RESUMO
Protein-oligonucleotide conjugates are increasingly used as detection probes in biological applications such as proximity sensing and spatial biology. The preparation of high-quality conjugate probes as starting reagents is critical for achieving good and consistent performance, which we demonstrate via the DNA proximity assay (DPA) for the one-pot quantification of protein targets. We first established a complete conjugation and anion-exchange chromatography purification workflow to reproducibly obtain pure subpopulations of protein probes carrying a discrete number of oligonucleotide strands. A systematic study using the purified conjugate sub-populations confirmed that the order of conjugate (number of oligonucleotides per protein) and its purity (the absence of the unconjugated antibody) were important for ensuring optimal and reproducible assay performance. The streamlined workflow was then successfully used to conjugate a pair of universal DPA initiator oligonucleotides onto a wide range of binders including antibodies, nanobodies, and antigens which enabled the versatile detection of different types of proteins such as cytokines, total antibodies, and specific antibody isotypes. The good assay robustness (the inter-assay coefficient of variation lower than 5%) and linear calibration curve was achieved across all targets with just a single mix-and-incubate reaction step and a short reaction time of 30 min. We anticipate the streamlined protein-oligonucleotide probe preparation workflow developed in this work to have broad utility across applications leveraging the specificity of protein bio-recognition with the programmability of DNA hybridization.
Assuntos
DNA , Oligonucleotídeos , Oligonucleotídeos/química , Proteínas/análise , Anticorpos/química , Hibridização de Ácido NucleicoRESUMO
Associative toehold is a powerful concept enabling efficient combinatorial computation in DNA circuit. A longer association length boosts circuit kinetics and equilibrium signal but results in higher leak rate. We reconcile this trade-off by using a hairpin lock design to dynamically elongate the effective associative toehold length in response to the input target. Design guidelines were established to achieve robust elongation without incurring additional leakages. Three hairpin initiators with different combinations of elongated associative toehold (4 â 6 nt, 5 â 8 nt and 6 â 9 nt) were shortlisted from the design framework for further discussion. The circuit performance improved in terms of reaction kinetics, equilibrium signal generated and limit of detection. Overall, the elongated associative toehold served as a built-in function to stabilize and favour the forward, desired reaction when triggered.
Assuntos
Pareamento de Bases , DNA/química , Cinética , TermodinâmicaRESUMO
Point mutations are a common form of genetic variation and have been identified as important disease biomarkers. Conventional methods for analyzing point mutations, e.g., polymerase chain reaction (PCR), are based on differences in thermal stability of the DNA duplex, which require extensive optimization of the reaction condition and nontrivial design of sequence-selective primers. This motivated the design of molecular translators to convert molecular inputs into generic output sequences, which allows for the target recognition and signal generation regions to be designed independently. In this work, we propose a translator design based on the concept of split proximity circuit (SPC) to achieve both high sequence selectivity and assay robustness using a universal reaction condition, i.e., room temperature and constant ionic concentration. We discussed the design aspects of the SPC recognition regions and demonstrated its plug-and-play capability to discriminate different point mutations for both DNA (seven G6PD mutations) and RNA (let-7 microRNA family members) targets while retaining the same signal generation region. Despite its simple design and nonstringent assay condition requirements, the SPC retained good analytical performance to detect subnanomolar target concentration within a reasonable time of an hour.
Assuntos
DNA/análise , MicroRNAs/análise , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , DNA/genética , Glucosefosfato Desidrogenase/genética , Humanos , Limite de Detecção , MicroRNAs/genéticaRESUMO
We used N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamide as an exogenous agent to modulate the formation of DNA assemblies at specific double-stranded sequences. The concept was demonstrated on the hybridization chain reaction that forms linear DNA. Through a series of melting curve analyses, we demonstrated that the binding of Py-Im polyamide positively influenced both the HCR initiation and elongation steps. In particular, Py-Im polyamide was found to drastically stabilize the DNA duplex such that its thermal stability approached that of an equivalent hairpin structure. Also, the polyamide served as an anchor between hairpin pairs in the HCR assembly, thus improving the originally weak interstrand stability. We hope that these proof-of-concept results can inspire future use of Py-Im polyamide as a molecular tool to modulate the formation of DNA assemblies.
