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 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.

A Fluorogenic DNAzyme for A Thermally Stable Protein Biomarker from Fusobacterium nucleatum, a Human Bacterial Pathogen.

Fusobacterium nucleatum has been correlated to many poor human conditions including oral infections, adverse pregnancies and cancer, and thus molecular tools capable of detecting this human pathogen can be used to develop diagnostic tests for them. Using a new selection method targeting thermally stable proteins without a counter-selection step, we derived an fluorogenic RNA-cleaving DNAzyme, named RFD-FN1, that can be activated by a thermally stable protein target that is unique to F. nucleatum subspecies. High thermal stability of protein targets is a very desirable attribute for DNAzyme-based biosensing directly with biological samples because nucleases found inherently in these samples can be heat-inactivated. We further demonstrate that RFD-FN1 can function as a fluorescent sensor in both human saliva and human stool samples. The discovery of RFD-FN1 paired with a highly thermal stable protein target presents opportunities for developing simpler diagnostic tests for this important pathogen.

A DNAzyme-Based Colorimetric Paper Sensor for Helicobacter pylori.

The reliable detection of pathogenic bacteria in complex biological samples using simple assays or devices remains a major challenge. Herein, we report a simple colorimetric paper device capable of providing specific and sensitive detection of Helicobacter pylori (H. pylori), a pathogen strongly linked to gastric carcinoma, gastric ulcers, and duodenal ulcers, in stool samples. The sensor molecule, an RNA-cleaving DNAzyme obtained through in vitro selection, is activated by a protein biomarker from H. pylori. The colorimetric paper sensor, designed on the basis of the RNA-cleaving property of the DNAzyme, is capable of sensitive detection of H. pylori in human stool samples with minimal sample processing and provides results in minutes. It remains fully functional under storage at ambient temperature for at least 130 days. This work lays a foundation for developing DNAzyme-enabled paper-based point-of-care diagnostic devices for monitoring pathogens in complex samples.

Colorimetric Detection of Uranyl Using a Litmus Test.

Ingestion of water containing toxic contaminants above levels deemed safe for human consumption can occur unknowingly since numerous common contaminants in drinking water are colorless and odorless. Uranyl is particularly problematic as it has been found at dangerous levels in sources of drinking water. Detection of this heavy metal-ion species in drinking water currently requires sending a sample to a laboratory where trained personnel use equipment to perform the analysis and turn-around times can be long. A pH-responsive colorimetric biosensor was developed to enable detection of uranyl in water which coupled the uranyl-specific 39E DNAzyme as a recognition element, and an enzyme capable of producing a pH change as the reporter element. The rapid colorimetric assay presented herein can detect uranyl in lake and well water at concentrations relevant for environmental monitoring, as demonstrated by the detection of uranyl at levels below the limits set for drinking water by major regulatory agencies including the World Health Organization (30 µg/L). This simple and inexpensive DNAzyme-based assay enabled equipment-free visual detection of 15 µg/L uranyl, using both solution-based and paper-based pH-dependent visualization strategies.

Detection of DNA Amplicons of Polymerase Chain Reaction Using Litmus Test.

We report on a new colorimetric DNA detection method that takes advantage of the power of polymerase chain reaction (PCR) and the simplicity of the classic litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in the increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of the bacterium Clostridium difficile that are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients.

RNA Protection is Effectively Achieved by Pullulan Film Formation.

RNA is a functionally versatile polymer but suffers from susceptibility to spontaneous and RNase-catalyzed degradation. This vulnerability makes it difficult to preserve RNA for extended periods of time, thus limiting its use in various contexts, including practical applications as functional nucleic acids. Here we present a simple method to preserve RNA by pullulan (a complex sugar produced by Aureobasidium pullulans fungus) film formation. This strategy can markedly suppress both spontaneous and RNase degradation. Importantly, the pullulan film readily dissolves in aqueous solution, thus allowing retrieval of fully functional RNA species. In order to illustrate the advantage of this protective method in a practical application, we engineered a simple paper sensor containing a bacteria-detecting RNA-cleaving DNAzyme. This detection capability of the device was unchanged after storage at room temperature for six months.

Integrating Deoxyribozymes into Colorimetric Sensing Platforms.

