Stem-loop Research Articles

Plasmon-emitter coupling in cytosine-rich hairpin DNA-templated silver nanoclusters: Thermal reversibility, white light emission, and dynamics inside live cells.

Bio-templated luminescent noble metal nanoclusters (NCs) have attracted great attention for their intriguing physicochemical properties. Continuous efforts are being made to prepare NCs with high fluorescence quantum yield (QY), good biocompatibility, and tunable emission properties for their widespread practical applications as new-generation environment-friendly photoluminescent materials in materials chemistry and biological systems. Herein, we explored the unique photophysical properties of silver nanoclusters (AgNCs) templated by cytosine-rich customized hairpin DNA. Our results indicate that a 36-nucleotide containing hairpin DNA with 20 cytosine (C20) in the loop can encapsulate photostable red-emitting AgNCs with an absolute QY of ∼24%. The luminescent properties in these DNA-templated AgNCs were found to be linked to the coupling between the surface plasmon and the emitter. These AgNCs exhibited excellent thermal sensitivity and were employed to produce high-quality white light emission with an impressive color rendering index of 90 in the presence of dansyl chloride. In addition, the as-prepared luminescent AgNCs possessing excellent biocompatibility can effectively mark the nuclear region of HeLa cells and can be employed as a luminescent probe to monitor the cellular dynamics at a single molecular resolution.

Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A: Impacts on rescue of sequence-modifying viruses

Senecavirus A (SVA) has a positive-sense, single-stranded RNA genome. Its 5´ untranslated region harbors an internal ribosome entry site (IRES), comprising 10 larger or smaller stem-loop structures (including a pseudoknot) that have been demonstrated to be well conserved. However, it is still unclear whether each stem-loop subdomain, such as a single stem or loop, is also highly conserved. To clarify this issue in the present study, a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis (SDM) on the IRES. The SDM-modified scenarios included: (1) stem-formed complementary sequences exchanging with each other; (2) loop transversion; (3) loop transition; and (4) point mutations. All cDNA clones were separately transfected into cells for rescuing viable viruses, whereas only four SVAs of interest could be recovered, and were genetically stable during 20 passages. One progeny grew significantly slower than the other three did. The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity. Our previous study indicated that a single motif from any of the ten stem structures, if completely mutated, would cause the failure of virus recovery. Interestingly, our present study revealed three stem structures, whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs. Moreover, one apical loop was demonstrated to have the ability to tolerate its own full-length transition, also having no impact on the recovery of sequence-modifying SVA. The present study suggested that not every stem-loop structure was strictly conserved in its conformation, while the full-length IRES itself was well conserved. This provides a new research direction on interaction between the IRES and many factors.

Conservation and distribution of the DRACH motif for potential m6A sites in avian leukosis virus subgroup J.

N6-methyladenosine (m6A) methylation is an internal post-transcriptional modification that has been linked to viral multiplication and pathogenicity. To elucidate the conservation patterns of potential 5'-DRACH-3' motifs in avian leukosis virus subgroup J (ALV-J), 149 ALV-J strains (139 isolates from China; ALV-J prototype HPRS-103 from the UK; and 9 strains from the USA, Russia, India, and Pakistan) available in GenBank before December 2023 were retrieved. According to the prediction results of the SRAMP web-server, these ALV-J genomes contained potential DRACH motifs, with the total number ranging from 43 to 64, which were not determined based on the isolation region and time. Conservative analysis suggested that 37 motifs exhibited a conservation of >80%, including 17 motifs with a grading above "high confidence." Although these motifs were distributed in the U5 region of LTRs and major coding regions, they were enriched in the coding regions of p27, p68, p32, and gp85. The most common m6A-motif sequence of the DRACH motif in the ALV-J genome was GGACU. The RNA secondary structure of each conserved motif predicted by SRAMP and RNAstructure web-server was mainly of two types-A-U pair (21/37) and hairpin loop (16/37)-based on the core adenosine. Considering the systematic comparative analysis performed in this study, future thorough biochemical research is warranted to determine the role of m6A modification during the replication and infection of ALV-J. These conservation and distribution analysis of the DRACH motif for potential m6A sites in ALV-J would provide a foundation for the future intervention of ALV-J infection and m6A modification.

Self-assembly of a AuNPs/Ti3C2 MXene hydrogel for cascade amplification of microRNA-122 biosensing.