Assuntos
DNA/síntese química , Imidazóis/química , Nylons/química , Pirróis/química , DNA/química , Conformação de Ácido NucleicoRESUMO
Guanine activation of fluorescence in DNA templated silver nanoclusters (AgNCs) is an interesting physical phenomenon which has yet to be fully understood to date. While the individual role of cytosine and guanine has been established, there is still a knowledge gap on how the AgNC-DNA system switches from dark to bright state. Here, we present evidence on the universal role of the DNA spacer sequence in physically separating two Ag+-binding cytosine sites to maintain the dark state while holding them together for structural re-organization by the guanine-rich strand to activate the bright state. The extent of turn-on signal could be modulated by adjusting the spacer length and composition. The ATATA spacer sequence was found to have negligible dark state fluorescence and a turn-on effect of 2440-fold, which was almost five times of the highest factor reported to date.
Assuntos
DNA/química , Nanoestruturas , Prata/química , Sequência de Bases , Citosina/química , DNA/análise , Fluorescência , Nucleotídeos/química , Moldes GenéticosRESUMO
Cell surface receptors play an important role in mediating cell communication and are used as disease biomarkers and therapeutic targets. We present a one-pot molecular toolbox, which we term the split proximity circuit (SPC), for the autonomous detection and visualization of cell surface receptor clusters. Detection was powered by antibody recognition and a series of autonomous DNA hybridization to achieve localized, enzyme-free signal amplification. The system under study was the human epidermal growth factor receptor (HER) family, that is, HER2:HER2 homodimer and HER2:HER3 heterodimer, both in cell lysate and in situ on fixed whole cells. The detection and imaging of receptors were carried out using standard microplate scans and confocal microscopy, respectively. The circuit operated specifically with minimal leakages and successfully captured the receptor expression profiles on three cell types without any intermediate washing steps.
Assuntos
DNA/química , Receptor ErbB-2/análise , Receptor ErbB-3/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia Confocal , Células Tumorais CultivadasRESUMO
DNA circuit is a versatile and highly-programmable toolbox which can potentially be used for the autonomous sensing of dynamic events, such as biomolecular interactions. However, the experimental implementation of in silico circuit designs has been hindered by the problem of circuit leakage. Here, we systematically analyzed the sources and characteristics of various types of leakage in a split proximity circuit which was engineered to spatially probe for target sites held within close proximity. Direct evidence that 3'-truncated oligonucleotides were the major impurity contributing to circuit leakage was presented. More importantly, a unique strategy of translocating a single nucleotide between domains, termed 'inter-domain bridging', was introduced to eliminate toehold-independent leakages while enhancing the strand displacement kinetics across a three-way junction. We also analyzed the dynamics of intermediate complexes involved in the circuit computation in order to define the working range of domain lengths for the reporter toehold and association region respectively. The final circuit design was successfully implemented on a model streptavidin-biotin system and demonstrated to be robust against both circuit leakage and biological interferences. We anticipate that this simple signal transduction strategy can be used to probe for diverse biomolecular interactions when used in conjunction with specific target recognition moieties.
Assuntos
Simulação por Computador , DNA/metabolismo , Engenharia Genética , Biotina/metabolismo , Modelos Moleculares , Oligonucleotídeos/metabolismo , Estreptavidina/metabolismoRESUMO
Biomolecular interactions have important cellular implications, however, a simple method for the sensing of such proximal events is lacking in the current molecular toolbox. We designed a dynamic DNA circuit capable of recognizing targets in close proximity to initiate a pre-programmed signal transduction process resulting in localized signal amplification. The entire circuit was engineered to be self-contained, i.e. it can self-assemble onto individual target molecules autonomously and form localized signal with minimal cross-talk. α-thrombin was used as a model protein to evaluate the performance of the individual modules and the overall circuit for proximity interaction under physiologically relevant buffer condition. The circuit achieved good selectivity in presence of non-specific protein and interfering serum matrix and successfully detected for physiologically relevant α-thrombin concentration (50 nM-5 µM) in a single mixing step without any further washing. The formation of localized signal at the interaction site can be enhanced kinetically through the control of temperature and probe concentration. This work provides a basic general framework from which other circuit modules can be adapted for the sensing of other biomolecular or cellular interaction of interest.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Aptâmeros de Nucleotídeos/metabolismo , Sítios de Ligação , DNA/metabolismo , Cinética , Transdução de Sinais , Biologia Sintética/métodos , Trombina/metabolismoRESUMO
Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin-biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 µL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.
