Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes

The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and were ligated using a thermostable ligase enzyme only when the genomic DNA carried this mutation. The allele-specific primers also carried complementary stem structures with end-labels (donor/acceptor fluorescent dyes, Cy5/Cy5.5, respectively), which formed a molecular beacon following ligation. We coupled ligase detection reaction (LDR) with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the molecular beacon probes formed upon ligation. LDR-spFRET provided the necessary specificity and sensitivity to detect single-point mutations in as little as 600 copies of human genomic DNA directly without PCR at a level of 1 mutant per 1000 wild type sequences using 20 LDR thermal cycles. We also demonstrate the ability to rapidly discriminate single base differences in the K-ras gene in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA (600 copies). Real-time LDR-spFRET detection of point mutations in the K-ras gene was accomplished in PMMA microfluidic devices using sheath flows.     

Real-time monitoring of double-stranded DNA cleavage using molecular beacons

Traditional methods to assay enzymatic cleavage of DNA are discontinuous, time-consuming and laborious. Here, we report a new approach for real-time monitoring of double-stranded DNA cleavage by restriction endonuclease based on nucleic acid ligation using molecular beacon. Upon cleavage of DNA, the cleavage product can be ligated by DNA ligase, which results in a fluorescence enhancement of the molecular beacon. This method permits real-time monitoring of DNA cleavage and makes it easy to characterize the activity of restriction endonuclease and to study the cleavage reaction kinetics.     

Enzyme‐Regulated Activation of DNAzyme: A Novel Strategy for a Label‐Free Colorimetric DNA Ligase Assay and Ligase‐Based Biosensing

The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label‐free and DNAzyme‐based strategy to detect DNA ligase activity. This novel strategy relies on the ligation‐trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin‐DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL^?1 and a detection limit of 0.2 U mL^?1. Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a “split DNA machine” to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL^?1.     

A splicing model‐based DNA‐computing approach on microfluidic chip

DNA computing is a new computation form based on DNA biochemical reactions, which is mainly composed of sticker and splicing computation models. In this work, a microfluidic chip‐based approach was established for splicing model‐based DNA computing. A finite automaton with two input symbols (a, b) and three states (S0, S1, and S2) was applied in the pattern recognition for isosceles triangles. The DNA computation processes of automaton were realized through DNA digestion, ligation, DNA separation, and detection on the microfluidic chip. The established approach is efficient, controllable, and easy to integrate, which paves the way for the building of complete biomolecular computers in the future.     

Nick sealing by T4 DNA ligase on a modified DNA template: tethering a functional molecule on D-threoninol

Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis.     

Conversion of a ribozyme to a deoxyribozyme through in vitro evolution

An RNA ligase ribozyme was converted to a corresponding deoxyribozyme through in vitro evolution. The ribozyme was prepared as a DNA molecule of the same sequence, and had no detectable activity. A population of randomized variants of this DNA was constructed and evolved to perform RNA ligation at a rate similar to that of the starting ribozyme. When the deoxyribozyme was prepared as an RNA molecule of the same sequence, it had no detectable activity. Thus, the evolutionary transition from an RNA to a DNA enzyme represents a switch, rather than a broadening, of the chemical basis for catalytic function. This transfer of both information and function is relevant to the transition between two different genetic systems based on nucleic acid-like molecules, as postulated to have occurred during the early history of life on Earth.     

Ligating DNA with DNA

Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond. Using in vitro selection, we have isolated a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes. We have used the action of two catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage. The two-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation. The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction. The initial rate constant (k(obs)) of the optimized deoxyribozyme ligase was found to be 1 x 10(-)(4) min(-)(1). Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10(5)-fold faster than that generated by a simple DNA template. The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence. However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated.     

Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

Enzyme-regulated activation of DNAzyme: a novel strategy for a label-free colorimetric DNA ligase assay and ligase-based biosensing

The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40?U?mL(-1) and a detection limit of 0.2?U?mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01?U?mL(-1).     

Isolation of novel ribozymes that ligate AMP-activated RNA substrates

Background: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5′-5′-pyrophosphate ‘capped’ RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5′-monophosphate (AMP) may be a vestige of ‘RNA world’ catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated.Results: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10¹⁵ RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5′-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of ∼ 5 × 10⁵ over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3′-5′-phosphodiester bonds and were highly specific for activation by AMP at the ligation site.Conclusions: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.     

基于分子信标实时监测大肠杆菌DNA连接酶催化的DNA连接过程

报道了一种对DNA连接过程进行实时监测的方法,利用分子信标核酸探针作为DNA连接反应的模板和检测探针,实时监测了 E.coli DNA连接酶催化的DNA连接反应,克服了传统的凝胶电泳技术操作复杂、周期长及无法实时监测DNA连接过程的缺点,为核酸连接过程的实时监测和连接酶催化机理的研究提供了更为丰富的信息.在此基础上,发展了一种快速、准确测定 E.coli DNA连接酶的方法,线性响应范围为4.0×10^-6～2.0×10^-4U/μL,检测下限为4.0×10^-6U/μL.     

Tethered DNA hairpins facilitate electrochemical detection of DNA ligation

A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.     

