Stem-loop Research Articles

Stability-Based Proteomics for Investigation of Structured RNA-Protein Interactions.

RNA-protein interactions are essential to RNA function throughout biology. Identifying the protein interactions associated with a specific RNA, however, is currently hindered by the need for RNA labeling or costly tiling-based approaches. Conventional strategies, which commonly rely on affinity pull-down approaches, are also skewed to the detection of high affinity interactions and frequently miss weaker interactions that may be biologically important. Reported here is the first adaptation of stability-based mass spectrometry methods for the global analysis of RNA-protein interactions. The stability of proteins from rates of oxidation (SPROX) and thermal protein profiling (TPP) methods are used to identify the protein targets of three RNA ligands, the MALAT1 triple helix (TH), a viral stem loop (SL), and an unstructured RNA (PolyU), in LNCaP nuclear lysate. The 315 protein hits with RNA-induced conformational and stability changes detected by TPP and/or SPROX were enriched in previously annotated RNA-binding proteins and included new proteins for hypothesis generation. Also demonstrated are the orthogonality of the SPROX and TPP approaches and the utility of the domain-specific information available with SPROX. This work establishes a novel platform for the global discovery and interrogation of RNA-protein interactions that is generalizable to numerous biological contexts and RNA targets.

Smartphone‐Based Free‐to‐Total Prostate Specific Antigen Ratio Detection System Using a Colorimetric Reaction Integrated with Proximity‐Induced Bio‐Barcode and CRISPR/Cas12a Assay

AbstractThe free‐to‐total prostate‐specific antigen (f/t‐PSA) ratio is of great significance in the accurate diagnosis of prostate cancer. Herein, a smartphone‐based detection system is reported using a colorimetric reaction integrated with proximity‐induced bio‐barcode and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a assay for f/t‐PSA ratio detection. DNA/antibody recognition probes are designed to bind f‐PSA or t‐PSA and induce the release of the DNA bio‐barcode. The CRISPR/Cas12a system is activated by the DNA bio‐barcode to release Ag+ from the C‐Ag+‐C structure of the hairpin DNA. The released Ag+ is used to affect the tetramethylbenzidine (TMB)‐H2O2‐based colorimetric reaction catalyzed by Pt nanoparticles (NPs), as the peroxidase‐like activity of the Pt NPs can be efficiently inhibited by Ag+. A smartphone with a self‐developed app is used as an image reader and analyzer to analyze the colorimetric reaction and provide the results. A limit of detection of 0.06 and 0.04 ng mL−1 is achieved for t‐PSA and f‐PSA, respectively. The smartphone‐based method showed a linear response between 0.1 and 100 ng mL−1 of t‐PSA or f‐PSA. In tests with clinical samples, the smartphone‐based method successfully diagnosed prostate cancer patients from benign prostatic hyperplasia patients and healthy cases with high sensitivity and specificity.

Bifidobacterial GH146 β-l-arabinofuranosidase for the removal of β1,3-l-arabinofuranosides on plant glycans

l-Arabinofuranosides with β-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a β-l-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 β-l-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-β1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-β1,3-Araf structures but not those containing Araf-β1,2-Araf and Araf-β1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two β-sandwich domains. A hairpin structure with two β-strands was observed in Bll3HypBA1, to extend from a β-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a β1,3-specific β-l-arabinofuranosidase.Key points• β1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar.• β-l-Arabinofuranosidases are widely present in intestinal bacteria.• Bll3HypBA1 is the first enzyme characterized as a β1,3-linkage-specific β-l-arabinofuranosidase.

A reagent-based label free electrochemiluminescence biosensor for ultrasensitive quantification of low-abundant chloramphenicol

