d engineering of an enzyme-powered three dimensional DNA nanomachine for discriminating single nucleotide variants † (original) (raw)
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Chemical science, 2018
Single nucleotide variants (SNVs) are important both clinically and biologically because of their profound biological consequences. Herein, we engineered a nicking endonuclease-powered three dimensional (3D) DNA nanomachine for discriminating SNVs with high sensitivity and specificity. Particularly, we performed a simulation-guided tuning of sequence designs to achieve the optimal trade-off between device efficiency and specificity. We also introduced an auxiliary probe, a molecular fuel capable of tuning the device in solution noncovalent catalysis. Collectively, our device produced discrimination factors comparable with commonly used molecular probes but improved the assay sensitivity by ∼100 times. Our results also demonstrate that rationally designed DNA probes through computer simulation can be used to quantitatively improve the design and operation of complexed molecular devices and sensors.
Nature Communications
Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain re...
Rational Design of DNA Nanoarchitectures
Angewandte Chemie International Edition, 2006
DNA has many physical and chemical properties that make it a powerful material for molecular constructions at the nanometer length scale. In particular, its ability to form duplexes and other secondary structures through predictable nucleotide-sequencedirected hybridization allows for the design of programmable structural motifs which can self-assemble to form large supramolecular arrays, scaffolds, and even mechanical and logical nanodevices. Despite the large variety of structural motifs used as building blocks in the programmed assembly of supramolecular DNA nanoarchitectures, the various modules share underlying principles in terms of the design of their hierarchical configuration and the implemented nucleotide sequences. This Review is intended to provide an overview of this fascinating and rapidly growing field of research from the structural design point of view. From the Contents 1. Introduction 1857 2. General Considerations of DNA-Sequence Design 1858 3. One-Dimensional DNA Strands for Assembly and Immobilization of Non-Nucleic Acid Compounds 1859 4. Design and Assembly of DNA Motifs 1860 5. Three-Dimensional Structures from DNA 1866 6. Applications of DNA Nanoarchitectures 1868 7. Conclusions and Perspectives 1872 DNA Nanoarchitectures Angewandte Chemie Udo Feldkamp is a research assistant at the University of Dortmund (Germany). He was born in Duisburg and studied Computer Science in Kaiserslautern and Dortmund, where he also completed his PhD thesis on computer-aided DNA sequence design under the supervision of Prof. Wolfgang Banzhaf. His research still focuses on DNA-based nanotechnology and DNA computing, but he is also interested in other fields of bioinformatics and in computational intelligence. Christof M. Niemeyer has been Professor of Chemistry (chair of Biological and Chemical Microstructuring) at the University of Dortmund (Germany) since 2002. He studied chemistry at the University of Marburg and completed his PhD on organometallic chemistry at the Max-Planck-Institut für Kohlenforschung in Mülheim/Ruhr with Prof. Manfred T. Reetz. He then did postdoctoral research at the Center for Advanced Biotechnology in Boston (USA) with Prof. Charles R. Cantor, and received his habilitation at the University of Bremen. He is interested in semisynthetic DNA-protein and nanoparticle-conjugates as well as their applications in life sciences, catalysis, and molecular nanotechnology.
Sequence-specific recognition of DNA nanostructures
Methods, 2014
DNA is the most exploited biopolymer for the programmed self-assembly of objects and devices that exhibit nanoscale-sized features. One of the most useful properties of DNA nanostructures is their ability to be functionalized with additional non-nucleic acid components. The introduction of such a component is often achieved by attaching it to an oligonucleotide that is part of the nanostructure, or hybridizing it to single-stranded overhangs that extend beyond or above the nanostructure surface. However, restrictions in nanostructure design and/or the self-assembly process can limit the suitability of these procedures. An alternative strategy is to couple the component to a DNA recognition agent that is capable of binding to duplex sequences within the nanostructure. This offers the advantage that it requires little, if any, alteration to the nanostructure and can be achieved after structure assembly. In addition, since the molecular recognition of DNA can be controlled by varying pH and ionic conditions, such systems offer tunable properties that are distinct from simple Watson-Crick hybridization. Here, we describe methodology that has been used to exploit and characterize the sequence-specific recognition of DNA nanostructures, with the aim of generating functional assemblies for bionanotechnology and synthetic biology applications.
