Abstract:
:LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T-A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50-100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Jacobsen N,Nielsen PS,Jeffares DC,Eriksen J,Ohlsson H,Arctander P,Kauppinen Sdoi
10.1093/nar/gnh056keywords:
subject
Has Abstractpub_date
2004-04-19 00:00:00pages
e64issue
7eissn
0305-1048issn
1362-4962pii
32/7/e64journal_volume
32pub_type
杂志文章abstract::The hammerhead self-cleaving motif occurs in a variety of RNAs that infect plants and consists of three non-conserved helices connected by a highly conserved central core. A variant hammerhead, called the extended hammerhead, is found in satellite 2 transcripts from a variety of caudate amphibians. The extended hammer...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/25.11.2189
更新日期:1997-06-01 00:00:00
abstract::During protein synthesis, the nascent peptide chain traverses the peptide exit tunnel of the ribosome. We monitor the co-translational movement of the nascent peptide using a fluorescent probe attached to the N-terminus of the nascent chain. Due to fluorophore quenching, the time-dependent fluorescence signal emitted ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky740
更新日期:2018-12-14 00:00:00
abstract::Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditio...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.19.3805
更新日期:1995-10-11 00:00:00
abstract::As protein-protein interactions are crucial in most biological processes, it is valuable to understand how and where protein pairs interact. We developed a web server HOMCOS (Homology Modeling of Complex Structure, http://biunit.naist.jp/homcos) to predict interacting protein pairs and interacting sites by homology mo...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn218
更新日期:2008-07-01 00:00:00
abstract::Protein structures often show similarities to another which would not be seen at the sequence level. Given the coordinates of a protein chain, the SALAMI server at www.zbh.uni-hamburg.de/salami will search the protein data bank and return a set of similar structures without using sequence information. The results page...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkp431
更新日期:2009-07-01 00:00:00
abstract::The infection of Pseudomonas aeruginosa by the giant bacteriophage phiKZ is resistant to host RNA polymerase (RNAP) inhibitor rifampicin. phiKZ encodes two sets of polypeptides that are distantly related to fragments of the two largest subunits of cellular multisubunit RNAPs. Polypeptides of one set are encoded by mid...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv1095
更新日期:2015-12-02 00:00:00
abstract::Expression of the wild-type p53 tumor suppressor gene has been found to play an important role in the regulation of cellular proliferation and differentiation. In addition, in many transformed cells and primary tumors, the gene has undergone allelic deletions and mutant forms of the p53 gene are expressed at elevated ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.2.345
更新日期:1993-01-25 00:00:00
abstract::Ribonucleic acids are highly conserved essential parts of cellular life. RNA function is determined to a large extent by its hydrodynamic behaviour. The presented study proposes a strategy to predict the hydrodynamic behaviour of RNA single strands on the basis of the polymer size. By atom-level shell-modelling of hig...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq808
更新日期:2011-02-01 00:00:00
abstract::E2F transcription factors (E2F1-8) are known to coordinately regulate the expression of a plethora of target genes, including those coding for microRNAs (miRNAs), to control cell cycle progression. Recent work has described the atypical E2F factor E2F7 as a transcriptional repressor of cell cycle-related protein-codin...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw146
更新日期:2016-07-08 00:00:00
abstract::Co-option of cis-regulatory modules has been suggested as a mechanism for the evolution of expression sites during development. However, the extent and mechanisms involved in mobilization of cis-regulatory modules remains elusive. To trace the history of non-coding elements, which may represent candidate ancestral cis...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt030
更新日期:2013-04-01 00:00:00
abstract::The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated fr...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/28.8.1802
更新日期:2000-04-15 00:00:00
abstract::The positions of the trfA and trfB promoters of broad host range IncP plasmid RK2 (identical to RP1, RP4, R68 and R18 ) were identified by RNA polymerase protection studies, and the nucleotide sequences of the promoter regions determined. A mutation within the trfA promoter sequence is associated with loss of kilD act...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/12.8.3619
更新日期:1984-04-25 00:00:00
abstract::HOX11 is a homeobox-containing oncogene of specific T-cell leukemias. We determined the DNA binding specificity of the Hox11 protein by using a novel technique of random oligonucleotide selection developed in this study. The optimal Hox11 binding sequence, GGCGGTAAGTGG, contained a core TAAGTG motif that is consistent...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.11.1928
更新日期:1995-06-11 00:00:00
abstract::We have previously mapped a repression domain from the active transcriptional repressor E4BP4 to a 65 amino acid segment near the C-terminus of the polypeptide. Here we show that the E4BP4 repression domain interacts specifically with the TBP binding repressor protein Dr1. Mutants that affect the ability of E4BP4 to b...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/24.18.3607
更新日期:1996-09-15 00:00:00
abstract::Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG). To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recomb...