RESUMO
DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.
Assuntos
Técnicas Biossensoriais , DNA , DNA/genética , DNA/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas Biossensoriais/métodosRESUMO
We report the impact of fluorophore-quencher labelling on the thermodynamics of hairpin opening by testing five fluorophores and two quenchers labelled at the end and/or internal positions. Two counter strategies were introduced, i.e. label the hairpin probe at an internal position or append an external hairpin stem on the trigger strand to promote coaxial stacking hybridization. The observations remained valid for complex hairpin opening operations such as hybridization chain reaction.
Assuntos
DNA , Corantes Fluorescentes , DNA/genética , Hibridização de Ácido Nucleico , TermodinâmicaRESUMO
MicroRNAs (miRNAs) hold immense potential as disease biomarkers, yet their short lengths and high sequence homology pose unique challenges in detection. Conventional methods such as the gold standard qRT-PCR and other isothermal amplification methods require sophisticated primer designs and use of enzymes which add uncertainties to the assay robustness. In this work, we demonstrate the use of a plug-and-play molecular detection platform, termed split proximity circuit (SPC), to achieve a selectivity comparable to qRT-PCR in differentiating point mutations using several miRNAs as proof-of-concept models. The analytical sensitivity of SPC has been improved by a hundred-fold over our previous work and matches/outperforms the enzyme-free assays reported in the literature by evolving the core signal-generating domains. Key design changes include improved hybridization chain reaction (HCR) hairpin sequences and the incorporation of a turn-on fluorescence signal based on fluorophore-quencher format. The core domains were then kept constant while redesigning the target recognition region to be complementary to various target sequences, all of which yield similar analytical performance. Notably, SPC maintained robust signal recovery with low variance even in complex biological matrices. With its enzyme-free and single room temperature operation, SPC presents a promising platform for quick and easy miRNA quantification.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Biomarcadores , Técnicas Biossensoriais/métodos , DNA , MicroRNAs/análise , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Mutação PuntualRESUMO
We established four-point guidelines for the sequence design of hairpin monomers in hybridization chain reaction (HCR). This enabled greater flexibility to customize specific hairpin sequences for use with the readout platform of interest. Using shorter hairpin stem length, a one-pot signal amplification system was demonstrated by incorporating distance-sensitive Förster resonance energy transfer (FRET) readout.
RESUMO
Single-nucleotide polymorphism (SNP) is an important biomarker for disease diagnosis, treatment monitoring, and development of personalized medicine. Recent works focused primarily on ultrasensitive detection, while the need for rapid and label-free single-nucleotide discrimination techniques, which are crucial criteria for translation into clinical applications, remains relatively unexplored. In this work, we developed a novel SNP detection assay that integrates two complementary nanotechnology systems, namely, a highly selective nanoparticle-DNA detection system and a single-particle sensitive nanopore readout platform, for rapid detection of single-site mutations. Discrete nanoparticle-DNA structures formed in the presence of perfectly matched (PM) or single-mismatched (SM) targets exhibited distinct size differences, which were resolved on a size-tunable nanopore platform to generate corresponding "yes/no" readout signals. Leveraging the in situ reaction monitoring capability of the nanopore platform, we demonstrated that real-time single-nucleotide discrimination of a model G487A mutation, responsible for glucose-6-phosphate dehydrogenase deficiency, can be achieved within 30 min with no false positives. Semiquantification of DNA samples down to picomolar concentration was carried out using a simple parameter of particle count without the need for sample labeling or signal amplification. The unique combination of nanoparticle-based detection and nanopore readout presented in this work brings forth a rapid, specific, yet simple biosensing strategy that can potentially be developed for point-of-care application.