Biosensors are analytical devices that have found a variety of applications in medical diagnostics, food quality control, environmental monitoring and biodefense. In recent years, functional nucleic acids, such as aptamers and nucleic acid enzymes, have shown great potential in biosensor development due to their excellent ability in target recognition and catalysis. Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules with catalytic activity and can be isolated to recognize a wide range of analytes through the process of in vitro selection. By using various signal transduction mechanisms, DNAzymes can be engineered into fluorescent, colorimetric, electrochemical and chemiluminescent biosensors. Among them, colorimetric sensors represent an attractive option as the signal can be easily detected by the naked eye. This reduces reliance on complex and expensive equipment. In this review, we will discuss the recent progress in the development of colorimetric biosensors that make use of DNAzymes and the prospect of employing these sensors in a range of chemical and biological applications.

Colorimetric Detection of Bacteria Using Litmus Test.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

In vitro selection of RNA-cleaving DNAzymes for bacterial detection.

DNAzymes refer to single-stranded DNA molecules with catalytic activity and can be isolated from synthetic random-sequence DNA pools using the technique of in vitro selection. DNAzymes that cleave RNA, known as "RNA-cleaving DNAzymes", represent one of the best-studied classes of DNAzymes and have been widely used for the development of biosensors and bioassays for various analytes. We have been interested in developing RNA-cleaving DNAzymes as bacterial sensors and these DNAzymes are engineered to perform three linked functions: recognition of a bacterial biomarker, RNA cleavage, and fluorescence generation. These fluorogenic DNAzymes emit fluorescence automatically in the presence of a bacterium of interest and can be used to set up a simple "mix-and-read" assay to detect this bacterium. In this article, we will discuss this DNAzyme system and present a proven strategy for isolating highly specific bacteria-responding DNAzyme probes from random-sequence DNA pools. We will also provide an in vitro selection protocol successfully used to derive RNA-cleaving fluorogenic DNAzyme probes that are capable of recognizing a targeted strain of Clostridium difficile.

Topological DNA Assemblies Containing Identical or Fraternal Twins.

DNA catenanes are assemblies made up of two or more DNA rings linked together through mechanical bonds, and they are desirable for engineering unique nanoscale devices. However, current methods of synthesizing DNA catenanes rely on the formation of strong linking duplexes between component units to enable interlocking and thus do not permit the synthesis of complex single-stranded DNA structures with freely functioning units. We have recently reported DNA sequences that can thread through a DNA circle without the formation of a linking duplex. Here we show that these unique DNA molecules can be further used to make intricate symmetric or asymmetric DNA [3]catenanes, single-stranded DNA assemblies made up of a central mother ring interlocked to two identical or fraternal twin daughter rings, which have never been reported before. These addressable freely functioning interlocked DNA rings should facilitate the design of elaborate nanoscale machines based on DNA.

A Catalytic DNA Activated by a Specific Strain of Bacterial Pathogen.

Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD-CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD-CD1 was derived by an in vitro selection approach where a random-sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross-reactivities to unintended C. difficile strains and other bacteria species. RFD-CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that in vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain-specific recognition of bacterial pathogens.

Sequence Mutation and Structural Alteration Transform a Noncatalytic DNA Sequence into an Efficient RNA-Cleaving DNAzyme.

We have previously shown that through test-tube molecular evolution, an arbitrarily chosen noncatalytic DNA sequence can be evolved into a catalytic DNA (DNAzyme) with significant RNA-cleaving activity. In this study, we aim to address the question of whether the catalytic activity of such a DNAzyme can be further optimized using in vitro selection. Several cycles of selective enrichment starting with a partially randomized DNA library have resulted in the isolation of many sequence variations that show notably improved catalytic activity. Bioinformatic analysis and activity examination of several DNAzyme-substrate constructs have led to two interesting findings about sequence mutations and the secondary structure of this DNAzyme: (1) three crucial mutations have transformed the DNAzyme into 8-17, a DNAzyme that has been discovered in multiple previous in vitro selection experiments, and (2) other mutations have allowed this special 8-17 variant to make structural fine-tuning in order to cleave an arbitrarily chosen RNA-containing substrate with a defined sequence. Our study not only showcases the combined power of directed molecular evolution and in vitro selection techniques in turning a noncatalytic nucleic acid sequence into an efficient enzyme, but it also raises the question of whether mother nature has used a similar approach to evolve natural enzymes.

Evolution of an Enzyme from a Noncatalytic Nucleic Acid Sequence.

