within a singular microstructure by manipulating the types and ratios of the circular DNA templates. The Multi‐RNAi‐MSs were converted into nanosized complexes by polyelectrolyte condensation to manipulate their physicochemical properties (size, shape, and surface charge) for favorable delivery, while maintaining the multifunctional properties of the siRNAs for combined therapeutic effects. These Multi‐RNAi‐MS systems have great potential in RNAi‐mediated biomedical applications, for example, for the treatment of cancer, genetic disorders, and viral infections.

Review of Continuous Circular Capsulorhexis Technique “New Technology for Better Sizing and Centration”

The cataract surgery has been modified over the time and the creation of a centered and precise capsulotomy can be challenging for surgeons in training. The term continuous circular capsulorhexis (CCC) described in 1987 is the actual method used by cataract surgeons. The efficacy of this technique depends on its centration, location, and size. It has been reported that the optimum CCC should be small enough to overlap 360 degrees of the IOL optic periphery to reduce posterior capsule opacification. Many devices have been created to improve the accuracy of CCC. Corneal and anterior capsular markers as well as substances to stain the anterior capsule (trypan blue) are the most used instruments while performing CCC. Different Image Guided Systems (Verion and Callisto Eye System) developed in the last years help surgeons by projecting a circular template of determined size on the anterior capsule intraoperatively. Laser assisted capsulotomy (LenSx) has been shown to be better centered than manual capsulorhexis with highly predictable diameters. Several factors when deciding which device to use must be taken into account: the availability of equipment, there producibility of the technique, the surgeon’s experience and the cost of the instruments. Thus, devices that help surgeons to increase accuracy are the best alternative for better sizing and centration of CCC, resulting in fewer complications and better postoperative prognosis for the patients.

Biosensing by Tandem Reactions of Structure Switching, Nucleolytic Digestion, and DNA Amplification of a DNA Assembly.

ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3'-5' digestion of single-stranded but not double-stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure-switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre-primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure-switching event that causes nucleolytic conversion of the pre-primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.

Biosensing by Tandem Reactions of Structure Switching, Nucleolytic Digestion, and DNA Amplification of a DNA Assembly

Abstractϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.

Optimal DNA templates for rolling circle amplification revealed by in vitro selection.

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC-rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.

A novel strategy to analyze L-tryptophan through allosteric Trp repressor based on rolling circle amplification.

Rolling circle amplification (RCA) has been considered as a powerful tool for nucleic acids detection. Here, a novel repressor-RCA-based method for L-tryptophan (L-Trp) detection was developed. This method utilizes the specific interaction between the RCA circular template and the Trp repressor protein (TrpR) involved in trp operon of Escherichia coli (E. coli). In the absence of L-Trp, the TrpR protein could not bind to the RCA template, and the RCA process can be continued. When L-Trp is present, the activated TrpR will bind to the operon sequence on the RCA template and inhibit the RCA reaction. Thus, the concentration of L-Trp is correlated directly with the fluorescent RCA signals. We succeeded in detecting L-Trp in a single step in simple homogeneous reaction system. The detection limit was estimated to be 0.77 μM (S/N=3) with good linearity. The method can unambiguously distinguish L-Trp from other 19 standard amino acids and L-Trp analogs. This strategy is also promising for detecting many small molecules such as other amino acids and carbohydrates.

A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans.

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.

Ultrasensitive genotyping with target-specifically generated circular DNA templates and RNA FRET probes.

A novel RNA FRET probe that can produce target-dependent signal amplification with the catalysis of RNase H has been developed for detection of rolling circle amplification (RCA) products with greatly improved sensitivity.

Image Edge Extraction Based on SUSAN Operator

A new edge extraction method was put forward based on the SUSAN operator, according to the problems of poor anti-noise ability and edge detection incomplete of the conventional differential detection operator. The circular template and the center of the circle (template nuclear) were used in this method, the numbers of pixels was calculated through the comparison pixels value of template with the other points of pixels in the template circle, and then compared with the threshold, so as to the edge of images was extracted. The results showed that this method had high precision, and could be able to fully extract the edge of images. It is an effective method of extracting the edge of images.

