Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells

There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell‐to‐cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence‐specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end‐channel electrodes, and completes the entire process in <5 min, including lysis, purification, fractionation, and delivery to DNA and RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross‐contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence‐specific quantitation using off‐chip RT‐qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively.     

Bifunctional RNA-Targeting Deoxyribozyme Nanodevice as a Potential Theranostic Agent

Theranostic approaches rely on simultaneous diagnostic of a disease and its therapy. Here, we designed a DNA nanodevice, which can simultaneously report the presence of a specific RNA target through an increase in fluorescence and cleave it. High selectivity of RNA target recognition under near physiological conditions was achieved. The proposed approach can become a basis for the design of DNA nanomachines and robots for diagnostics and therapy of viral infections, cancer, and genetic disorders.     

Programming Rotary Motions with a Hexagonal DNA Nanomachine

Biological macromolecular machines perform impressive mechanical movements. F-adenosine triphosphate (ATP) synthase uses a proton gradient to generate ATP through mechanical rotations. Here, a programmed hexagonal DNA nanomachine, in which a three-armed DNA nanostructure (TAN) can perform stepwise rotations in the confined nanospace powered by DNA fuels, is demonstrated. The movement of TAN can precisely go through a 60° rotation, which is confirmed by atomic force microscopy, and each stepwise directional rotating is monitored by fluorescent measurements. Moreover, the rotary nanomachine is used to spatially organize cascade enzymes: glucose oxidase (GOx) and horseradish peroxidase (HRP) in four different arrangements. The multistep regulations of the biocatalytic activities are achieved by employing TAN rotations. This work presents a new prototype of rotary nanodevice with both angular and directional control, and provides a nanoscale mechanical engineering platform for the reactive molecular components, demonstrating that DNA-based framework may have significant roles in futuristic nanofactory construction.     

DNA纳米机器:梦想照进现实

可进体内治病救人的纳米机器人一直是人们梦寐以求的未来科技和医疗手段.最近,国家纳米科学中心的丁宝全、聂广军等在这个方向取得了重要的突破,成功开发了基于DNA纳米机器的癌症免疫治疗疫苗.他们首先利用DNA折纸术构筑了一个可精确负载抗原和佐剂的管状结构,通过皮下注射递送至淋巴结,经由内吞在树突细胞内涵体内发生pH响应性的锁链打开,暴露抗原和佐剂,从而激活树突细胞,产生抗原特异性的T细胞,有效杀伤肿瘤细胞.该疫苗不仅可以有效抑制肿瘤的生长和复发,还诱导特异性记忆效应,可持续产生特异性的保护.这提供了一个精准递送分子药物的平台,让人看到成功发展纳米机器人的曙光,有望给医学和医疗保健带来重要变革.     

Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA

Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme‐based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ‐based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.     

结直肠癌组织中K-ras基因突变的毛细管电泳检测

通过对PCR扩增的76例结直肠癌组织及癌旁正常组织DNA基因组共152个样本纯化变性后,采用毛细管电泳-激光诱导荧光检测(CE-LIF)结合单链构象多态性(SSCP)分析方法检测了人结直肠癌组织及癌旁正常组织中K-ras基因第12/13位密码子突变。所检测的76例结直肠癌患者中有30例患者存在基因突变,并对异常片段进行测序验证,测序证实以碱基G→A点突变为主。结果表明所建立的CE-LIF技术结合SSCP分析检测K-ras基因突变的方法高效、快速、灵敏、准确,适合于临床上大样本结直肠癌中K-ras基因突变分析,对选择抗结直肠癌药物有一定的指导作用。     

A DNA Nanodevice for Cancer Vaccination

To realize effective cancer immunotherapy, Ding et al. constructed a structurally well-defined DNA-based nanodevice to quantitatively assemble cancer cell-specific antigen and multiple adjuvants as a cancer vaccine. This nanodevice vaccine can efficiently accumulate in the draining lymph nodes and respond to the endosomal acidic environment of dendritic cells to release the antigen and adjuvants. These active payloads stimulate dendritic cells maturation and antigen presentation to elicit a robust, antigen-specific cytotoxic T-lymphocyte response to kill cancer cells. This work has been published online in the Nature Materials on Sept.7, 2020.     