A three-dimensional (3D) self-assembled AuNPs/Ti3C2 MXene hydrogel (AuNPs/Ti3C2 MXH) nanocomposite was prepared for the fabrication of a novel microRNA-122 electrochemical biosensor. The 3D hydrogel structure was gelated from two-dimensional MXene nanosheets with the assistance of graphite oxide and ethylenediamine. MXene hydrogels supported the in situ formation of Au nanoparticles (AuNPs) that predominantly exploring the (111) facet, and these AuNPs are utilized as carriers for hairpin DNA (hpDNA) probes, facilitating DNA hybridization. MXene acted as both a reductant and stabilizer, significantly improving the electrochemical signal. In addition, the conjugation of PAMAM dendrimer-encapsulated AuNPs and H-DNA worked as an ideal bridge to connect targets and efficient electrochemical tags, providing a high amplification efficiency for the sensing of microRNA-122. A linear relationship between the peak currents and the logarithm of the concentrations of microRNA-122 from 1.0 × 10-2 to 1.0 × 102 fM(I = 1.642 + 0.312 lgc, R2 = 0.9891), is obtained. The detection limit is 0.8 × 10-2 fM (S/N = 3). The average recovery for human serum detection ranged from 97.32 to 101.4% (RSD < 5%).

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

A dual-signal ratiometric electrochemical aptasensor based on Thi/Au/ZIF-8 and catalytic hairpin assembly for ultra-sensitive detection of aflatoxin B1.

Adual-signal ratiometric electrochemical aptasensor has been developed for AFB1 detection using thionine/Au/zeolitic imidazolate framework-8 (Thi/Au/ZIF-8) nanomaterials and catalytic hairpin assembly (CHA) reaction. Thi/Au/ZIF-8 combined with DNA hairpin 2 (H2) was used as a signal probe. [Fe(CN)6]3-/4- was served as another signal probe, and the IThi/Au/ZIF-8/I[Fe(CN)6]3-/4- ratio was for the first timeutilized to quantify AFB1. AFB1-induced CHA was used to expand the ratio of electrical signals. In the presence of AFB1, H2/Thi/Au/ZIF-8 bound to the electrode via CHA, enhanced the current signal of Thi/Au/ZIF-8. H2 contained the DNA phosphate backbone hindered [Fe(CN)6]3-/4- redox reaction and resulted in a lower [Fe(CN)6]3-/4- current signal. This aptasensor exhibited high specificity for AFB1, a linear range of 0.1pgmL-1 to 100ngmL-1, and a detection limit of 0.089pgmL-1. It demonstrated favorable sensitivity, selectivity, stability, and repeatability. The aptasensor was suitable for detecting AFB1 in peanuts and black tea and holds potential for real sample applications.

Hyperbranched RCA-mediated construction of a dendritic DNA nanostructure for ratiometric fluorescence biosensing

As a robust isothermal strand amplification technique, rolling circle amplification (RCA) is popularly adopted for constructing various homogeneous fluorescence biosensing strategies. However, the limited functional unit decoration on linear RCA products and the slow homogeneous reaction kinetics usually restrict their performance. Herein, by designing a target biorecognition-triggered hyperbranched RCA reaction to construct an Mg2+-dependent DNAzyme (MNAzyme)-decorated dendritic DNA nanostructure, a novel ratiometric fluorescence biosensing method was proposed. The catalyzed cleavage of MNAzymes toward a short stand labeled with the FAM fluorophore and its quencher realized a “signal-on” fluorescence output. Meanwhile, a “signal-off” fluorescence output of 2-aminopurine (2-AP) labeled at a DNA hairpin was realized through the DNA hybridization with the nanostructure. Besides efficient acceleration of homogeneous reaction kinetics, these fluorescence responses were dramatically amplified by the high decoration of MNAzymes on the dendritic nanostructure and its strong signal suppression toward 2-AP. Together with the exonuclease III-amplified target biorecognition reaction, a very low detection limit of 0.53 fg mL–1 and a six-order magnitude linear range were endowed for kanamycin antibiotic assay. This excellent analytical performance and its high selectivity, and good operability determine promising prospect of the method for practical applications.

Microsatellite break-induced replication generates highly mutagenized extrachromosomal circular DNAs.

