Acquisition of Phage Sensitivity by Bacteria through Exchange of Phage Receptors.

Bacteriophages (phages) typically exhibit a narrow host range, yet they tremendously impact horizontal gene transfer (HGT). Here, we investigate phage dynamics in communities harboring phage-resistant (R) and sensitive (S) bacteria, a common scenario in nature. Using Bacillus subtilis and its lytic phage SPP1, we demonstrate that R cells, lacking SPP1 receptor, can be lysed by SPP1 when co-cultured with S cells. This unanticipated lysis was triggered in part by phage lytic enzymes released from nearby infected cells. Strikingly, we discovered that occasionally phages can invade R cells, a phenomenon we termed acquisition of sensitivity (ASEN). We found that ASEN is mediated by R cells transiently gaining phage attachment molecules from neighboring S cells and provide evidence that this molecular exchange is driven by membrane vesicles. Exchange of phage attachment molecules could even occur in an interspecies fashion, enabling phage adsorption to non-host species, providing an unexplored route for HGT. VIDEO ABSTRACT.

High degree of coordination and division of labor among subunits in a homomeric ring ATPase.

Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor.

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.

Noncatalytic aspartate at the exonuclease domain of proofreading DNA polymerases regulates both degradative and synthetic activities.

Most replicative DNA polymerases (DNAPs) are endowed with a 3'-5' exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III. The importance of this aspartate has been assessed by site-directed mutagenesis at the corresponding Asp121 of the family B ϕ29 DNAP. Substitution of this residue by either glutamate or alanine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, both on ssDNA and dsDNA. The polymerization activity of these mutants was also affected due to a defective translocation following nucleotide incorporation. Alanine substitution for the homologous Asp90 in family A T7 DNAP showed essentially the same phenotype as ϕ29 DNAP mutant D121A. This functional conservation, together with a close inspection of ϕ29 DNAP/DNA complexes, led us to conclude a pivotal role for this aspartate in orchestrating the network of interactions required during internal proofreading of misinserted nucleotides.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

Preparation and characterization of manganese, cobalt and zinc DNA nanoflowers with tuneable morphology, DNA content and size.

Recently reported DNA nanoflowers are an interesting class of organic-inorganic hybrid materials which are prepared using DNA polymerases. DNA nanoflowers combine the high surface area and scaffolding of inorganic Mg2P2O7 nanocrystals with the targeting properties of DNA, whilst adding enzymatic stability and enhanced cellular uptake. We have investigated conditions for chemically modifying the inorganic core of these nanoflowers through substitution of Mg2+ with Mn2+, Co2+ or Zn2+ and have characterized the resulting particles. These have a range of novel nanoarchitectures, retain the enzymatic stability of their magnesium counterparts and the Co2+ and Mn2+ DNA nanoflowers have added magnetic properties. We investigate conditions to control different morphologies, DNA content, hybridization properties, and size. Additionally, we show that DNA nanoflower production is not limited to Ф29 DNA polymerase and that the choice of polymerase can influence the DNA length within the constructs. We anticipate that the added control of structure, size and chemistry will enhance future applications.

Limited reverse transcriptase activity of phi29 DNA polymerase.

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

Biomineralized Metal-Organic Framework Nanoparticles Enable Enzymatic Rolling Circle Amplification in Living Cells for Ultrasensitive MicroRNA Imaging.

The enzymatic amplification strategy in living cells faces challenges of highly efficient intracellular codelivery of amplification reagents including DNA polymerase. In this work, we develop biomineralized metal-organic framework nanoparticles (MOF NPs) as a carrier system for intracellular codelivery of ϕ29 DNA polymerase (ϕ29DP) and nucleic acid probes and realize a polymerization amplification reaction in living cells. A pH-sensitive biodegradable MOF NP of zeolitic imidazolate framework-8 (ZIF-8) is utilized to encapsulate ϕ29DP and adsorb nucleic acid probes. After uptake into cells, the encapsulated ϕ29DP and surface-adsorbed DNA probes are released and escaped from endolysosomes. In the presence of ϕ29DP and deoxyribonucleotide triphosphates (dNTPs), the intracellular miRNA-21 triggers a rolling circle amplification (RCA) reaction and the autonomous synthesized Mg2+-dependent DNAzyme cleaves the fluorogenic substrate, providing a readout fluorescence signal for the monitoring of miRNA-21. This is the first example of the intracellular RCA reaction in living cells. Therefore, the proposed method provides new opportunities for achieving enzymatic amplification reaction in living cells.

