Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Messenger RNA enrichment using synthetic oligo(T) click nucleic acids.

ABSTRACT: Enrichment of mRNA is a key step in a number of molecular biology techniques, particularly in the rapidly growing field of transcriptomics. Currently, mRNA is isolated using oligo(thymine) DNA (oligo(dT)) immobilized on solid supports, which binds to the poly(A) tail of mRNA to pull the mRNA out of solution through the use of magnets or centrifugal filters. Here, a simple method to isolate mRNA by complexing it with synthetic click nucleic acids (CNAs) is described. Oligo(T) CNA bound efficiently to mRNA, and because of the insolubility of CNA in water, >90% of mRNA was readily removed from solution using this method. Simple washing, buffer exchange, and heating steps enabled mRNA's enrichment from total RNA, with a yield of 3.1 ± 1.5% of the input total RNA by mass, comparable to the yield from commercially available mRNA enrichment beads. Further, the integrity and activity of mRNA after CNA-facilitated pulldown and release was evaluated through two assays. In vitro translation of EGFP mRNA confirmed the translatability of mRNA into functional protein and RT-qPCR was used to amplify enriched mRNA from total RNA extracts and compare gene expression to results obtained using commercially available products.

SUBMITTER: Anderson AJ

PROVIDER: S-EPMC7891491 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Autoantibody Profiling in Lupus Patients using Synthetic Nucleic Acids.

Project description:Autoantibodies to nuclear components of cells (antinuclear antibodies, ANA), including DNA (a-DNA), are widely used in the diagnosis and subtyping of certain autoimmune diseases, including systemic lupus erythematosus (SLE). Despite clinical use over decades, precise, reproducible measurement of a-DNA titers remains difficult, likely due to the substantial sequence and length heterogeneity of DNA purified from natural sources. We designed and tested a panel of synthetic nucleic acid molecules composed of native deoxyribonucleotide units to measure a-DNA. ELISA assays using these antigens show specificity and reproducibility. Applying the ELISA tests to serological studies of pediatric and adult SLE, we identified novel clinical correlations. We also observed preferential recognition of a specific synthetic antigen by antibodies in SLE sera. We determined the probable basis for this finding using computational analyses, providing valuable structural information for future development of DNA antigens. Synthetic nucleic acid molecules offer the opportunity to standardize assays and to dissect antibody-antigen interactions.

| S-EPMC5883037 | biostudies-literature

Quantification of plasma HIV RNA using chemically engineered peptide nucleic acids.

Project description:The remarkable stability of peptide nucleic acids (PNAs) towards enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device. Here we report the development of chemically engineered PNAs for the quantitative detection of HIV RNA at clinically relevant levels that are competitive with current PCR-based assays. Using a sandwich hybridization approach, chemical groups were systematically introduced into a surface PNA probe and a reporter PNA probe to achieve quantitative detection for HIV RNA as low as 20 copies per millilitre of plasma. For the surface PNA probe, four cyclopentane groups were incorporated to promote stronger binding to the target HIV RNA compared with PNA without the cyclopentanes. For the reporter PNA probe, 25 biotin groups were attached to promote strong signal amplification after binding to the target HIV RNA. These general approaches to engineer PNA probes may be used to detect other RNA target sequences.

| S-EPMC4187110 | biostudies-literature

Rapid differentiation of hiPSCs into functional oligodendrocytes using an OLIG2 synthetic modified messenger RNA.

Project description:Transcription factors (TFs) have been introduced to drive the highly efficient differentiation of human-induced pluripotent stem cells (hiPSCs) into lineage-specific oligodendrocytes (OLs). However, effective strategies currently rely mainly on genome-integrating viruses. Here we show that a synthetic modified messenger RNA (smRNA)-based reprogramming method that leads to the generation of transgene-free OLs has been developed. An smRNA encoding a modified form of OLIG2, in which the serine 147 phosphorylation site is replaced with alanine, OLIG2S147A, is designed to reprogram hiPSCs into OLs. We demonstrate that repeated administration of the smRNA encoding OLIG2 S147A lead to higher and more stable protein expression. Using the single-mutant OLIG2 smRNA morphogen, we establish a 6-day smRNA transfection protocol, and glial induction lead to rapid NG2+ OL progenitor cell (OPC) generation (>70% purity) from hiPSC. The smRNA-induced NG2+ OPCs can mature into functional OLs in vitro and promote remyelination in vivo. Taken together, we present a safe and efficient smRNA-driven strategy for hiPSC differentiation into OLs, which may be utilized for therapeutic OPC/OL transplantation in patients with neurodegenerative disease.

| S-EPMC9568531 | biostudies-literature

Monkeypox viral nucleic acids detected using both DNA and RNA extraction workflows.

Project description:Molecular methods have been used to detect human pathogens in wastewater with sampling typically performed at wastewater treatment plants (WWTP) and upstream locations within the sewer system. A wastewater-based surveillance (WBS) program was established at the University of Miami (UM) in 2020, which included measurements of SARS-CoV-2 levels in wastewater from its hospital and within the regional WWTP. In addition to the development of a SARS-CoV-2 quantitative PCR (qPCR) assay, qPCR assays to detect other human pathogens of interest were also developed at UM. Here we report on the use of a modified set of reagents published by the CDC to detect nucleic acids of Monkeypox virus (MPXV) which emerged during May of 2022 to become a concern worldwide. Samples collected from the University hospital and from the regional WWTP were processed through DNA and RNA workflows and analyzed by qPCR to detect a segment of the MPXV CrmB gene. Results show positive detections of MPXV nucleic acids in the hospital and wastewater treatment plant wastewater which coincided with clinical cases in the community and mirrored the overall trend of nationwide MPXV cases reported to the CDC. We recommend the expansion of current WBS programs' methods to detect a broader range of pathogens of concern in wastewater and present evidence that viral RNA in human cells infected by a DNA virus can be detected in wastewater.

| S-EPMC10213602 | biostudies-literature

Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions.