Extrachromosomal circular DNAs (eccDNAs) are produced from all regions of the eucaryotic genome. We used inverse PCR of non-B microsatellites capable of forming hairpin, triplex, quadruplex and AT-rich structures integrated at a common ectopic chromosomal site to show that these non-B DNAs generate highly mutagenized eccDNAs by replication-dependent mechanisms. Mutagenesis occurs within the non-B DNAs and extends several kilobases bidirectionally into flanking and nonallelic DNA. Each non-B DNA exhibits a different pattern of mutagenesis, while sister clones containing the same non-B DNA also display distinct patterns of recombination, microhomology-mediated template switching and base substitutions. Mutations include mismatches, short duplications, long nontemplated insertions, large deletions and template switches to sister chromatids and nonallelic chromosomes. Drug-induced replication stress or the depletion of DNA repair factors Rad51, the COPS2 signalosome subunit or POLη change the pattern of template switching and alter the eccDNA mutagenic profiles. We propose an asynchronous capture model based on break-induced replication from microsatellite-induced DNA double strand breaks to account for the generation and circularization of mutagenized eccDNAs and the appearance of genomic homologous recombination deficiency (HRD) scars. These results may help to explain the appearance of tumor eccDNAS and their roles in neoantigen production, oncogenesis and resistance to chemotherapy.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

Utility of oligonucleotide in upregulating circular RNA production in a cellular model

Circular RNAs (circRNAs), are a covalently closed, single-stranded RNA without 5′- and 3′-termini, commonly stem from the exons of precursor mRNAs (pre-mRNAs). They have recently garnered interest, with studies uncovering their pivotal roles in regulating various aspects of cell functions and disease progressions. A notable feature of circRNA lies in the mechanism of its biogenesis involving a specialized form of splicing: back-splicing. A splicing process that relies on interactions between introns flanking the circularizing exon to bring the up and downstream splice sites in proximity through the formation of a prerequisite hairpin structure, allowing the spliceosomes to join the two splice sites together to produce a circular RNA molecule. Based on this mechanism, we explored the feasibility of facilitating the formation of such a prerequisite hairpin structure by utilizing a newly designed oligonucleotide, CircuLarIzation Promoting OligoNucleotide (CLIP-ON), to promote the production of circRNA in cells. CLIP-ON was designed to hybridize with and physically bridge two distal sequences in the flanking introns of the circularizing exons. The feasibility of CLIP-ON was confirmed in HeLa cells using a model pre-mRNA, demonstrating the applicability of CLIP-ON as a trans-acting modulator to upregulate the production of circRNAs in a cellular environment.

Label-free and sensitive fluorescent detection of Escherichia coli O157: H7 in milk based on cascade strand displacement amplification and G-quadruplex-thioflavin T

The development of a simple, rapid, sensitive, and accurate method for detecting Escherichia coli O157:H7 in complex matrices is critical for ensuring food safety. In this study, a fluorescent biosensor employing aptamer (Apt) -functionalized magnetic beads and cascade strand displacement amplification (SDA) was devised for the detection of E. coli O157:H7 in milk samples. E. coli O157:H7 is captured by the aptamer, causing the complementary DNA (cDNA) to not bind to the aptamer and to be released into the supernatant. Following magnetic separation, the cDNA present in the supernatant initiates the opening of the hairpin (HP) structures. This enables primers to attach to the opened HP, triggering the initial SDA process, which generates a substantial quantity of double-stranded DNA (dsDNA) and recycles cDNA to open more HP structures. The dsDNA activates a second SDA, producing single-stranded DNA (ssDNA) for template binding and instigating a third SDA process. Triple SDA leads to the production of numerous G-quadruplex sequences and the formation of a G-quadruplex structure in the presence of K+. The inclusion of ThT into the G-quadruplex significantly enhances fluorescence. Under optimal conditions, this approach can detect E. coli O157:H7 at levels as low as 101 CFU/mL in pure cultures within 2.5 h, with a broad linear detection range of 101–106 CFU/mL. This method exhibits remarkable specificity towards the target bacteria over non-target bacteria. Furthermore, the technique has been effectively utilized for the detection of E. coli O157:H7 in milk samples. Notably, the method involves only two main steps, identification and amplification, significantly reducing contamination risks and minimizing operational steps.