영상등록을 위한 Mutual Information 기반의 원형 템플릿 정합

본 논문에서는 영상 등록을 위한 유사도 계산에 사용되는 원형 템플릿의 설계 방법을 제안한다. 원형 템플릿은 영상의 이동 및 회전 변환에 불변한 성질을 가지고 있어 기준 영상 및 관측 영상 사이에 이동 및 회전 변환이 존재하더라도 영상 등록 제어점을 정확하게 정합하는 장점이 있다. 기준 영상의 제어점을 중심으로 일정한 거리 이내에 다수의 원주를 구성하고 각 원주 위에 일정한 간격으로 위치하는 화소들로 이루어지는 원형 템플릿을 생성하고 이를 이차원 이산 극좌표 행렬(Discrete Polar Coordinate Matrix, DPCM)으로 구성한다. 관측 영상에서도 동일한 형태의 원형 템플릿을 생성하고 탐색 범위 내의 각 위치에서 관측 영상의 원형 템플릿을 0도에서 360도 범위 내에서 일정 각도 간격으로 회전시키면서 극좌표 행렬을 생성하고 기준 영상의 극좌표 행렬과의 유사도를 Mutual Information을 이용해서 계산한다. 탐색 범위 내의 각 위치와 회전 각도에 대한 Mutual Information이 최대가 되는 화소를 정합쌍으로 결정한다. 제안한 알고리즘은 서로 다른 두 시기에 촬영한 KOMPSAT-2 영상에 적용하여 영상의 회전 변화 조건하에서 우수한 정합 성능을 보임을 확인하였다. This paper presents a method for designing circular template used in similarity measurement for image registration. Circular template has translation and rotation invariant property, which results in correct matching of control points for image registration under the condition of translation and rotation between reference and sensed images. Circular template consisting of the pixels located on the multiple circumferences of the circles whose radii vary from zero to a certain distance, is converted to two-dimensional Discrete Polar Coordinate Matrix (DPCM), whose elements are the pixels of the circular template. For sensed image, the same type of circular template and DPCM are created by rotating the circular template repeatedly by a certain degree in the range between 0 and 360 degrees and then similarity is calculated using mutual information of the two DPCMs. The best match is determined when the mutual information for each rotation angle at each pixel in search area is maximum. The proposed algorithm was tested using KOMPSAT-2 images acquired at two different times and the results indicate high accurate matching performance under image rotation.

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.

Rapidly estimating numerosity independent of size-related distance or occlusion

Finding objects in natural environments presents a challenge for even simple objects with known properties. Because of occlusion and distance, the number and extent (visual angle) of visible surfaces do not directly convey the number of objects seen. Are those three reddish surfaces one nearby partially occluded apple or three distant apples? Resolving such ambiguities seems effortless for human observers. To benchmark human performance, we asked subjects to indicate which of two briefly pictured trees had more apples. Trees were generated in a virtual environment, and pairs were constructed with variations in occlusion, distance, or number of visible apples. Preliminary results show good performance (>95% correct) on the task, despite some less fruit-laden trees having double the visible apple area of their partners. Subjects did not simply respond to the extent of visible apple surfaces; their judgments were robust to variations in occlusion or visual angle. We believe this is the result of a multi-stage process similar to Xu and Chun's (TiCS, 2009) "individuation and identification" stages, where proto-objects with low-level features are first spatially localized then further refined into identifiable objects. We created a simple algorithm to investigate this hypothesis. First, it segments pixels with "tree-like" color (RGB values) by blurring and thresholding. It then tags apple-colored pixels within these tree-like regions. These pixels are convolved with a circular template whose radius depends on the area of the tree-like region. Local maximums give locations of apples that might explain the observed pixels, given the estimated tree size. This system can distinguish between a cluster of patches representing a single heavily-occluded apple nearby, versus multiple apples at a distance. It performs comparably to humans on our task, suggesting that subjects group proto-objects derived from visible patches into individual apples as a basis for numerosity judgments. Meeting abstract presented at VSS 2014

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA.

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3′-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.

Rolling Circle Amplification Immunoassay Combined with Gold Nanoparticle Aggregates for Colorimetric Detection of Protein

A highly sensitive and novel colorimetric rolling circle amplification (RCA) immunoassay for detecting C-reactive protein (CRP) has been developed. In the assay, a CRP capture antibody was immobilized on magnetic beads and a CRP detection antibody was conjugated with single-stranded DNA (ssDNA) using N-[ε-maleimidocaproyloxy] sulfosuccinimide ester. Along with the addition of CRP, a "sandwich" structure was formed. Subsequently, the ssDNA was used as a primer to initiate the RCA reaction in the presence of the circular template, phi29 DNA polymerase and deoxynucleotide triphosphates. The RCA product obtained by magnetic separation, and long tandem repeated sequences mediated the aggregation of gold nanoparticles (AuNPs), which could be observed by the naked eye or quantified using absorption spectra with a detection limit of 30 fg mL(-1) and a linear response range from 10 ng mL(-1) to 1 pg mL(-1). This assay offers the advantages of isothermal conditions, low cost and label-free quantification that could be hopeful for ultrasensitive and robust visual protein detection.