The mechanism by which enzymes arose from both abiotic and biological worlds remains an unsolved natural mystery. We postulate that an enzyme can emerge from any sequence of any functional polymer under permissive evolutionary conditions. To support this premise, we have arbitrarily chosen a 50-nucleotide DNA fragment encoding for the Bos taurus (cattle) albumin mRNA and subjected it to test-tube evolution to derive a catalytic DNA (DNAzyme) with RNA-cleavage activity. After only a few weeks, a DNAzyme with significant catalytic activity has surfaced. Sequence comparison reveals that seven nucleotides are responsible for the conversion of the noncatalytic sequence into the enzyme. Deep sequencing analysis of DNA pools along the evolution trajectory has identified individual mutations as the progressive drivers of the molecular evolution. Our findings demonstrate that an enzyme can indeed arise from a sequence of a functional polymer via permissive molecular evolution, a mechanism that may have been exploited by nature for the creation of the enormous repertoire of enzymes in the biological world today.

An Efficient Catalytic DNA that Cleaves L-RNA.

Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples.

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.

Translating bacterial detection by DNAzymes into a litmus test.

Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world.

Engineering interlocking DNA rings with weak physical interactions.

Catenanes are intriguing molecular assemblies for engineering unique molecular devices. The resident rings of a catenane are expected to execute unhindered rotation around each other, and to do so, they must have weak physical interactions with each other. Due to sequence programmability, DNA has become a popular material for nanoscale object engineering. However, current DNA catenanes, particularly in the single-stranded (ss) form, are synthesized through the formation of a linking duplex, which makes them less ideal as mobile elements for molecular machines. Herein we adopt a random library approach to engineer ssDNA [2] catenanes (two interlocked DNA rings) without a linking duplex. Results from DNA hybridization, double-stranded catenane synthesis and rolling circle amplification experiments signify that representative catenanes have weak physical interactions and are capable of operating as independent units. Our findings lay the foundation for exploring free-functioning interlocked DNA rings for the design of elaborate nanoscale machines based on DNA.

Arrest of rolling circle amplification by protein-binding DNA aptamers.

Certain DNA polymerases, such as ϕ29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super-long single-stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand-displacement ability of these polymerases. In this work, the ability of Ï•29DNAP to carry out RCA over circular templates containing a protein-binding DNA aptamer sequence was investigated. It was found that protein-aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein-binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.

A paper-based surface-enhanced resonance Raman spectroscopic (SERRS) immunoassay using magnetic separation and enzyme-catalyzed reaction.

In this study, a novel paper-based SERRS immunoassay based on magnetic separation and alkaline phosphatase (ALP) enzyme catalyzed hydrolysis reaction was developed. By using modified antibodies conjugated to magnetic beads, capture of mouse IgG followed by addition of ALP-labeled antibodies would form a sandwich-like immunoconjugate. After magnetic separation, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), a low SERRS active compound, was added as the substrate for ALP to generate a high SERRS response. Detection was conducted on a silver colloid/PVP/filter paper SERS substrate by spotting a pre-aggregated silver colloid sol onto polyvinyl pyrrolidone (PVP) modified filter paper using a semi-automatic TLC sample applicator. The optimization of the highly SERS active paper-based substrate, dynamic hydrolysis process of BCIP, quantitative detection of IgG, and selectivity of the assay was studied in detail. By taking advantage of magnetic separation in order to decrease the background interference, the selective enzyme reaction involved in producing a highly SERRS active product could detect mouse IgG from 1 to 500 ng mL(-1) with a LOD of 0.33 ng mL(-1).

Lighting Up RNA-Cleaving DNAzymes for Biosensing.

The development of the in vitro selection technique has allowed the isolation of functional nucleic acids, including catalytic DNA molecules (DNAzymes), from random-sequence pools. The first-ever catalytic DNA obtained by this technique in 1994 is a DNAzyme that cleaves RNA. Since then, many other RNase-like DNAzymes have been reported from multiple in vitro selection studies. The discovery of various RNase DNAzymes has in turn stimulated the exploration of these enzymatic species for innovative applications in many different areas of research, including therapeutics, biosensing, and DNA nanotechnology. One particular research topic that has received considerable attention for the past decade is the development of RNase DNAzymes into fluorescent reporters for biosensing applications. This paper provides a concise survey of the most significant achievements within this research topic.