Fully resolved simulation of spherical and non-spherical particles in a turbulent channel flow

This paper investigates the behavior of finite-size particles in a turbulent channel flow using a custom direct numerical simulation solver within the FOAM-Extend framework. The solver integrates the cut-cell immersed boundary module with a Lagrangian particle-tracking subroutine capable of simulating the motion of both spherical and non-spherical particles. The study investigates the complex interactions between particles and turbulent structures, offering insights into how particle shape and orientation affect their behavior within the flow field. Additionally, it examines the collision dynamics of two spherical particles in a turbulent channel. The simulations reveal that particle shape significantly influences particle trajectories, rotation, and their interactions with turbulent structures. The Q-criterion visualization showed the creation of hairpin and vortex ring structures shed by the particles. Finally, the close proximity and collision of particles was shown to significantly modify the flow pattern and particle dynamics.

Three-way junction structure-mediated reverse transcription-free exponential amplification reaction for pathogen RNA detection.

Since RNA is an important biomarker of many infectious pathogens, RNA detection of pathogenic organisms is crucial for disease diagnosis and environmental and food safety. By simulating the base mismatch during DNA replication, this study presents a novel three-way junction structure-mediated reverse transcription-free exponential amplification reaction (3WJ-RTF-EXPAR) for the rapid and sensitive detection of pathogen RNA. The target RNA served as a switch to initiate the reaction by forming a three-way junction (3WJ) structure with the ex-trigger strand and the ex-primer strand. The generated trigger strand could be significantly amplified through EXPAR to open the stem-loop structure of the molecular beacon to emit fluorescence signal. The proofreading activity of Vent DNA polymerase, in combination with the unique structure of 2+1 bases at the 3'-end of the ex-primer strand, could enhance the role of target RNA as a reaction switch to reduce non-specific amplification and ensure excellent specificity to differentiate target pathogen from those causing similar symptoms. Furthermore, detection of target RNA showed a detection limit of 1.0×104 copies/mL, while the time consumption was only 20 min, outperforming qRT-LAMP and qRT-PCR, the most commonly used RNA detection methods in clinical practice. All those indicates the great application prospects of this method in clinical diagnostic.

Logically integrating exo/endogenous gated DNA trackers for precise microRNA imaging via synergistic manipulation

Nanomaterials provide an ideal platform for biomolecular display due to their dimensions approach the molecular scale, facilitating binding behavior akin to that observed in solution-based processes. DNA nanoprobes hold great promise as miniature detectives capable of detecting miRNAs within cells. However, current nanoprobes face a challenge in achieving the required precision for accurate miRNA detection, particularly within the intricate confines of the cellular microenvironment, due to interference with biological autofluorescence, off-target effects, and a lack of spatiotemporal control. Here, we have designed a dual-stimuli responsive DNA tracker, synergistically utilizing specific intracellular cues and external triggers, which enables spatiotemporal-controlled and precise detection and imaging of miRNAs "on demand". The tracker, which combines zeolitic imidazolate framework-67 (ZIF-67) and unique hairpin DNA structures, effectively anchored onto the ZIF-67 through electrostatic interactions, remains in a dormant state until activated by abundant cellular ATP, resulting in the release of the hairpin structures that include a PC linker incorporated into the loop region. Subsequent irradiation triggers specific recognition of the target miRNA. The newly developed HP-PC-BT@ZIF-67 tracker demonstrates precise spatiotemporal miRNA detection and exhibits excellent biocompatibility, enabling specific miRNA recognition "on demand" within cancer cells. This research presents a reliable miRNA imaging platform in the intricate cellular environment, opening up the possibilities for precise biomedical analysis and disease diagnosis.

The RNA secondary structure of androgen receptor-FL and V7 transcripts reveals novel regulatory regions.

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor belonging to the steroid hormone nuclear receptor family. Due to its roles in regulating cell proliferation and differentiation, AR is tightly regulated to maintain proper levels of itself and the many genes it controls. AR dysregulation is a driver of many human diseases including prostate cancer. Though this dysregulation often occurs at the RNA level, there are many unknowns surrounding post-transcriptional regulation of AR mRNA, particularly the role that RNA secondary structure plays. Thus, a comprehensive analysis of AR transcript secondary structure is needed. We address this through the computational and experimental analyses of two key isoforms, full length (AR-FL) and truncated (AR-V7). Here, a combination of in-cell RNA secondary structure probing experiments (targeted DMS-MaPseq) and computational predictions were used to characterize the static structural landscape and conformational dynamics of both isoforms. Additionally, in-cell assays were used to identify functionally relevant structures in the 5' and 3' UTRs of AR-FL. A notable example is a conserved stem loop structure in the 5'UTR of AR-FL that can bind to Poly(RC) Binding Protein 2 (PCBP2). Taken together, our results reveal novel features that regulate AR expression.