Project description:Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of host-pathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens.

| S-EPMC6889467 | biostudies-literature

Inhibition of pre-mRNA splicing by synthetic branched nucleic acids.

Project description:The cellular transformation of a precursor mRNA (pre-mRNA) into its mature or functional form proceeds by way of a splicing reaction, in which the exons are ligated to form the mature linear RNA and the introns are excised as branched or lariat RNAs. We have prepared a series of branched compounds (bRNA and bDNA), and studied the effects of such molecules on the efficiency of mammalian pre-mRNA splicing in vitro. Y-shaped RNAs containing an unnatural L-2'-deoxycytidine unit (L-dC) at the 3' termini are highly stabilized against exonuclease hydrolysis in HeLa nuclear extracts, and are potent inhibitors of the splicing pathway. A bRNA containing internal 2'-O-methyl ribopyrimidine units and L-dC at the 3' ends was at least twice as potent as the most potent of the bRNAs containing no 2' modifications, with an IC50 of approximately 5 micro M. Inhibitory activity was maintained in a branched molecule containing an arabino-adenosine branchpoint which, unlike the native bRNAs, resisted cleavage by the lariat- debranching enzyme. The data obtained suggest that binding and sequestering of a branch recognition factor by the branched nucleic acids is an early event, which occurs prior to the first chemical step of splicing. Probably, an early recognition element preferentially binds to the synthetic branched molecules over the native pre-mRNA. As such, synthetic bRNAs may prove to be invaluable tools for the purification and identification of the putative branchpoint recognition factor.

| S-EPMC275466 | biostudies-literature

Rapid differentiation of hiPSCs into functional oligodendrocytes using an OLIG2 synthetic modified messenger RNARapid differentiation of hiPSCs into functional oligodendrocytes using an OLIG2 synthetic modified messenger RNA

Project description:We show that a synthetic modified messenger RNA (smRNA)-based reprogramming method that leads to the generation of transgene-free OLs has been developed. An smRNA encoding a modified form of OLIG2, a key TF in OL development, in which the serine 147 phosphorylation site is replaced with alanine, OLIG2S147A, is designed to reprogram hiPSCs into OLs. We demonstrate that repeated administration of the smRNA encoding OLIG2 S147A lead to higher and more stable protein expression. Using the single-mutant OLIG2 smRNA morphogen, we establish a 6-day smRNA transfection protocol, and glial induction lead to rapid NG2+ OL progenitor cell (OPC) generation (> 70% purity) from hiPSC-derived neural progenitor cells (NPCs). The smRNA-induced NG2+ OPCs can mature into functional OLs in vitro and promote remyelination in vivo. Proteomic analysis of OLIG2-binding proteins indicates that OLIG2 is bound by the heat shock protein 70 (HSP70) complex. The HSP70 complex is bound more strongly to OLIG2 with the modified phosphorylation site than to wild-type OLIG2.

2023-03-10 | PXD036975 | Pride

Murine protein which binds preferentially to oligo-C-rich single-stranded nucleic acids.

Project description:Two single-stranded nucleic acid binding proteins mCBP and mCTBP were identified by means of their binding to a potential recombination hotspot in LTRs of mouse retro-transposons. Both are nuclear proteins of 35 and 55 kDa respectively. mCBP binds preferentially to oligo dC, mCTBP to oligo dCdT. mCBP was purified and its cDNA was isolated and sequenced.

| S-EPMC308089 | biostudies-other

Publisher Correction: Rapid differentiation of hiPSCs into functional oligodendrocytes using an OLIG2 synthetic modified messenger RNA.

Project description: Not available

| S-EPMC9633832 | biostudies-literature

Delivery of nucleic acids using nanomaterials.

Project description:The increasing number of approved nucleic acid therapeutics demonstrates the potential for the prevention and treatment of a broad spectrum of diseases. This trend underscores the significant impact and promise of nucleic acid-based treatments in the field of medicine. Nevertheless, employing nucleic acids as therapeutics is challenging due to their susceptibility to degradation by nucleases and their unfavorable physicochemical characteristics that hinder delivery into cells. Appropriate vectors play a pivotal role in improving nucleic acid stability and delivering nucleic acids into specific cells. The maturation of delivery systems has led to breakthroughs in the development of therapeutics based on nucleic acids such as DNA, siRNA, and mRNA. Non-viral vectors have gained prominence among the myriad of nanomaterials due to low immunogenicity, ease of manufacturing, and simplicity of cost-effective, large-scale production. Here, we provide an overview of the recent advancements in nanomaterials for nucleic acid delivery. Specifically, we give a detailed introduction to the characteristics of polymers, lipids, and polymer-lipid hybrids, and provide comprehensive descriptions of their applications in nucleic acid delivery. Also, biological barriers, administration routes, and strategies for organ-selective delivery of nucleic acids are discussed. In summary, this review offers insights into the rational design of next-generation delivery vectors for nucleic acid delivery.

| S-EPMC10719232 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data