A giant virus genome is densely packaged by stable nucleosomes

The doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap DNA to form remarkably similar nucleosomes. By releasing Marseillevirus chromatin from virions into solution and performing genome-wide nuclease digestion and chemical cleavage assays, we find that the higher-order organization of Marseillevirus chromatin differs greatly from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions, without linker DNA or phasing along genes. Likewise, we observe that most nucleosomes reconstituted onto 3-copy tandem repeats of a nucleosome positioning sequence are tightly packed and fully wrapped. We also document repeat generation and instability during viral passage in amoeboid culture. Dense promiscuous packing of fully wrapped nucleosomes rather than 'beads-on-a-string' with genic punctuation suggests a viral genome protection function for doublet histones.

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.

Altered cfDNA Fragmentation Profile in Hypomethylated Regions as Diagnostic Marker in Breast Cancer

Backgroud: Breast cancer, the most common malignancy in women, has been proved to have both altered plasma cell-free DNA (cfDNA) methylation and fragmentation profiles, nevertheless, simultaneously detecting both of them for breast cancer diagnosis has never been reported. Moreover, although fragmentation pattern of cfDNA is determined by nuclease digestion of chromatin, structure of which may be affected by DNA methylation, whether cfDNA methylation and fragmentation are biologically related or not still remains unclear.Methods: Improved cfMeDIP-seq were utilized to characterize both cfDNA methylation and fragmentation profiles in 25 plasma samples from both healthy individuals and patients with breast cancer. The feasibility of using cfDNA fragmentation profile in hypo- and hyper- methylated regions as diagnostic markers for breast cancer was evaluated. Results: Mean size of cfDNA fragments ranging from 100 to 220 base pairs (bp) was found to increase from 170.06 (Input libraries) to 173.04 (IP libraries) bp in healthy individuals, which was not observed in patients with breast cancer (170.51 to 170.71 bp). Furthermore, mean size of cfDNA fragments mapped to hypomethylated regions decreased more win patients with breast cancer (4.60 bp, 172.33 bp in hypermethylated regions to 167.73 bp in hypomethylated regions) than healthy individuals (2.87 bp, 174.54 bp in hypermethylated regions to 171.67 bp in hypomethylated regions). The feasibility of using abnormality of short cfDNA fragments ratio in hypomethylated genomic regions for diagnosis of breast cancer in validation cohort was evaluated. 7 out of 11 patients were detected as having breast cancer (63.6% sensitivity), whereas no healthy individuals were mis-detected (100% specificity). Conclusion: We identified enriched short cfDNA fragments after 5mC-immunoprecipitation (IP) in patients with breast cancer, and demonstrated the enriched short cfDNA fragments might originated from hypomethylated genomic regions. Furthermore, we proved the feasibility of using differentially methylated regions (DMRs)-dependent cfDNA fragmentation profile for breast cancer diagnosis.

Differentially methylated regions (DMRs)-dependent cfDNA fragmentation profile for diagnosis of breast cancer

Background Breast cancer, the most common malignancy in women, has been proved to have both altered plasma cell-free DNA (cfDNA) methylation and fragmentation profile, nevertheless, simultaneously detecting both of them for breast cancer diagnosis has never been reported. Moreover, although it is known that fragmentation pattern of cfDNA is determined by nuclease digestion of chromatin, structure of which may be affected by DNA methylation, whether cfDNA methylation and fragmentation are biologically related or not still remains unclear.Methods A total of 25 plasm samples from both healthy individuals and patients with breast cancer were divided into discovery cohort and validation cohort. Improved cfMeDIP-seq were utilized to simultaneously profile both cfDNA methylation and fragments size, short fragments ratio were investigated in differentially methylated regions (DMRs). The feasibility of using cfDNA fragmentation profile in hypo- and hyper- methylated regions as a diagnostic marker for breast cancer was evaluated.Results Mean cfDNA fragments size ranging from 100 to 220 base pairs (bp) was found to increase from 170.06 (Input libraries) to 173.04 (IP libraries) bp in healthy individuals, which was not observed in patients with breast cancer (170.51 to 170.71 bp). Furthermore, mean size of cfDNA fragments mapped to hypomethylated regions decreased more in patients with breast cancer (4.60 bp, 172.33 bp in hypermethylated regions to 167.73 bp in hypomethylated regions) than healthy individuals (2.87 bp, 174.54 bp in hypermethylated regions to 171.67 bp in hypomethylated regions). An approach called ‘correlation assessment of DMRs-dependent cfDNA fragmentation profile’ was developed to evaluate the feasibility of using abnormality of short cfDNA fragments ratio in hypomethylated genomic windows for diagnosis of breast cancer in validation cohort. 7 out of 11 patients were detected as having breast cancer (63.6% sensitivity), whereas no healthy individuals were mis-detected (100% specificity).Conclusion We identified enriched short cfDNA fragments after 5mC-immunoprecipitation (IP) in patients with breast cancer, and demonstrated the enriched short cfDNA fragments might originated from hypomethylated genomic regions. Furthermore, we proved the feasibility of using differentially methylated regions (DMRs)-dependent cfDNA fragmentation profile for breast cancer diagnosis.

DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies

Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.

Hepatocellular-Targeted mRNA Delivery Using Functionalized Selenium Nanoparticles In Vitro

Selenium’s (Se) chemopreventative and therapeutic properties have attracted attention in nanomedicine. Se nanoparticles (SeNPs) retain these properties of Se while possessing lower toxicity and higher bioavailability, potentiating their use in gene delivery. This study aimed to formulate SeNPs for efficient binding and targeted delivery of FLuc-mRNA to hepatocellular carcinoma cells (HepG2) in vitro. The colorectal adenocarcinoma (Caco-2) and normal human embryonic kidney (HEK293) cells that do not have the asialoorosomucoid receptor (ASGPR) were utilized for comparison. SeNPs were functionalized with chitosan (CS), polyethylene glycol (PEG), and lactobionic acid (LA) for ASGPR targeting on HepG2 cells. Nanoparticles (NPs) and their mRNA-nanocomplexes were characterized by Fourier transform infra-red (FTIR) and UV-vis spectroscopy, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). Gel and fluorescence-based assays assessed the NP’s ability to bind and protect FLuc-mRNA. Cytotoxicity was determined using the -(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, while transgene expression was evaluated using the luciferase reporter gene assay. All NPs appeared spherical with sizes ranging 57.2–130.0 nm and zeta potentials 14.9–31.4 mV. NPs bound, compacted, and protected the mRNA from nuclease digestion and showed negligible cytotoxicity in vitro. Targeted gene expression was highest in the HepG2 cells using the LA targeted NPs. These NPs portend to be efficient nanocarriers of nucleic acids and warrant further investigation.