Whole-genome mapping of APOBEC mutagenesis in metastatic urothelial carcinoma identifies driver hotspot mutations and a novel mutational signature

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes mutate specific DNA sequences and hairpin-loop structures, challenging the distinction between passenger and driver hotspot mutations. Here, we characterized 115 whole genomes of metastatic urothelial carcinoma (mUC) to identify APOBEC mutagenic hotspot drivers. APOBEC-associated mutations were detected in 92% of mUCs and were equally distributed across the genome, while APOBEC hotspot mutations (ApoHMs) were enriched in open chromatin. Hairpin loops were frequent targets of didymi (twins in Greek), two hotspot mutations characterized by the APOBEC SBS2 signature, in conjunction with an uncharacterized mutational context (Ap[C>T]). Next, we developed a statistical framework that identified ApoHMs as drivers in coding and non-coding genomic regions of mUCs. Our results and statistical framework were validated in independent cohorts of 23 non-metastatic UCs and 3,744 samples of 17 metastatic cancers, identifying cancer-type-specific drivers. Our study highlights the role of APOBEC in cancer development and may contribute to developing novel targeted therapy options for APOBEC-driven cancers.

Boosting the sensitivity for tau protein detection with a bifunctional nanoenzyme signal amplifier

The trace amounts of tau protein in the blood are considered a significant biomarker in the early diagnosis of Alzheimer's disease. Herein, an electrochemical biosensor has been developed that utilizes a bifunctional nanoenzyme signal amplifier (Nanosa), which incorporates hybridization chain reaction (HCR) amplification and peroxide-mimicking nanoenzymes enhancement to enable ultrasensitive detection of tau. The Nanosa utilizes a peroxide-active MoS2/Au substrate on which DNA aptamer and hairpin probes are fixed as signal amplifiers capable of triggering HCR. The Fc molecules were initially aggregated at the electrode interface through HCR amplification, and were tested by adding an electrolyte solution containing hydrogen peroxide (H2O2). Under the catalysis of a peroxide nanoenzyme, the electrochemical signal of Fc molecules was further enhanced. The bifunctional Nanosa can not only achieve the pre-enrichment of Fc molecules at the electrode interface but also catalyze the enhancement of its electrochemical signal. The well-designed and optimized poly adenine tail not only as an electrode coupling agent but also as an excellent antifouling coating. The ingenious design of the Nanosa-based biosensing strategy enables the ultrasensitive analysis of tau with a broad linear range from 0.1 pg/mL to 100 ng/mL and a lower LOD of 33.4 fg/mL. Meanwhile, the outstanding analytical performance of this Nanosa-based biosensing strategy enables its successful application in determining tau levels in real plasma samples, and showed good agreement with results obtained using a commercial ELISA kit.

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

Label-free and portable detection of HIV-DNA by a handheld luminometer

BackgroundThe human immunodeficiency virus (HIV) remains a major worldwide health problem. Nowadays, many methods have been developed for quantitative detecting human immunodeficiency virus DNA (HIV-DNA), such as fluorescence and colorimetry. However, these methods still have the disadvantages of being expensive and requiring professional technicians. Early diagnosis of pathogens is increasingly dependent on portable instruments and simple point-of-care testing (POCT). Therefore, it is meaningful and necessary to develop portable and cheap methods for detecting disease markers. ResultsIn this work, a label-free chemiluminescence (CL) method was developed for detecting HIV-DNA via a handheld luminometer. To achieve label-free target detection, the CL catalyst, G-triplex-hemin DNAzyme (G3-hemin DNAzyme), was in-situ assembled in the presence of HIV-DNA. For improving sensitivity, HIV-DNA induced the cyclic strand displacement reaction (SDR), which can form three G3-hemin DNAzymes in one cycle. So, the chemiluminescence reaction between luminol and H2O2 was highly effectively catalyzed, and the CL intensity was linearly related with the concentration of HIV-DNA in the range of 0.05–10 nM with a detection limit of 29.0 pM. Due to the high specificity of hairpin DNA, single-base mismatch can be discriminated, which ensured the specific detection of HIV-DNA. SignificanceIn-situ formation of G3-hemin DNAzyme led to label-free and selective detection without complex synthesis and functionalization. Therefore, it offers a cheap, selective, sensitive and portable method for detecting disease-related genes, which is promising for POCT of clinical diagnosis in resource-limited settings.