Today, great attentions have been attracted on chloramphenicol (CAP) to its nonnegligible biotoxicity to human. It is most important and urgent to quantify the low-abundant chloramphenicol by generating simple and efficient analysis approach for the detection of chloramphenicol. Herein, we proposed a novel reagent-based label free electrochemiluminescence (ECL) biosensor based on AuNPs@CoFe-PBA nanomaterials as highly efficient catalysts for luminol/hydrogen peroxide (H2O2) system and target indirectly induced activation of the CRISPR/Cas 12a amplification system for simple and efficient quantification of the low-abundant chloramphenicol. Typically, AuNPs@CoFe-PBA nanomaterials as a novel catalyst for luminol/H2O2 system were enclosed in the X-type DNA hydrogel to greatly inhibit their catalytic performance due to the blocking effect of DNA hydrogel. In presence of target CAP, it induced DNA structure transformation of the aptamer of chloramphenicol to generate a toehold to present catalyzed hairpin assembly (CHA) amplification with the help of two self-designed hairpin structure DNA to perform the hybridizer of the two hairpins, which could activate the CRISPR/Cas 12a system to carry out the DNA cleavage. Its activity breaks the structure of DNA hydrogels and releases AuNPs@CoFe-PBA nanomaterials, which enhances the ECL signals of the luminol/H2O2 systems for simple and sensitive analysis of chloramphenicol. Most importantly, all of the target induced amplifications were performed in the detection solution and the ECL reactions were generated on the bare electrode without any modification to construct a reagent-based label-free analysis system simply with high efficiency for quantification of the low-abundant chloramphenicol. The results showed that there was a good linear relationship between ECL intensity and CAP concentration in range from 10 pg/mL to 1 ug/mL with a detection limit of 7.75 pg/mL, providing great potentials to quantify the low-abundant chloramphenicol and some other biomarkers for early disease diagnosis, food safety testing and environmental analysis.

High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

Computational studies of pressure-induced effects on DNA hairpin structures

Computational studies of pressure-induced effects on DNA hairpin structures

Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) are two long noncoding RNAs upregulated in multiple cancers, marking these RNAs as therapeutic targets. While traditional small-molecule and antisense-based approaches are effective, we report a locked nucleic acid (LNA)-based approach that targets the MALAT1 and MENβ triple helices, structures comprised of a U-rich internal stem-loop and an A-rich tract. Two LNA oligonucleotides resembling the A-rich tract (i.e., A9GCA4) were examined: an LNA (L15) and a phosphorothioate LNA (PS-L15). L15 binds tighter than PS-L15 to the MALAT1 and MENβ stem loops, although both L15 and PS-L15 enable RNA•LNA-RNA triple-helix formation. Based on UV thermal denaturation assays, both LNAs selectively stabilize the Hoogsteen interface by 5-13 °C more than the Watson-Crick interface. Furthermore, we show that L15 and PS-L15 displace the A-rich tract from the MALAT1 and MENβ stem loop and methyltransferase-like protein 16 (METTL16) from the METTL16-MALAT1 triple-helix complex. Human colorectal carcinoma (HCT116) cells transfected with LNAs have 2-fold less MALAT1 and MENβ. This LNA-based approach represents a potential therapeutic strategy for the dual targeting of MALAT1 and MENβ.

Precursor-directed biosynthesis and biological activity of tripropeptin Cpip, a new tripropeptin C analog containing pipecolic acid.

Tripropeptin C, a non-ribosomal cyclic lipopeptide containing three proline residues, exhibits excellent efficacy in the mouse-methicillin-resistant Staphylococcus aureus septicemia model. Since tripropeptins contain L-prolyl-D-proline and, as a result, are known to form a hairpin structure in proteins, it was of interest to determine whether this substructure contributes to their antibacterial activity. In this study, prolines in tripropeptin C were replaced with pipecolic acid(s) using precursor-directed biosynthesis. Only a new tripropeptin analog, tripropeptin Cpip, which has one L-pipecolic acid in place of L-proline, was isolated. The in vitro antimicrobial activity of the new analog was approximately two to four times weaker activity against Gram-positive bacteria, including drug-resistant species, compared with that of tripropeptin C. These results suggest that the L-prolyl-D-proline substructure plays an important role in the observed potency of tripropeptins.

A photoelectrochemical aptasensor for omethoate determination based on a photocatalysis of CeO2@MnO2 heterojunction for glucose oxidation

