DNA Oligonucleotide Probes Research Articles

A smart-responsive Y-shaped DNA scaffold drug conjugate achieving precise and synergetic tumor chemo-gene therapy via promoting tumor apoptosis and activating the anti-tumor immunity

Constructing precise delivery systems to enhance the antitumor efficacy of drugs represents a pressing clinical practice need. The characteristics of suitable physiological stability, flexible tunable size, and highly programmable architecture make functional DNA scaffolds an ideal solution to this requirement. Here, we designed DNA oligonucleotide probes Y1, Y2 and Y3 to construct a smart-responsive Y-shaped DNA scaffold drug conjugate (sYDD). This sYDD incorporates two AS1411 aptamers, an apurinic/apyrimidinic (AP) site, a B-cell lymphoma-2 (Bcl-2) antisense oligonucleotide (ASO) and a doxorubicin (DOX)-carrying sequence. The Bcl-2 ASO and DOX within sYDD could be responsively released in tumor cells that exhibited high expression of apurinic/apyrimidinic endonuclease 1 (APE1) and microRNA-21 (miR-21). And the in vitro and in vivo experiments demonstrated that sYDD exhibited substantial antitumor efficiency. Moreover, the flow cytometry and RNA sequencing results proved that the proposed sYDD effectively activated anti-tumor immune responses. This strategy provides an alternative approach for designing synergistic chemo-gene drug conjugates, which facilitates the development of precision tumor treatment modalities.

Novel RNA genosensor based on highly stable gold nanoparticles decorated phosphorene nanohybrid with graphene for highly sensitive and low-cost electrochemical detection of coconut cadang-cadang viroid.

Coconut cadang-cadang viroid (CCCVd) is an infectious single-stranded RNA (ssRNA) pathogen, which leads directly to the death of a large number of coconut palm trees and heavy economic loss to coconut farmers. Herein, a novel electrochemical impedance RNA genosensor is presented based on highly stable gold nanoparticles (AuNPs) decorated phosphorene (BP) nanohybrid with graphene (Gr) for highly sensitive, low-cost, and label-free detection of CCCVd. BP-AuNPs are environmentally friendly prepared by ultrasonic-assisted liquid-phase exfoliation of black phosphorus, accompanying direct reduction of chloroauric acid. Gr/BP-AuNPs are facilely prepared by the in situ growth of AuNPs onto the BP surface and its nanohybrid with Gr to improve environmental stability of BP. Gr/BP-AuNP-based RNA genosensor is fabricated by immobilizing the thiol-functionalized single-stranded DNA (ssDNA) oligonucleotide probe onto the surface of Gr/BP-AuNP-modified glassy carbon electrode via gold-thiol interactions, which served as an electrochemical genosensing platform for the label-free impedance detection of CCCVd by hybridization between the functionalized ssDNA probe and the complementary CCCVd ssRNA sequence in a wide linear range from 1.0 × 10-11 to 1.0 × 10-7M with a low limit of detection of 2.8 × 10-12M. This work supplies an experimental support and theoretical direction for the fabrication of RNA biosensors based on graphene-like materials and potential application for a specific diagnosis of plant RNA viral disease in Arecaceae planting industry.

Nanoribbon Biosensor-Based Detection of microRNA Markers of Prostate Cancer.

Prostate cancer (PC) is one of the major causes of death among elderly men. PC is often diagnosed later in progression due to asymptomatic early stages. Early detection of PC is thus crucial for effective PC treatment. The aim of this study is the simultaneous highly sensitive detection of a palette of PC-associated microRNAs (miRNAs) in human plasma samples. With this aim, a nanoribbon biosensor system based on "silicon-on-insulator" structures (SOI-NR biosensor) has been employed. In order to provide biospecific detection of the target miRNAs, the surface of individual nanoribbons has been sensitized with DNA oligonucleotide probes (oDNA probes) complementary to the target miRNAs. The lowest concentration of nucleic acids, detectable with our biosensor, has been found to be 1.1 × 10-17 M. The successful detection of target miRNAs, isolated from real plasma samples of PC patients, has also been demonstrated. We believe that the development of highly sensitive nanotechnology-based biosensors for the detection of PC markers is a step towards personalized medicine.

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC.

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Multi-Probe Nano-Genomic Biosensor to Detect S. aureus from Magnetically-Extracted Food Samples.