Using molecular beacon to monitor activity of E. coli DNA ligase

NAD(+)-dependent DNA ligase has been widely used in gene diagnostics for disease-associated mutation detection and has proved to be necessary for screening bactericidal drugs targeted to DNA ligases. However, further research has been restricted since conventional ligase assay technology is limited to gel electrophoresis, which is discontinuous, time-consuming and laborious. An innovative approach is developed for monitoring the activity of E. coli DNA ligase catalyzing nucleic acid ligation in the report. This approach utilizes a molecular beacon hybridized with two single-stranded DNA (ssDNA) segments to be ligated to form a hybrid with a nick, and could therefore be recognized by the enzyme. Ligation of the two ssDNA segments would cause conformation changes of the molecular beacon, leading to significant fluorescence enhancement. Compared to gel electrophoresis, this approach can provide real time information about ligase, is more time efficient, and is easier to use. The effect of quinacrine, a drug for malaria, on the activity of the ligase is detected, thereby certifying the capability of the method for developing novel antibacterial drugs targeted at NAD(+)-dependent ligase. The fidelity of strand joining by the ligase is examined based on this approach. The effects of external factors on activity of the ligase are analyzed, and then an assay of E. coli DNA ligase is performed with a broad linear range of 4.0 x 10(-4) Weiss Unit mL(-1) to 0.4 Weiss Unit mL(-1) and the detection limit of 4.0 x 10(-4) Weiss Unit mL(-1).     

Computer simulation studies of the influence of side alkyl chain on glass transition behavior of carbazole trimer

In this work,all-atom molecular dynamics simulations were employed to study the influence of the side alkyl chain on glass transition behavior of several carbazole trimers(CT) in a temperature range from 423 to 183 K.The glass transition temperatures were obtained from the break in the slope of the volume-temperature curves and found to agree with the experimental values.The short time dynamics of four CT molecules were probed by usingvelocity autocorrelation functions and mean-square displacements.The current studies showed that the dynamics of CT systems can be easily interpreted through the cage effect.Furthermore,the investigation of the torsional autocorrelation function and P_(2-state)/P_(3-state) functions showed that the rotational barriers of side chains can slow down the conformational relaxation and lead to stronger temperature dependence of conformational relaxation.The relaxation time,characteristic time of P_(2-state)(t) and P_(3-state)(t) functions were all found to have Arrhenius-type temperature dependence.     

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.     

Destabilizing universal linkers for signal amplification in self-ligating probes for RNA

Recent studies have established the utility of oligonucleotide ligation methods in the detection of DNAs and RNAs in solution and in cellular imaging. Notably, the ligated full-length oligonucleotide products commonly bind to the target nucleic acid much more tightly than do the two starting half-probes, which effectively limits the resulting signals to one per target. Here, we report on a molecular strategy for destabilizing ligated products in template-promoted self-ligation reactions, thus yielding multiple signals per target. A new universal linker design is described in which a dabsyl leaving group is placed on a short alkane tether. This allows the placement of an electrophile at the end of any DNA sequence, in contrast to earlier ligation strategies, and it also speeds reaction rates by a factor of 4-5. This new class of molecular linker/activator yields as much as 92-fold amplification of signals in DNA and RNA detection, and proceeds without enzymes, added reagents, or thermal cycling. The linker is shown to destabilize the ligation product without destabilizing the transition state for ligation. This lowers product inhibition, and the target DNA or RNA thus becomes a catalyst for isothermally generating multiple signals for its detection. This enhanced signal generation is demonstrated in solution experiments and in solid supported assays.     

Nanoliter droplet array for microRNA detection based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR

In this paper, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of microRNA was developed. By employing T4 RNA ligase 2 instead of T4 DNA ligase, we designed simplified stem-loop probes to perform microRNA-templated DNA ligation and reduced the non-specific ligation of T4 DNA ligase. SYBR green I dye was employed instead of TaqMan probes in present miniaturized real-time PCR systems. Specifically, we optimized the dosage of SYBR Green I dye in nanoliter droplet and verified the performance of this system by detecting synthetic mir-122 with a 6 logs dynamic range (from 1.5 × 10⁵ to 1.5 × 10¹⁰ copies). Linear relationship of the standard curve (R² = 0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122 across five mouse tissues and the result was consistent with that TaqMan microRNA assay. We think this miniaturized real-time PCR platform reduced the detection cost considerably, thus showing the great potential to quantitative biology.     

Template-free, polymerase-free DNA polymerization

A combination of a DNA ligase and a restriction endonuclease provides a DNA polymerase activity, which might suggest a novel strategy for polymer synthesis.     

Bioelectronic Interface Connecting Reversible Logic Gates Based on Enzyme and DNA Reactions

It is believed that connecting biomolecular computation elements in complex networks of communicating molecules may eventually lead to a biocomputer that can be used for diagnostics and/or the cure of physiological and genetic disorders. Here, a bioelectronic interface based on biomolecule‐modified electrodes has been designed to bridge reversible enzymatic logic gates with reversible DNA‐based logic gates. The enzyme‐based Fredkin gate with three input and three output signals was connected to the DNA‐based Feynman gate with two input and two output signals—both representing logically reversible computing elements. In the reversible Fredkin gate, the routing of two data signals between two output channels was controlled by the control signal (third channel). The two data output signals generated by the Fredkin gate were directed toward two electrochemical flow cells, responding to the output signals by releasing DNA molecules that serve as the input signals for the next Feynman logic gate based on the DNA reacting cascade, producing, in turn, two final output signals. The Feynman gate operated as the controlled NOT gate (CNOT), where one of the input channels controlled a NOT operation on another channel. Both logic gates represented a highly sophisticated combination of input‐controlled signal‐routing logic operations, resulting in redirecting chemical signals in different channels and performing orchestrated computing processes. The biomolecular reaction cascade responsible for the signal processing was realized by moving the solution from one reacting cell to another, including the reacting flow cells and electrochemical flow cells, which were organized in a specific network mimicking electronic computing circuitries. The designed system represents the first example of high complexity biocomputing processes integrating enzyme and DNA reactions and performing logically reversible signal processing.