PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9222504

Interacting RNA species identified by combinatorial selection.

B Cho·D C Taylor·H B Nicholas·F J Schmidt
View original source ↗Export metadata ↓

Abstract

RNA molecules were selected from a random sequence library for their ability to bind to an RNA stem-loop target. Oligonucleotides with extensive Watson-Crick complementarity to the RNA ligand were selected against by inclusion of a blocking oligodeoxynucleotide in the binding phase of the selection protocol. After 18 generations of SELEX (systematic evolution of ligands by exponential enrichment) a single RNA family was predominant in the binding population. The winning aptamer RNA bound the target RNA with an apparent Kd = 70 nM. Structural mapping and Fe(II)-EDTA protection indicated that the target RNA interacted with small unpaired loops in the aptamer structure.

Explore related subjects

Base CompositionBase SequenceBinding SitesDNA, ComplementaryEndoribonucleasesLigandsMolecular Sequence DataNucleic Acid ConformationRNARNA, CatalyticRibonuclease PStructure-Activity RelationshipJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

B Cho, D C Taylor, H B Nicholas, F J Schmidt. 1997. Interacting RNA species identified by combinatorial selection.. https://doi.org/10.1016/s0968-0896(97)00046-1

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Sp1 is essential for p16 expression in human diploid fibroblasts during senescence.

BACKGROUND: p16(INK4a) tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16(INK4a) gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from -466 to -451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts. METHODOLOGY/PRINCIPAL FINDINGS: Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16(INK4a) gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16(INK4a). Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16(INK4a) gene expression. In addition, the enhanced binding of Sp1 to p16(INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level. CONCLUSIONS/SIGNIFICANCE: All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16(INK4a) expression during cell aging.

Base Composition↗
PubMed2007

Application of CE for determination of DNA base composition.

DNA base composition expressed as mol% of guanine plus cytosine (% GC) or GC content is a key parameter of bacterial taxonomy and genomic analyses. Direct chemical determination methods such as HPLC as well as indirect methods based on physical properties of deoxyribonucleic acid (DNA), melting point (T(m)), and buoyant density (B(d)) have been conventionally applied to determine the GC content. However, these methods require relatively large amounts of sample DNA, time, and labor. We have developed a protocol to determine the GC content by fine separation of nucleosides with CZE. Genomic DNAs with known GC content from 23 bacterial strains were determined by CE at the optimized conditions of 27 degrees C, 20 kV in 50 mM of NaHCO(3) (pH 9.0) and 70 mM SDS added. Nucleosides from <1 microg of DNA hydrolyzed with nuclease-P1 and bacterial alkaline phosphatase were separated in a 75 microm wide and 80 cm long silica capillary. The nucleoside peak areas were determined at 254 nm in less than 12 min. The CE-based determination of GC content requires only small amounts of DNA, and thus should be applicable to environmental genomics (metagenomics), as >90% of environmental micro-organisms are nonculturable and produce only small amounts of genomic DNA.

Base Composition↗