One of the most prevalent causes of foodborne illnesses worldwide is staphylococcal food poisoning. This study aimed to provide a robust method to extract the bacteria Staphylococcus aureus from food samples using glycan-coated magnetic nanoparticles (MNPs). Then, a cost-effective multi-probe genomic biosensor was designed to detect the nuc gene of S. aureus rapidly in different food matrices. This biosensor utilized gold nanoparticles and two DNA oligonucleotide probes combined to produce a plasmonic/colorimetric response to inform users if the sample was positive for S. aureus. In addition, the specificity and sensitivity of the biosensor were determined. For the specificity trials, the S. aureus biosensor was compared with the extracted DNA of Escherichia coli, Salmonella enterica serovar Enteritidis (SE), and Bacillus cereus. The sensitivity tests showed that the biosensor could detect as low as 2.5 ng/µL of the target DNA with a linear range of up to 20 ng/µL of DNA. With further research, this simple and cost-effective biosensor can rapidly identify foodborne pathogens from large-volume samples.

Chromosome Painting Using Chromosome-Specific BAC Clones.

Chromosome painting (CP) refers to visualization of large chromosome regions, chromosome arms or entire chromosomes via fluorescence in situ hybridization (FISH) of chromosome-specific DNA sequences. For CP in crucifers (Brassicaceae), typically contigs of chromosome-specific bacterial artificial chromosomes (BAC) from Arabidopsis thaliana are applied as painting probes on chromosomes of A. thaliana or other species (comparative chromosome painting, CCP). CP/CCP enables to identify and trace particular chromosome regions and/or chromosomes throughout all mitotic and meiotic stages as well as corresponding interphase chromosome territories. However, extended pachytene chromosomes provide the highest resolution of CP/CCP. Fine-scale chromosome structure, structural chromosome rearrangements (such as inversions, translocations, centromere repositioning), and chromosome breakpoints can be investigated by CP/CCP. BAC DNA probes can be accompanied by other types of DNA probes, such as repetitive DNA, genomic DNA, or synthetic oligonucleotide probes. Here, we describe a robust step-by-step protocol of CP and CCP which proved to be efficient across the family Brassicaceae, but which is also applicable to other angiosperm families.

A dual-mode strategy based on β-galactosidase and target-induced DNA polymerase protection for transcription factor detection using colorimetry and a glucose meter.

In this work, we report a novel dual-mode method for the highly specific and sensitive detection of transcription factors (TFs) via the integration of Klenow polymerase protection induced by target-specific recognition, cascade-signal amplification using the hybridization chain reaction (HCR) and CRISPR/Cas12a system, and dual-signal transduction mediated by β-galactosidase (β-gal) and two substrates. A dual-mode signal-sensing interface was constructed by immobilizing the oligo DNA probe (P1) tethered β-gal in a 96-well plate. A hairpin H1 with the ability to initiate HCRs was designed to contain the TF binding site. The binding between the TF and H1 protected the H1 from being extended by the Klenow fragment. After thermal denaturation, the reserved H1 launched the HCR and the HCR products activated CRISPR/Cas12a to cleave P1 and reduce the β-gal on the sensing interface, and thus the contents of the TFs and the corresponding signals mediated by the catalysis of β-gal showed a correlation. This work was the first attempt at utilizing β-gal for dual-signal transduction. It is a pioneering study to utilize the HCR-CRISPR/Cas12a system for dual-mode TF sensors. It revealed that DNA polymerase protection through the binding of TF and DNA could be applied as a new pattern to develop TF sensors.

Fluorescence in situ Localization of Pri-miRNAs in Isolated Arabidopsis thaliana Nuclei.

Here, we present an approach combining fluorescence in situ hybridization (FISH) and immunolabeling for localization of pri-miRNAs in isolated nuclei of A. thaliana. The presented method utilizes specific DNA oligonucleotide probes, modified by addition of digoxigenin-labeled deoxynucleotides to its 3' hydroxyl terminus by terminal deoxynucleotidyl transferase (TdT). The probes are then detected by immunolabeling of digoxigenin (DIG) using specific fluorescent-labeled antibodies to visualize hybridized probes. Recently, we have applied this method to localize pri-miRNA156a, pri-miRNA163, pri-miRNA393a, and pri-miRNA414 in the nuclei isolated from leaves of 4-week-old A. thaliana. The present approach can be easily implemented to analyze nuclear distribution of diverse RNA classes, including mRNAs and pri-miRNAs in isolated fixed cells or nuclei from plant.

Conferring the Midas Touch on Integrative Taxonomy: A Nanogold-Oligonucleotide Conjugate-Based Quick Species Identification Tool