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain.

To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

Robust and highly specific fluorescence sensing of Salmonella typhimurium based on dual-functional phi29 DNA polymerase-mediated isothermal circular strand displacement polymerization.

A simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization (ICSDP). The strategy relies on target-triggered formation of a mature primer that initiates the cyclic strand displacement polymerization reaction with the aid of dual functional phi29; thus, amplified detection of the target can be achieved. To our knowledge, this work is the first report where dual functional phi29-assisted ICSDP has been employed for fluorescence sensing of pathogenic bacteria. It is worth noting that a hairpin pre-primer is introduced that can be trimmed into a mature primer for initiating ICSDP via the 3' → 5' proofreading exonuclease activity of phi29, which contributes to the ultrahigh specificity of the strategy owing to the elimination of the unwished nonspecific extension. On the basis of the present amplification strategy, our biosensor exhibits excellent specificity and sensitivity toward S. typhimurium with an excellent detection limit as low as 1.5 cfu mL-1. In addition, the strategy offers the advantages of a simplified operation, shortened analysis time, and highly sensitive detection of pathogens with only a one-step reaction. Furthermore, by redesigning the corresponding binding molecules, the proposed strategy can be easily extended for the detection of a wide spectrum of analytes. Hence, the dual functional phi29-assisted ICSDP strategy indeed creates a robust and convenient fluorescence sensing platform for the identification of pathogenic bacteria and related food safety analysis.

Primer remodeling amplification-activated multisite-catalytic hairpin assembly enabling the concurrent formation of Y-shaped DNA nanotorches for the fluorescence assay of ochratoxin A.

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.

A non-canonical multisubunit RNA polymerase encoded by the AR9 phage recognizes the template strand of its uracil-containing promoters.

AR9 is a giant Bacillus subtilis phage whose uracil-containing double-stranded DNA genome encodes distant homologs of ß and ß' subunits of bacterial RNA polymerase (RNAP). The products of these genes are thought to assemble into two non-canonical multisubunit RNAPs - a virion RNAP (vRNAP) that is injected into the host along with phage DNA to transcribe early phage genes, and a non-virion RNAP (nvRNAP), which is synthesized during the infection and transcribes late phage genes. We purified the AR9 nvRNAP from infected B. subtilis cells and characterized its transcription activity in vitro. The AR9 nvRNAP requires uracils rather than thymines at specific conserved positions of late viral promoters. Uniquely, the nvRNAP recognizes the template strand of its promoters and is capable of specific initiation of transcription from both double- and single-stranded DNA. While the AR9 nvRNAP does not contain homologs of bacterial RNAP α subunits, it contains, in addition to the ß and ß'-like subunits, a phage protein gp226. The AR9 nvRNAP lacking gp226 is catalytically active but unable to bind to promoter DNA. Thus, gp226 is required for promoter recognition by the AR9 nvRNAP and may represent a new group of transcription initiation factors.

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

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

An Adaptable Platform for Directed Evolution in Human Cells.

The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small-molecule adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small-molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.

Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal Amplification with an Aerolysin Nanopore.

Sensitive detection of cancer cells at extremely low concentrations would greatly facilitate the screening and early diagnosis of cancer. Herein, we present a novel nanopore-based strategy for ultrasensitive detection of Ramos cells (human Burkitt's lymphoma cells), by combining the enzymatic signal amplification with an aerolysin nanopore sensor. In this assay, an aptamer for Ramos cells was prehybridized with a short complementary DNA. The presence of target cells causes the target-aptamer complex to unwind to free the complementary DNA, which would subsequently trigger the enzymatic cycling amplification. This process eventually generated a large number of output DNA, which could quantitatively produce characteristic current events when translocated through aerolysin. The proposed method exhibits excellent sensitivity, and as few as 5 Ramos cells could be detected. With good selectivity, the approach can allow for the determination of cancer cells in human serum, offering a powerful tool for biomedical research and clinical diagnosis.