In the present work, we developed a photoelectrochemical aptasensor to determine omethoate (OMT) based on the dual signal amplification of CeO2@MnO2 photocatalysis for glucose oxidation and exonuclease I-assisted cyclic catalytic hydrolysis. CeO2@MnO2 heterojunction material prepared by hydrothermal method was linked with captured DNA (cDNA) and then assembled on the ITO conductive glass to form ITO/CeO2@MnO2-cDNA, which exhibited significant photocurrent response and good photocatalytic performance for glucose oxidation under visible light irradiation, providing the feasibility for sensitive determining OMT. After binding with the aptamer of OMT (apt), the formation of rigid double stranded cDNA/apt kept CeO2@MnO2 away from ITO surface, which ensured a low photocurrent background for the constructed ITO/CeO2@MnO2-cDNA/apt aptasensor. In the presence of target OMT, the restoration of the cDNA hairpin structure and the exonuclease I-assisted cyclic catalytic hydrolysis led to the generation and amplification of measurement photocurrent signals, and allowed the aptasensor to have an ideal quantitative range of 0.01–10.0 nM and low detection limit of 0.0027 nM. Moreover, the aptasensor has been applied for selective determination of OMT in real samples with good precision of the relative standard deviation less than 6.2 % and good accuracy of the recoveries from 93 % to 108 %. What's more, the aptasensor can be used for other target determination only by replacing the captured DNA and corresponding aptamer.

Carbon-nanotube field-effect transistors for resolving single-molecule aptamer-ligand binding kinetics.

Small molecules such as neurotransmitters are critical for biochemical functions in living systems. While conventional ultraviolet-visible spectroscopy and mass spectrometry lack portability and are unsuitable for time-resolved measurements in situ, techniques such as amperometry and traditional field-effect detection require a large ensemble of molecules to reach detectable signal levels. Here we demonstrate the potential of carbon-nanotube-based single-molecule field-effect transistors (smFETs), which can detect the charge on a single molecule, as a new platform for recognizing and assaying small molecules. smFETs are formed by the covalent attachment of a probe molecule, in our case a DNA aptamer, to a carbon nanotube. Conformation changes on binding are manifest as discrete changes in the nanotube electrical conductance. By monitoring the kinetics of conformational changes in a binding aptamer, we show that smFETs can detect and quantify serotonin at the single-molecule level, providing unique insights into the dynamics of the aptamer-ligand system. In particular, we show the involvement of G-quadruplex formation and the disruption of the native hairpin structure in the conformational changes of the serotonin-aptamer complex. The smFET is a label-free approach to analysing molecular interactions at the single-molecule level with high temporal resolution, providing additional insights into complex biological processes.

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

Enzyme-free sensitive SERS biosensor for the detection of thalassemia-associated microRNA-210 using a cascade dual-signal amplification strategy

Backgroundβ-thalassemia is a blood disorder caused by autosomal mutations. Gene modulation therapy to activate the γ-globin gene to induce fetal hemoglobin (HbF) synthesis has become a new option for the treatment of β-thalassemia. MicroRNA-210 (miR-210) contributes to studying the mechanism regulating γ-globin gene expression and is a potential biomarker for rapid β-thalassemia screening. Traditional miRNA detection methods perform well but necessitate complex and time-consuming miRNA sample processing. Therefore, the development of a sensitive, accurate, and simple miRNA level monitoring method is essential. ResultsWe have developed a non-enzymatic surface-enhanced Raman scattering (SERS) biosensor utilizing a signal cascade amplification of catalytic hairpin assembly reaction (CHA) and proximity hybridization-induced hybridization chain reaction (HCR). Au@Ag NPs were used as the SERS substrate, and methylene blue (MB)- modified DNA hairpins were used as the SERS tags. The SERS assay involved two stages: implementing the CHA-HCR cascade signal amplification strategy and conducting SERS measurements on the resulting product. The HCR was started by the products of target-triggered CHA, which formed lengthy nicked double-stranded DNA (dsDNA) on the Au@Ag NPs surface to which numerous SERS tags were attached, leading to a significant increase in the SERS signal intensity. High specificity and sensitivity for miR-210 detection was achieved by monitoring MB SERS intensity changes. The suggested SERS biosensor has a low detection limit of 5.13 fM and is capable of detecting miR-210 at concentration between 10 fM and 1.0 nM. SignificanceThe biosensor can detect miR-210 levels in the erythrocytes of β-thalassemia patients, enabling rapid screening for β-thalassemia and suggesting a novel approach for investigating the regulation mechanism of miR-210 on γ-globin gene expression. In the meantime, this innovative technique has the potential to detect additional miRNAs and to become an important tool for the early diagnosis of diseases and for biomedical research.

Thermodynamic-assisted highly effective target converting strategy for sensitive electrochemical detection of trace lead (II) ion

In the present work, a thermodynamic-assisted target-converting strategy was designed by utilizing new 2D DNA nanoprobes (DNPs) to develop electrochemical biosensors for the sensitive determination of trace Pb2+. Herein, a stable DNP with a special structure was prepared through the hybridization reaction between three single-strand DNA (RS1, RS2, and P). Once the existence of Pb2+, the DNP will crumble and then release RS1 and RS2 due to the strong thermodynamic instability of the RS1/RS2 duplex. since RS1 and RS2 were designed in the same sequence in the free domain, they can be simultaneously used as effective output DNA, enhancing the conversion rate of Pb2+to ssDNA up to 1:2. Following the same principle, ferrocene-labeled bipedal DNPs (Fc-bDNPs) are prepared for stable electrochemical signal output. In the presence of RS1, RS2, and a hairpin DNA strand (H1), the ferrocene-labeled DNA strands (S3) escape from the Fc-bDNPs due to strand-displacement reaction, accompanying release of the two ssDNA outputs that can simultaneously trigger another strand-displacement reaction. Consequently, the one Pb2+input makes multiple Fc-labeled DNA strands far away from the electrode surface, resulting in a dramatic decrease in the response current of Fc. The developed biosensors show a linear relationship between current value and logarithmic concentration of Pb2+ ranging from 1 pM to 40 nM with a detection limit of 0.30 pM.

Ultrasensitive electrochemiluminescence biosensor based on dual quenching effects of silver nanoclusters and multiple cycling amplification for detection of ATP

In this work, an electrochemiluminescence (ECL) biosensor based on dual ECL quenching effects of silver nanoclusters (Ag NCs) and multiple cycling amplification was designed to achieve ultrasensitive detection of ATP. The specific recognition of target ATP to aptamer initiated multiple cycling amplification, and a small amount of target was converted into a large number of DNA product chains (S1) by amplification. After S1 opened hairpin DNA 2 (HP2), Ag NCs approached the surface of CdS quantum dots (QDs) modified-electrode by complementary DNA, resulting in a significant decrease of ECL intensity from CdS QDs. The quenching principle is as follows. Firstly, the absorption spectrum of Ag NCs overlaps well with the ECL emission spectrum of CdS QDs, leading to effective ECL resonance energy transfer (ECL-RET); Secondly, Ag NCs could catalyze electrochemical reduction of K2S2O8, leading to consumption of ECL co-reactant and reducing ECL of QDs. The double-ECL quenching achieved ultrasensitive biosensing detection of ATP with a wide range from 1 aM to 1 pM. This present work reported new principle of double-quenching QDs ECL by Ag NCs, and developed a novel ECL biosensor by combining with multiple cycle amplification technique, which has great contribution to the development of QDs ECL and biosensing applications.

Shared hairpin structure electrochemiluminescence biosensor based on Au@Ni-Co metal organic frameworks for simultaneous detection of Pb(II) and S.aureus

The excessive content of lead (Pb(II)) and Staphylococcus aureus (S.aureus) seriously harms the quality of aquatic products. In this paper, a highly sensitive electrochemiluminescence (ECL) biosensor was constructed using the synergistic effect of Au NPs@Nickel-Cobalt-Metal-organic frameworks (Au@Ni-Co-MOFs) and double potential resolution function of urchin-like Au@luminol and Cadmium sulfide quantum dots (CdS QDs) for synchronous detection of Pb(II) and S.aureus in aquatic products. Au@Ni-Co-MOFs as the base material, its cube structure can improve the surface active area and sensitivity of the sensor, providing more catalytic active sites for the two functional probes. Urchin-like Au@luminol binding aptamer DNA2 specifically recognizes Pb(II), CdS QDs binding aptamer DNA3 specifically recognizes S.aureus, which collaboratively catalyzed hydrogen peroxide reduction to produce two electrochemiluminescence signals. The shared hairpin structure DNA1 binds stably to Au@Ni-Co-MOFs via the Au-S bond, and the two functional probes are complementary paired with the DNA1 respectively to ensure the specificity of the aptamer. According to the ECL intensity changes of different potentials signal sources, the synchronous detection of Pb(II) and S.aureus with different concentrations is realized. The sensor realizes the detection of two targets in aquatic products and provides a new strategy for the simultaneous detection of multiple targets.

RNA Interference Effectors Selectively Silence the Pathogenic Variant GNAO1 c.607 G > A In Vitro.

RNA interference (RNAi)-based therapeutics hold the potential for dominant genetic disorders, enabling sequence-specific inhibition of pathogenic gene products. We aimed to direct RNAi for the selective suppression of the heterozygous GNAO1 c.607 G > A variant causing GNAO1 encephalopathy. By screening short interfering RNA (siRNA), we showed that GNAO1 c.607G>A is a druggable target for RNAi. The si1488 candidate achieved at least twofold allelic discrimination and downregulated mutant protein to 35%. We created vectorized RNAi by incorporating the si1488 sequence into the short hairpin RNA (shRNA) in the adeno-associated virus (AAV) vector. The shRNA stem and loop were modified to improve the transcription, processing, and guide strand selection. All tested shRNA constructs demonstrated selectivity toward mutant GNAO1, while tweaking hairpin structure only marginally affected the silencing efficiency. The selectivity of shRNA-mediated silencing was confirmed in the context of AAV vector transduction. To conclude, RNAi effectors ranging from siRNA to AAV-RNAi achieve suppression of the pathogenic GNAO1 c.607G>A and discriminate alleles by the single-nucleotide substitution. For gene therapy development, it is crucial to demonstrate the benefit of these RNAi effectors in patient-specific neurons and animal models of the GNAO1 encephalopathy.

Identification and Validation of the miR156 Family Involved in Drought Responses and Tolerance in Tea Plants (Camellia sinensis (L.) O. Kuntze).

The microRNA156 (miR156) family, one of the first miRNA families discovered in plants, plays various important roles in plant growth and resistance to various abiotic stresses. Previously, miR156s were shown to respond to drought stress, but miR156s in tea plants (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. Herein, we identify 47 mature sequences and 28 precursor sequences in tea plants. Our evolutionary analysis and multiple sequence alignment revealed that csn-miR156s were highly conserved during evolution and that the rates of the csn-miR156 members' evolution were different. The precursor sequences formed typical and stable stem-loop structures. The prediction of cis-acting elements in the CsMIR156s promoter region showed that the CsMIR156s had diverse cis-acting elements; of these, 12 CsMIR156s were found to be drought-responsive elements. The results of reverse transcription quantitative PCR (RT-qPCR) testing showed that csn-miR156 family members respond to drought and demonstrate different expression patterns under the conditions of drought stress. This suggests that csn-miR156 family members may be significantly involved in the response of tea plants to drought stress. Csn-miR156f-2-5p knockdown significantly reduced the Fv/Fm value and chlorophyll content and led to the accumulation of more-reactive oxygen species and proline compared with the control. The results of target gene prediction showed that csn-miR156f-2-5p targeted SQUAMOSA promoter binding protein-like (SPL) genes. Further analyses showed that CsSPL14 was targeted by csn-miR156f-2-5p, as confirmed through RT-qPCR, 5' RLM-RACE, and antisense oligonucleotide validation. Our results demonstrate that csn-miR156f-2-5p and CsSPL14 are involved in drought response and represent a new strategy for increasing drought tolerance via the breeding of tea plants.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

RNA-based liposomes for oral cancer: From biophysical characterization to biological evaluation

Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated.Firstly, the RNA structure and binding interaction with ligands (C8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C8 forms a complex with RNA and adopts an open conformation upon RNA binding.Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C8 loaded in RNA-functionalized liposomes indicate the co-existence of free C8 in solution (inside the liposome) and C8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line.Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes.

A signal-on photoelectrochemical aptasensor based on ferrocene labeled triple helix DNA molecular switch for detection of antibiotic amoxicillin

A sensitive signal-on photoelectrochemical aptasensor for antibiotic determination was constructed based on the energy level matching between ferrocene and CuInS2. P-type CuInS2 microflower was complexed with reduced graphene oxide (CuInS2/rGO) to get photocathode current with good photoelectric conversion efficiency and stability. Then, hairpin DNA (HP) was covalently bonded to the electrode surface. A triple helix DNA (THMS) was used as a molecular switch. After the specific recognition between target and THMS in homogeneous solution, ferrocene labeled probe (Fc-T2) was released. Finally, Fc-T2 was captured by the HP, which leaded the obvious increase of photocurrent for the energy level matching between ferrocene and CuInS2. The increase of the photocurrent signal was proportional to the concentration of target amoxicillin (AMOX), the linear range was 100 fM-100 nM with detection limit of 19.57 fM. Meanwhile, the method has been successfully applied for milk and lake water samples analysis with satisfactory results.