Nanogold or functionalized gold nanoparticles (GNPs) have myriad applications in medical sciences. GNPs are widely used in the area of nanodiagnostics and nanotherapeutics. Applications of GNPs in taxonomic studies have not been studied vis-à-vis its extensive medical applications. GNPs have great potential in the area of integrative taxonomy. We have realized that GNPs can be used to visually detect animal species based on molecular signatures. In this regard, we have synthesized gold nanoparticles (<20 nm) and have developed a method based on interactions between thiolated DNA oligonucleotides and small-sized GNPs, interactions between DNA oligonucleotides and target DNA molecules, and self-aggregating properties of small-sized GNPs under high salt concentrations leading to a visible change in colour. Exploiting these intermolecular and interparticle interactions under aqueous conditions, in the present work, we have demonstrated the application of our procedure by using a DNA oligonucleotide probe designed against a portion of the mitochondrial genome of the codling moth Cydia pomonella. This method is accurate, quick, and easy to use once devised and can be used as an additional tool along with DNA barcoding. This tool can be used for distinguishing cryptic species, identification of morphovariants of known species, diet analysis, and identification of pest species in quarantine facilities without any need of performing repetitive DNA sequencing. We suggest that designing and selecting a highly specific DNA probe is crucial in increasing the specificity of the procedure. Present work may be considered as an effort to introduce nanotechnology as a new discipline to the extensive field of integrative taxonomy with which disciplines like palaeontology, embryology, anatomy, ethology, ecology, biochemistry, and molecular biology are already associated for a long time.

DNA coated CoZn-ZIF metal-organic frameworks for fluorescent sensing guanosine triphosphate and discrimination of nucleoside triphosphates

Imidazole-based metal-organic frameworks (MOFs) are easy to prepare as well-dispersed nanoparticles, which have attracted a lot of interest in sensing. Metal substitution is an effective way to regulate the composition and performance of MOFs. Herein, by tuning the contents of Co and Zn, a series of homobimetallic CoxZn100-x-ZIF (x = 0–100) were synthesized. Using a fluorescently-labeled DNA oligonucleotide probe, guanosine triphosphate (GTP) can readily displace the adsorbed DNA from Co50Zn50-ZIF, resulting in over 30-fold fluorescence enhancement with 1 mM GTP. Co80Zn20-ZIF could specifically recognize adenosine triphosphate (ATP), whereas Co65Zn35-ZIF and Co20Zn80-ZIF responded to both ATP and GTP. For comparison, Co50Ni50-ZIF and Co50Cu50-ZIF were also prepared, but none of them were selective for any of the molecules, indicating a synergetic effect of cobalt and zinc in Co50Zn50-ZIF for the selective recognition of GTP. This system can sensitively detect GTP with a detection limit of 0.13 μM. Moreover, based on the varying binding affinities of these CoZn-ZIFs towards different nucleoside triphosphates (NTPs), a ZIF fluorescent sensor array was also designed for the discrimination of the four types of NTPs.

Nitrogen-Doped Carbon Dots for Selective and Rapid Gene Detection of Human Papillomavirus Causing Cervical Cancer.

According to WHO, cervical cancer is considered as one of the most frequently diagnosed cancers and the fourth main source of cancer death in women in 2020 worldwide. Hence, there is a need for development of cervical cancer screening with new rapid and cost-effective methods. Although there are few methods available for HPV identification, these techniques are less sensitive, time-consuming, and costly. An ultra-sensitive, selective, and label-free DNA-based impedimetric electrochemical genosensor is developed in this study to detect HPV-18 for cervical cancer. Electrochemical analysis was performed for the characterization of the sensing platform and for the detection of analyte. A single-stranded 25mer oligonucleotide DNA probe was immobilized onto a nitrogen-doped carbon nanodot-modified ITO electrode. Furthermore, the hybridization event was measured by testing the complementary single stranded DNA sequence in the samples. The sensor could distinguish between complementary as well as non-complementary sequences. Herein, impedance quantification demonstrated a limit of detection of 0.405 fM. The developed genosensor showed high selectivity toward HPV-18 in the clinical samples. This sensing platform can be considered as a rapid and selective method for the screening of HPV-18.

Molecular cytogenetic study of Eranthis (Ranunculaceae)

45S and 5S ribosomal DNA were originally localized on chromosomes of five species of winter aconits,namely, Eranthis cilicica, E. hyemalis (section Eranthis), E. pinnatifida, E. stellata и E. tanhoensis (section Shibateranthis).Fluorescence in situ hybridization was performed with oligonucleotide DNA probes Oligo-pTa71-2 and Oligo-5S rDNAof wheat that are complementary to 45S and 5S ribosomal DNA. In addition, oligonucleotide DNA probe (Oligo-5.8SrDNA-Ran, 50 b) for localization of 45S rDNA was designed and tested. This probe is based on the 5.8S rDNA sequencesof some species of fam. Ranunculaceae taken from GenBank. A specific hybridization of the Oligo-5S rDNA and Oligo5.8S rDNA-Ran probes with the chromosomes of Eranthis was shown. The use of the Oligo-pTa71-2 probe did not localizeclusters of 45S rDNA on chromosomes of studied species.

MicroRNA of N-region from SARS-CoV-2: Potential sensing components for biosensor development.