Disulfide-Linked Allosteric Modulators for Multi-cycle Kinetic Control of DNA-Based Nanodevices

Nature employs sulfur switches, that is, redox-active disulfides, to kinetically control biological pathways in a highly efficient and reversible way. Inspired by this mechanism, we describe herein a DNA-based synthetic nanodevice that acts as a sulfur switch and can be temporally controlled though redox regulation. To do this, we rationally designed disulfide DNA strands (modulators) that hybridize to a ligand-binding DNA nanodevice and act as redox-active allosteric regulators inducing the nanodevice to release or load its ligand. Upon reduction, the allosteric modulator spontaneously de-hybridizes from the nanodevice and, as a result, its effect is transient. The system is reversible and has an unprecedented high tolerance to waste products and displays transient behavior for over 40 cycles without significant loss of efficiency. Kinetic control of DNA-based ligand-binding nanodevices through purely chemical reactions paves the way for temporal regulation of more complex chemical pathways.     

DNA Adduct Detection after Post-Labeling Technique with PCR Amplification (DNA-ADAPT–qPCR) Identifies the Pre-ribosomal RNA Gene as a Direct Target of Platinum–Acridine Anticancer Agents

To study the DNA damage caused by a potent platinum–acridine anticancer agent (PA) in cancer cells, an assay based on biorthogonal post-labeling using a click chemistry-enabled, azide-modified derivative (APA) was developed. The method involves biotinylation, affinity capture, and bead-based enrichment of APA-modified genomic DNA. The key steps of the assay were validated and optimized in model duplexes, including full-length plasmids, restriction fragments, and a DNA ladder. Native DNA treated with APA and subsequently subjected to post-labeling with a biotin affinity tag was enzymatically digested and fragments were analyzed by in-line LC–MS and MS/MS. The monofunctional–intercalative adducts formed by APA in 5′-pyrimidine/guanine sequences in double-stranded DNA were quantitatively biotinylated by strain-promoted 1,3-dipolar cycloaddition chemistry. When applied to DNA extracted from A549 lung cancer cells, the assay in combination with qPCR amplification demonstrates that platinum–acridines form adducts in the gene sequences encoding pre-ribosomal RNA, a potential pharmacological target of these agents.     

Biomimetic RNA‐Silencing Nanocomplexes: Overcoming Multidrug Resistance in Cancer Cells

RNA interference (RNAi) is an RNA‐dependent gene silencing approach controlled by an RNA‐induced silencing complex (RISC). Herein, we present a synthetic RISC‐mimic nanocomplex, which can actively cleave its target RNA in a sequence‐specific manner. With high enzymatic stability and efficient self‐delivery to target cells, the designed nanocomplex can selectively and potently induce gene silencing without cytokine activation. These nanocomplexes, which target multidrug resistance, are not only able to bypass the P‐glycoprotein (Pgp) transporter, due to their nano‐size effect, but also effectively suppress Pgp expression, thus resulting in successful restoration of drug sensitivity of OVCAR8/ADR cells to Pgp‐transportable cytotoxic agents. This nanocomplex approach has the potential for both functional genomics and cancer therapy.     

A DNA‐Mediated Chemically Induced Dimerization (D‐CID) Nanodevice for Nongenetic Receptor Engineering To Control Cell Behavior

Small‐molecule regulation is a powerful switching tool to manipulate cell signal transduction for a desired function; however, most available methods usually require genetic engineering to endow cells with responsiveness to user‐defined small molecules. Herein, we demonstrate a nongenetic approach for small‐molecule‐controlled receptor activation and consequent cell behavior manipulation that is based on DNA‐mediated chemically induced dimerization (D‐CID). D‐CID uses a programmable chemical‐responsive DNA nanodevice to trigger DNA strand displacement and induce the activation of c‐Met, a tyrosine kinase receptor cognate for hepatocyte growth factor, through dimerization. Through the use of various functional nucleic acids, including aptamers and DNAzymes, as recognition modules, the versatility of D‐CID in inducing c‐Met signaling upon addition of various small‐molecular or ionic cues, including ATP, histidine, and Zn²⁺, is demonstrated. Moreover, owing its multi‐input properties, D‐CID can be used to manipulate the behaviors of multiple cell populations simultaneously in a selective and programmable fashion.     

