RNA Target Site Research Articles

Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines.

Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.

Selective enrichment of RNA species for tight binding to Escherichia coli rho factor.

We have applied the SELEX procedure (systematic evolution of ligands by exponential enrichment) to obtain RNA molecules that bind tightly to the Escherichia coli transcription termination factor rho. The starting pool was a population of RNA molecules 77 nucleotides (nt) long, in which was embedded a cassette of 30 nt of randomized sequence. The apparent dissociation constant of this RNA pool for hexameric rho factor was about 1 microM. After eight rounds of selection by filter binding, with RNA in either 10-fold or 40 to 100-fold excess at each step, the dissociation constant of the selected RNA had dropped by more than 500-fold to about 1 nM. Analysis of 29 clonal isolates from the population revealed that five had KDs substantially weaker than 10 nM (presumably background carryover), 40% were C-rich (as might have been predicted from rho's known substrate binding), and 40% had a strikingly preserved potential hairpin, in most cases of 6 base pairs with a 3 nt CAA loop and preceded by a CCCCA consensus. The rho-dependent trp t' terminator region includes a related potential hairpin structure; however, it is energetically unfavorable. The implications of the sequence findings for elucidating both static and dynamic aspects of rho factor recognition and response to its RNA target site are discussed.

Direct genetic selection for a specific RNA-protein interaction.

The decision between lytic and lysogenic development of temperate DNA bacteriophages is determined largely by transcriptional regulation through DNA-binding proteins. To determine whether a heterologous RNA-binding activity could control the developmental fate of a DNA bacteriophage, a derivative of P22 was constructed in which the chosen developmental pathway is regulated by an RNA-binding molecule interacting with its RNA target site located in a phage mRNA. In the example presented, lysogenic development of the phage relies upon R17 coat protein expression in the susceptible host cell and the availability of a suitable coat protein binding site encoded by the phage genome. Through the analysis of phage mutants that are able to grow lytically in susceptible cells that express the coat protein, additional insights were obtained regarding the specific interaction of the R17 coat protein with its RNA binding site. This study also suggests a novel and extremely sensitive strategy for selecting RNA-binding activities in vivo.