Identification and characterization of an endolysin - Like from Bacillus subtilis.

Drug-resistant Gram-positive pathogens have been a rising risk in hospitals and food industries from the last decades. Here in, the potential of endolysin production in Dasht Desert Bacterial Culture Collection (DDBCC), against indicator bacteria, was investigated. DDBCC was screened against autoclaved-indicator bacteria; Streptococcus faecalis, Streptococcus pyogenes, Bacillus sp, Bacillus subtilis and Staphylococcus aureus as the substrates for the endolysin enzymes. The endolysins were produced in BHI medium followed by ammonium sulfate purification. Peptidoglycan hydrolytic activity was tested by zymogram method. Lysogenic bacteria were induced by 0.1 µg/ml mitomycin C for bacteriophages extraction. The lysogenic bacteria inhibited S. pyogenes, S. faecalis, Bacillus sp. and B. subtilis. The strain DDBCC10 was selected for further experiments on its higher and specific activity against the cell wall of S. faecalis. The highest activity for the endolysin was obtained at 50-60% ammonium sulfate saturation as 8 U/ml. Lys10, a 22 kDa enzyme, digested the cell wall of S. faecalis in 15 min while the whole phage from DDBCC10 could form plaque on S. faecalis and S. pyogenes. In a Transmission Electron Microscopy assay (TEM), the phage was distinguished as a member of Siphoviridae. Here; Lys10 is introduced as a new biocontrol agent against S. faecalis for therapeutics, disinfection, and food preservatives purposes at a much lower expense than recombinant endolysins.

Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains.

Single-stranded DNA (ssDNA) is a critical intermediate in all DNA transactions. Because ssDNA is more flexible than double-stranded (ds) DNA, interactions with ssDNA-binding proteins (SSBs) may significantly compact or elongate the ssDNA molecule. Here, we develop and characterize low-complexity ssDNA curtains, a high-throughput single-molecule assay to simultaneously monitor protein binding and correlated ssDNA length changes on supported lipid bilayers. Low-complexity ssDNA is generated via rolling circle replication of short synthetic oligonucleotides, permitting control over the sequence composition and secondary structure-forming propensity. One end of the ssDNA is functionalized with a biotin, while the second is fluorescently labeled to track the overall DNA length. Arrays of ssDNA molecules are organized at microfabricated barriers for high-throughput single-molecule imaging. Using this assay, we demonstrate that E. coli SSB drastically and reversibly compacts ssDNA templates upon changes in NaCl concentration. We also examine the interactions between a phosphomimetic RPA and ssDNA. Our results indicate that RPA-ssDNA interactions are not significantly altered by these modifications. We anticipate that low-complexity ssDNA curtains will be broadly useful for single-molecule studies of ssDNA-binding proteins involved in DNA replication, transcription, and repair.

Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

Characterization of Wall Teichoic Acid Degradation by the Bacteriophage ϕ29 Appendage Protein GP12 Using Synthetic Substrate Analogs.

The genetics and enzymology of the biosynthesis of wall teichoic acid have been the extensively studied, however, comparatively little is known regarding the enzymatic degradation of this biological polymer. The GP12 protein from the Bacillus subtilis bacteriophage ϕ29 has been implicated as a wall teichoic acid hydrolase. We have studied the wall teichoic acid hydrolase activity of pure, recombinant GP12 using chemically defined wall teichoic acid analogs. The GP12 protein had potent wall teichoic acid hydrolytic activity in vitro and demonstrated ∼13-fold kinetic preference for glycosylated poly(glycerol phosphate) teichoic acid compared with non-glycosylated. Product distribution patterns suggested that the degradation of glycosylated polymers proceeded from the hydroxyl terminus of the polymer, whereas hydrolysis occurred at random sites in the non-glycosylated polymer. In addition, we present evidence that the GP12 protein possesses both phosphodiesterase and phosphomonoesterase activities.

Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases.

Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2'-OMeRNA modification, which might be due to the size of the C2'-modified moieties. 2'-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction.