Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While ...nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations.
We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3'-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments.
We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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•AuNP-DNA polymer was pre-prepared as sensitivity enhancement probe (SEP) to improve sensitivity of strip for the first time.•Compared with the strip biosensor without SEP, the ...sensitivity of strip with SEP was increased by 40 times.•The strip biosensor enables visual detection of Pb2+ within 20 min with 2.5 nM LOD.
PS2M aptamer was selected as sensing element to construct Pb2+ strip biosensor, and for the first time, AuNP-DNA polymer was used as sensitivity enhancement probe (SEP) to improve sensitivity. Pb2+ separated the double-strand formed by PS2M and its complementary sequence at Anti-PS2M and induced PS2M to form G-quadruplex structure, then single-strand Anti-PS2M was released. When migrated along the strip, sequence at one end of Anti-PS2M hybridized with AuNP-DNA probe G immobilized on conjugate pad, when the composite migrated to test zone (TZ), other end of Anti-PS2M hybridized with biotin-DNA probe T on TZ to form sandwich structure. Subsequent SEP hybridized with the remaining oligonucleotides of AuNP-DNA probe G on TZ thus enhanced the signal. After optimization, the false positive result could be eliminated and the best signal value could be obtained. The results showed that the limit of detection of Pb2+ strip biosensor was 2.5 nM by adding the sensitivity enhancement probe, which was 40 times better than that strip without enhancement. The test strip showed good results in the direct detection of soil and lake samples, presenting good practical value. In addition, the cost of this strip biosensor was low, with good market prospect.
Purpose: The aim of the study is to understand the involvement of G-Quadruplex (G-Q) structures in altering the expression profile of WNT/epidermal growth factor receptor (EGFR) pathway receptor ...genes in chemo-tolerant Triple Negative Breast Cancer (TNBC) samples. Materials and Methods: At first, Gene Expression Omnibus datasets were mined where the expression profile of WNT/EGFR pathway genes in TNBC samples and MDA-MB-231, a TNBC cell line, were checked in response to doxorubicin, a chemotherapeutic drug. Next, to unveil the probable mechanism of regulation, the presence of G-Q structure was checked in in silico study and later validated by immunohistochemical analyses in our pool of sample. These observed results were correlated with patient's demography and survival status. Results: Expression of the receptors (FZD7, LRP6, EGFR) of the WNT/EGFR pathway were found to be differentially expressed in TNBC samples; further emphasized in our samples (n = 61). Notably, these G-Q structures were found in the promoter region of the WNT pathway receptor genes (FZD7, LRP6, and EGFR). Validating in our patient sample pool, a significant increase in G-Q immunostaining was observed in samples, after neoadjuvant chemotherapy (NACT) samples (n = 17) than the pretherapeutic samples (n = 44). Similar pattern of G-Q immunostaining was noticed in doxorubicin-treated MDA-MB-231 cell line. Intriguingly, low staining of G-Q among the pretherapeutic samples, but NACT TNBC samples, was found to be significantly correlated with lymph node metastasis. Conclusions: This study showed that the augmented immunostaining of G-Q structure might have an important involvement in regulating the expression pattern of the WNT/EGFR pathway genes in response to doxorubicin treatment of TNBC.
Thioflavin T (ThT), a typical probe for protein fibrils, also binds human telomeric G-quadruplexes with a fluorescent light-up signal change and high specificity against DNA duplexes. Cell ...penetration and low cytotoxicity of fibril probes having been widely established, modifying ThT and other fibril probes is an attractive means of generating new G-quadruplex ligands. Thus, elucidating the binding mechanism is important for the design of new drugs and fluorescent probes based on ThT. Here, we investigated the binding mechanism of ThT with several variants of the human telomeric sequence in the presence of monovalent cations. Fluorescence titrations and electrospray ionization mass spectrometry (ESI-MS) analyses demonstrated that each G-quadruplex unit cooperatively binds to several ThT molecules. ThT brightly fluoresces when a single ligand is bound to the G-quadruplex and is quenched as ligand binding stoichiometry increases. Both the light-up signal and the dissociation constants are exquisitely sensitive to the base sequence and to the G-quadruplex structure. These results are crucial for the sensible design and interpretation of G-quadruplex detection assays using fluorescent ligands in general and ThT in particular.
SNAIL1 is a key regulator of epithelial-mesenchymal transition (EMT) and its expression is associated with tumor progression and poor clinical prognosis of cancer patients. Compared to the studies of ...SNAIL1 stability and its transcriptional regulation, very limited knowledge is available regarding effective approaches to directly target SNAIL1. In this study, we revealed the potential regulation of SNAIL1 gene expression by G-quadruplex structures in its promoter. We first revealed that the negative strand of the SNAIL1 promoter contained a multi-G-tract region with high potential of forming G-quadruplex structures. In circular dichroism studies, the oligonucleotide based on this region showed characteristic molar ellipticity at specific wavelengths of G-quadruplex structures. We also utilized native polyacrylamide gel electrophoresis, gel-shift assays, immunofluorescent staining, dimethyl sulfate footprinting and chromatin immunoprecipitation studies to verify the G-quadruplex structures formed by the oligonucleotide. In reporter assays, disruption of G-quadruplex potential increased SNAIL1 promoter-mediated transcription, suggesting that G-quadruplexes played a negative role in SNAIL1 expression. In a DNA synthesis study, we detected G-quadruplex-mediated retardation in the SNAIL1 promoter replication. Consistently, we discovered that the G-quadruplex region of the SNAIL1 promoter is highly enriched for mutations, implicating the clinical relevance of G-quadruplexes to the altered SNAIL1 expression in cancer cells.
•G-quadruplex structures can be formed in the SNAIL1 promoter.•G-quadruplex negatively regulates SNAIL1 promoter-mediated transcription.•G-quadruplex formation retards DNA synthesis in the SNAIL1 promoter and potentially causes replication errors.
RNA G-quadruplex (rG4) structure and its association with rG4-binding proteins/peptides are important for its function. However, there is very limited study that investigates what factors are ...involved in rG4 that drive the rG4-protein/peptide interaction. Here we study and uncover the effect of RNA sequence context and stereochemistry on G-quadruplex-peptide interaction. Using rG4-binding RHAU53 peptide as an example, we report that the number of G-quartet, thermostability, overhanging nucleotides, and RNA base chirality have an impact on rG4-RHAU53 binding. Notably, our data also demonstrate that RHAU53 preferentially binds to 5′ G-quartet over 3’ G-quartet, and showcase that RHAU53 interacts with unnatural L-rG4 for the first time. Our findings reported here offer unique insights to the potential development of targeting tools that recognize rG4 structure and rG4-binding peptide/protein.
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•Effect of RNA sequence and stereochemistry on G-quadruplex-RHAU53 binding uncovered.•RHAU53 preferentially binds to 5′ G-quartet over 3′ G-quartet.•RHAU53 tends to interact with D-rG4 than L-rG4.•Biochemical and Biophysical Research Communications.
Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of ...the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.
Identification of a new G-quadruplex ligand having anti-telomerase activity would be a promising strategy for cancer therapy. The screened compound from ZINC database using docking studies was ...experimentally verified for its binding with three different telomeric G-quadruplex DNA sequences and anti-telomerase activity in A549 cells. Identified compound is an intrinsic fluorescent molecule, permeable to live cells and has a higher affinity to 22AG out of three different telomeric G-quadruplex DNA. It showed cytotoxicity and a significant reduction of telomerase activity in human A549 cells at a very low dose. So, this compound has a good anti-cancer effect.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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