Detection of Mutations in RNA Polymerase Beta Subunit Gene Encoding Resistance to Rifampin in Mycobacterium tuberculosis by DNA Microarray

Abstract

Rapid and efficient diagnosis is essential in the management of drug‐resistant tuberculosis. A DNA microarray technique based on differential hybridization method was described in the present study for detecting mutations in the RNA polymerase beta subunit (rpoB) gene of Mycobacterium tuberculosis (M. tuberculosis) cultures and in clinical specimens. The mutations in rpoB confer resistance to rifampin, an important first‐line antituberculosis drug. The differential hybridization approach was mainly based on the effect of a single base mismatch on the melting temperature of the hybridized DNA; therefore, any point mutation of rpoB gene resulting in the rifampin resistance can be detected efficiently. The development of the DNA microarray involves the design of dozens of oligonucleotide probes for identifying rifampin‐resistant and ‐sensitive strains. The method comprises isolating genomic DNA from the samples containing M. tuberculosis cells, amplifying rpoB gene coding sequence to produce fluorescently labelled product, and hybridization with the oligonucleotide arrays. The results demonstrated the capability of DNA microarray to provide important clinically relevant information about the rpoB gene of mycobacterial organisms. The DNA microarray offers a reliable diagnostic test for rapidly detecting multidrug resistance caused by gene mutations of mycobacteria.     

Electrochemical Detection of FAM134B Mutations in Oesophageal Cancer Based on DNA‐Gold Affinity Interactions

Inexpensive, simple and rapid DNA sensors capable of accurate and sensitive detection of cancer specific point mutations in DNA biomarkers are crucial for the routine screening of genetic mutations in cancer. Conventional approaches based on sequencing, mass spectroscopy, and fluorescence are highly effective, but they are tedious, slow and require labels and expensive equipment. Recent electrochemistry based approaches mostly rely on conventional DNA biosensing using recognition and transduction layers, and hence limited by the complicated steps of sensor fabrication associated with surface cleaning, self‐assembled monolayer formation, and target hybridization. Herein we report a relatively simple and inexpensive method for detecting point mutation in cancer by using the direct adsorption of purified DNA sequences onto an unmodified gold surface. The method relies on the base dependent affinity interaction of DNA with gold. Since the affinity interaction (adsorption) trend of DNA bases follows as adenine (A) > cytosine (C) > guanine (G)> thymine (T), two DNA sequences with different DNA base compositions (i. e., amplified mutated sequences will be distinctly different than its original sequence) will have different adsorption affinity towards gold. The amount of mutation sites on a DNA sequence is quantified by monitoring the electrochemical current as a function of the relative adsorption level of DNA samples onto a bare gold electrode. This method can successfully distinguish single point mutation in DNA from oesophageal cancer. We demonstrated the clinical utility of this approach by detecting different levels of mutations in tissue samples (n=9) taken from oesophageal cancer patients. Finally, the method was validated with High Resolution Melt (HRM) curve analysis and Sanger Sequencing.     

Inhibition of Influenza Virus Replication in MDCK Cells by Circular Dumbbell RNA/DNA Chimeras with Closed Alkyl Loop Structures

We have demonstrated that a new type of circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON) with two closed nucleotide or alkyl loop structures (hexa‐ethylene glycol) inhibits influenza virus A replication in MDCK cells. The enzymatic synthesis of circular dumbbell RNA/DNA chimeric oligonucleotides was achieved by enzymatically ligating a self‐complementary phosphorylated oligonucleotide with T4‐RNA ligase. The CDRDON‐Al, with two closed alkyl loop structures, showed higher nuclease resistance, hybridization, and cellular uptake than the anti‐S‐ODN and the CDRDON, with two closed nucleotide hairpin‐loop structures. The circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON‐Al‐PB2‐as), containing an AUG initiation‐codon sequence as the target of PB2, showed highly inhibitory effects on influenza A virus RNA expression. The limited toxicity of unmodified phosphodiester oligonucleotides and the sequence‐specific binding to target mRNA indicate that circular dumbbell RNA/DNA chimeric phosphodiester oligonucleotides can be used with intact cells, and may prevent viral replication in culture.     

Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction.

In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.     

Amplification, mutation, and sequencing of a six-letter synthetic genetic system

The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.