Multimodal Characterization of a Linear DNA-Based Nanostructure
ACS Nano, 2012
9 10 D eveloping methods for patterning 11 discrete particles and molecules at 12 the nanoscale has become an im-13 portant avenue for nanotechnological re-14 search as the limitations and inefficiencies 15 of conventional top-down patterning be-16 come ever more apparent. For such work, 17 investigators are increasingly turning to 18 self-assembly methodologies. While there 19 are many systems that allow for self-assem-20 bly of simple molecular structures, DNA-21 based technologies provide inherent ad-22 vantages due to the precise nature of 23 WatsonÀCrick base pairing and the molec-24 ular-level control one has over base se-25 quence. In the simplest examples, linear 26 DNA constructs have been assembled with 27 a wide variety of particle and molecular 28 attachments including fluorescent dyes, 29 semiconductor quantum dots (QDs), gold 30 nanoparticles (AuNPs), and proteins, clearly 31 demonstrating the potential of DNA functio-32 nalization chemistry for nanoscale control. 1À4 33 Greatly expanding the potential application 34 space, Seeman pioneered methods that use 35 DNA as a self-assembled structural material. 5 36 As he elegantly demonstrated, the use of 37 crossovers, tiles, and junctions permits a wide 38 range of structures to be realized. 6À10 Rothe-39 mund extended this methodology further 40 with DNA origami, 11 an approach for creating 41 arbitrary two-dimensional DNA structures 42 using a long scaffold strand and many smaller 43 staple strands. 12À16 In all implementations, 44 since each base or set of bases within the 45 DNA structure is uniquely addressable, there 46 is potential for a self-assembly approach that 47 allows for arbitrary particle or molecular pla-48 cement with a resolution approaching the 49 base-to-base separation distance of ∼3 Å. 50 In order to reach a full understanding of 51 the accuracy with which such DNA struc-52 tures form and the consequent precision 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 that can be realized in DNA-based pattern-77 ing, it is necessary to examine a variety of 78 assembled structures in intimate detail 79 using a diverse set of techniques. To date, 80 such a study has not been performed, nor is 81 it clear what exactly would constitute a "full" 82 characterization. In molecular biology, X-ray 83 crystallography is the gold standard in di-84 ABSTRACT Designer DNA structures have garnered much interest as a way of assembling novel nanoscale architectures with exquisite control over the positioning of discrete molecules or nanoparticles.
Organic & biomolecular chemistry, 2018
The labelling of DNA oligonucleotides with signalling groups that give a unique response to duplex formation depending on the target sequence is a highly effective strategy in the design of DNA-based hybridisation sensors. A key challenge in the design of these so-called base discriminating probes (BDPs) is to understand how the local environment of the signalling group affects the sensing response. The work herein describes a comprehensive study involving a variety of photophysical techniques, NMR studies and molecular dynamics simulations, on anthracene-tagged oligonucleotide probes that can sense single base changes (point variants) in target DNA strands. A detailed analysis of the fluorescence sensing mechanism is provided, with a particular focus on rationalising the high dependence of this process on not only the linker stereochemistry but also the site of nucleobase variation within the target strand. The work highlights the various factors and techniques used to respectively...
Single nucleotide recognition using a probes-on-carrier DNA chip
BioTechniques, 2019
Following the sequencing of the human genome, SNP analysis of individual patients has become essential for achieving the best drug response and ensuring optimal care. In this study, we developed a cost-effective probes-on-carrier DNA chip for the detection of SNPs. Our chips harbored three different probes against the TP53 gene, and were capable of detecting wild-type TP53 and SNPs such as rs121912651 and rs11540652. Four cell lines were used to validate the specificity of probe hybridization. Strong fluorescence intensity was observed in hybridized spots based on hybridization for perfect base pairing between complementary strands, whereas significantly lower fluorescence (p < 0.05) was observed in nonhybridized spots. These hybridization results indicated that the probes-on-carrier chip is suitable for SNP genotyping.
Detecting SNPs Using a Synthetic Nanopore
Nano Letters, 2007
We have discovered a voltage threshold for permeation through a synthetic nanopore of dsDNA bound to a restriction enzyme that depends on the sequence. Molecular dynamic simulations reveal that the threshold is associated with a nanonewton force required to rupture the DNA−protein complex. A single mutation in the recognition site for the restriction enzyme, i.e., a single nucleotide polymorphism (SNP), can easily be detected as a change in the threshold voltage. Consequently, by measuring the threshold voltage in a synthetic nanopore, it may be possible to discriminate between two variants of the same gene (alleles) that differ in one base.
DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA po-lymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active sub-strates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were com-plexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly dis-tinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome se-quencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete (1, 2). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Single-molecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). Although optical methods for single-molecule sequencing have been achieved and commercialized, the most successful, Pacific Biosciences' single molecule real-time (SMRT) sequencing by synthesis (SBS) approach, requires expensive instrumentation and the use of fluorescently tagged nucleotides (4, 5). In the last two decades, there has been great interest in taking advantage of nanopores, naturally occurring or solid-state ion channels, for polymer characterization and distinguishing the bases of DNA in a low-cost, rapid, single-molecule manner (6–9). Three nanopore sequencing approaches have been pursued: strand se-quencing in which the bases of DNA are identified as they pass sequentially through a nanopore (6, 7), exonuclease-based nanopore Significance Efficient cost-effective single-molecule sequencing platforms will facilitate deciphering complete genome sequences, determining haplotypes, and identifying alternatively spliced mRNAs. We demonstrate a single-molecule nanopore-based sequencing by synthesis approach that accurately distinguishes four DNA bases by electronically detecting and differentiating four different polymer tags attached to the terminal phosphate of the nucleotides during their incorporation into a growing DNA strand in the polymerase reaction. With nanopore detection, the distinct polymer tags are much easier to differentiate than natural nucleotides. After tag release, growing DNA chains consist of natural nucleotides allowing long reads. Sequencing is realized on an electronic chip containing an array of independently addressable electrodes, each with a single polymerase–nanopore complex, potentially offering the high throughput required for precision medicine.