journal_title:Nucleic acids research
pub_type: 评论,杂志文章
doi:10.1093/nar/gkg746
更新日期:2003-09-15 00:00:00
abstract::Characterization of the ability of human interferons (IFNs) to rapidly induce genes led to the identification of the first two members of the STAT (signal transducers and activators of transcription) family, Stat1 and Stat2. To study the unique role of this transcription factor in IFN signaling under more physiologica...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/27.21.4191
更新日期:1999-11-01 00:00:00
abstract::Microarrays are an effective tool for monitoring genome-wide gene expression levels. In current microarray analyses, the majority of genes on arrays are frequently eliminated for further analysis because the changes in their expression levels (ratios) are considered to be not significant. This strategy risks failure t...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl058
更新日期:2006-03-14 00:00:00
abstract::The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw105
更新日期:2016-06-02 00:00:00
abstract::The binding of transposase (Tnp) to the specific Tn5 end sequences is the first dedicated reaction during transposition. In this study, comparative DNA-binding analyses were performed using purified full-length Tnp and a C-terminal deletion variant (delta369) that lacks the putative dimerization domain. The shape of t...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/24.19.3790
更新日期:1996-10-01 00:00:00
abstract::Genomic DNA of calf thymus contains 1.5 times as much 5-methylcytosine as similar sperm DNA, but the major EcoRI repeat fragment from satellite I of thymus contains ten times as much 5-methylcytosine as the corresponding fragment from sperm DNA. Restriction enzyme analyses of the total DNA and the satellite I fragment...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/9.18.4537
更新日期:1981-09-25 00:00:00
abstract::The transcription of ribosomal genes in a cell line (Kc) derived from female D.melanogaster, has been investigated using hybridization probes prepared from restriction fragments of a cloned rDNA repeat with a 5 kb type I [Wellauer et al. (1978) Cell 14, 269-278] intron. Gels, of nuclear RNA that have been transferred ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/8.1.67
更新日期:1980-01-11 00:00:00
abstract::The MalaCards human disease database (http://www.malacards.org/) is an integrated compendium of annotated diseases mined from 68 data sources. MalaCards has a web card for each of ∼20 000 disease entries, in six global categories. It portrays a broad array of annotation topics in 15 sections, including Summaries, Symp...
journal_title:Nucleic acids research
pub_type: 杂志文章,评审
doi:10.1093/nar/gkw1012
更新日期:2017-01-04 00:00:00
abstract::Chromosomes in vertebrates are protected at both ends by telomere DNA composed of tandem (TTAGGG)n repeats. DNA replication produces a blunt-ended leading strand telomere and a lagging strand telomere carrying a single-stranded G-rich overhang at its end. The G-rich strand can form G-quadruplex structure in the presen...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkm203
更新日期:2007-01-01 00:00:00
abstract::Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic tra...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq030
更新日期:2010-06-01 00:00:00
abstract::An improved algorithm was elaborated to analyse tRNA interaction with aminoacyl-tRNA synthetase based on analysis of tRNA sequences. The fundamental element defining the interaction between the tRNA and the synthetase is not a single nucleotide but a nucleotide combination named a tile which comprises of a given nucle...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.8.1941
更新日期:1993-04-25 00:00:00
abstract::The endonuclease activity within the influenza virus cap-snatching process is a proven therapeutic target. The anti-influenza drug baloxavir is highly effective, but is associated with resistance mutations that threaten its clinical efficacy. The endonuclease resides within the N-terminal domain of the PA subunit (PAN...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa1294
更新日期:2021-01-19 00:00:00
abstract::The CD spectra of twelve DNA restriction fragments ranging in size from 12 to 360 base pairs are reported. Since the sequences of these fragments are known, it is possible to calculate their CD spectra from a set of nearest neighbor contributions derived from a combination of synthetic polydeoxyribonucleotides. While ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/9.13.3029
更新日期:1981-07-10 00:00:00
abstract::Direct labeling of RNA is an expedient method for labeling large quantities (e.g. micrograms) of target RNA for microarray analysis. We have developed an efficient labeling system that uses T4 RNA ligase to attach a 3'-biotinylated donor molecule to target RNA. Microarray analyses indicate that directly labeled RNA is...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gnh085
更新日期:2004-06-17 00:00:00
abstract::Accumulating evidence has demonstrated that transcriptional regulation is affected by DNA methylation. Understanding the perturbation of DNA methylation-mediated regulation between transcriptional factors (TFs) and targets is crucial for human diseases. However, the global landscape of DNA methylation-mediated transcr...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa041
更新日期:2020-03-18 00:00:00
abstract::A detailed scheme of the Peptidyl Transferase Centre of bacterial ribosomes is proposed by summarizing the literature data on the substrate specificity of the acceptor and donor sites. According to the proposed scheme only the elements of the donor and acceptor having a stable structure bind with the ribosome. The pre...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/2.12.2223
更新日期:1975-12-01 00:00:00