A graphene-based biosensing platform based on the release of DNA probes and rolling circle amplification.

We report a versatile biosensing platform capable of achieving ultrasensitive detection of both small-molecule and macromolecular targets. The system features three components: reduced graphene oxide for its ability to adsorb single-stranded DNA molecules nonspecifically, DNA aptamers for their ability to bind reduced graphene oxide but undergo target-induced conformational changes that facilitate their release from the reduced graphene oxide surface, and rolling circle amplification (RCA) for its ability to amplify a primer-template recognition event into repetitive sequence units that can be easily detected. The key to the design is the tagging of a short primer to an aptamer sequence, which results in a small DNA probe that allows for both effective probe adsorption onto the reduced graphene oxide surface to mask the primer domain in the absence of the target, as well as efficient probe release in the presence of the target to make the primer available for template binding and RCA. We also made an observation that the circular template, which on its own does not cause a detectable level of probe release from the reduced graphene oxide, augments target-induced probe release. The synergistic release of DNA probes is interpreted to be a contributing factor for the high detection sensitivity. The broad utility of the platform is illustrated though engineering three different sensors that are capable of achieving ultrasensitive detection of a protein target, a DNA sequence and a small-molecule analyte. We envision that the approach described herein will find useful applications in the biological, medical, and environmental fields.

Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology

This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

Label-free picomolar detection of Pb2+ using atypical icosahedra gold nanoparticles and rolling circle amplification

A novel electrochemical sensing system for Pb2+ has been developed based on atypical icosahedral gold nanoparticle (AI-AuNPs) modification and the improved rolling circle amplification (RCA). The newly synthesized AI-AuNPs which were applied to modify the gold electrode surface greatly enhanced the sensitivity and expanded the detection range due to its large specific surface area and a large density of irrational kinks and step atoms on the particle surface. Additionally, the improved RCA introduced a manipulated circular template DNA which can be hybridized with the primer obtained from the DNAzymes catalysis reaction in the presence of Pb2+ as a cofactor to simplify the RCA procedure, and the largely elongated RCA products at the same time efficiently blocked the electron transfer at the electrode surface, thus significantly increased the electrochemical signal. The design of the label free electrochemical impendence sensing strategy was evidenced to be a highly sensitive and selective method for the lead ions monitoring compared with the previous reports and the detection range was 1pM–1μΜ with a detection limit of 290fM (3δ rule). Furthermore, the proposed detection system was also environmental-friendly and inexpensive, which can be successfully used in real serum sample detection.

Gap-Directed Translesion DNA Synthesis of an Abasic Site on Circular DNA Templates by a Human Replication Complex

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged “minicircle” DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.

Diverse topological requirements for mitochondrial transcription (573.5)

That transcription of the three human mitochondrial promoters can be recapitulated in vitro using a limited set of proteins including the mitochondrial RNA polymerase (POLRMT) and two accessory factors, mitochondrial transcription factor A (TFAM) and transcription factor B2 (TFB2M). The complete set of mechanisms that regulate promoter selection are unknown. The objective of this study was to determine whether the topological state of template DNA plays a role in promoter selection. We used a plasmid‐based system for the in vitro analysis of transcription that recapitulates features of the mtDNA to illustrate the relationship between topology and the three mitochondrial promoters and the involvement of TFAM which can bend template DNA. Both heavy‐strand promoters (HSP1 and HSP2) can be simultaneously transcribed from a circular template but increasing TFAM concentrations suppress HSP2 transcription at concentrations that result in HSP1 activation. We identified a requirement for the use of supercoiled DNA at HSP2 for transcription from circular templates which is not present for HSP1 and LSP. We conclude that the mitochondrial promoters have distinct requirements for DNA topology and TFAM dosage in vitro providing a means for the differential regulation of mitochondrial gene expression.Grant Funding Source: NIH/NICHD