An oligonucleotide DNA probe has been developed for the application in the DNA electrochemical biosensor for the early diagnosis of coronavirus disease (COVID‐19). Here, the virus microRNA from the N‐gene of severe acute respiratory syndrome‐2 (SARS‐CoV‐2) was used for the first time as a specific target for detecting the virus and became a framework for developing the complementary DNA probe. The sequence analysis of the virus microRNA was carried out using bioinformatics tools including basic local alignment search tools, multiple sequence alignment from CLUSTLW, microRNA database (miRbase), microRNA target database, and gene analysis. Cross‐validation of distinct strains of coronavirus and human microRNA sequences was completed to validate the percentage of identical and consent regions. The percent identity parameter from the bioinformatics tools revealed the virus microRNAs’ sequence has a 100% match with the genome of SARS‐CoV‐2 compared with other coronavirus strains, hence improving the selectivity of the complementary DNA probe. The 30 mer with 53.0% GC content of complementary DNA probe 5′ GCC TGA GTT GAG TCA GCA CTG CTC ATG GAT 3′ was designed and could be used as a bioreceptor for the biosensor development in the clinical and environmental diagnosis of COVID‐19.

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores.

The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).

Molecular identification of a novel intracellular proteobacteria from scallop Chlamys farreri

The scallop Chlamys farreri is an important bivalve species cultured in the north of China. With the large expansion of culturing, mass mortalities have occurred persistently and caused great economic loss since 1996. An intracellular Rickettsiales-like prokaryote (RLP) was proposed as the causative agent. To identify the RLP, a partial 16S rDNA sequence of 1319 base pairs was amplified, cloned and sequenced. Phylogenetic analysis indicates that the prokaryote is a member of the α subclass of the proteobacteria. In the phylogenetic tree, it forms an independent and novel clade, which shares an evolutionary line of descent with a group of uniquely obligate intracellular organism comprised of Caedibacter caryophilus, an endosymbiont of Acanthamoeba sp., and the necrotizing hepatopancreatitis (NHP) bacterium of shrimp. Based on the obtained 16S rDNA sequence, three specific RNA targeted oligonucleotide DNA probes were designed, synthesized and labeled with digoxigenin for in situ hybridization detection. Results showed that the probes specifically bonded to the RLP inclusion in infected C. farreri tissues, indicating the amplified, cloned and sequenced 16S rDNA originated from the intracellular RLP parasitizing the C. farreri. We propose the name Sinorickettsia chlamys sp.nov. to C. farreri RLP.

DNA pom-pom nanostructure as a multifunctional platform for pathogenic bacteria determination and inactivation

Pathogenic bacteria levels are significantly related with disease control, clinical diagnosis, and even environmental monitoring. It is becoming highly urgent to achieve ultrasensitive detection of pathogenic bacteria and efficient combat of bacterial infection. Toward this end, we have assembled a DNA Pom-Pom nanostructure (PP–N) based multifunctional platform for pathogenic bacteria determination and inactivation. In particular, one DNA oligonucleotide probe that serve as a trigger was specifically designed for the autonomous cross-opening of metastable DNA hairpin probes and long dsDNA structure formation, achieving a catalytic self-assembly of DNA nanostructure. Numerous DNA strands in this PP-N assembly provide sufficient interaction sites for functional domains and connector, showing high programmability, excellent biostability, as well as selective target recognition. With these properties, the fluorescence dyes modified PP-N platform showed excellent bacteria analysis with both excellent selectivity and ultrasensitive determination limit as low as 2.0 CFU/mL. Furthermore, the aptamer-functionalized and antibiotics loaded PP-N platform demonstrate excellent merits of high antibiotics-loading capacity and negligible cytotoxicity to targets. Therefore, this DNA PP-N assembly based multifunctional platform promise its great application in targeted sensing, combating bacterial infection, and potential clinic therapy.

FISH in Suspension or in Adherent Cells.

Fluorescence in situ hybridization (FISH) enables the detection and enumeration of microorganisms in a diversity of samples. Short-length oligonucleotide DNA probes complementary to 16S or 23S rRNA sequences are generally used to target different phylogenetic levels. The protocol for the application of FISH to aggregated or suspended cells in mixed microbial communities is described in this chapter, with a special emphasis on environmental samples.

Computational design of probes to detect bacterial genomes by multivalent binding

Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target-probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.

Transcript specific mRNP capture from Drosophila egg-chambers for proteomic analysis

mRNA binding proteins (RBPs) play a major role in post-transcriptional control of gene expression. To understand the complex regulatory processes regulating a specific mRNA during its life-time, a comprehensive view of the bound RBPs is essential. Here, we describe a method for transcript-specific isolation of endogenous ribonucleoprotein complexes (RNPs) from Drosophila egg-chambers. The method, which is based on in-solution hybridization of short biotinylated antisense DNA oligonucleotide probes to multiple segments of a transcript of interest allows unbiased identification of associated proteins by quantitative proteomics.