Louisiana State University and Agricultural & Mechanical College

University of Kentucky Research Foundation

Boehringer Ingelheim Vetmedica Gmbh

University of Warsaw

Uniwersytet Warszawski

Robert E Rhoads

Edward Darzynkiewicz

Joanna Kowalska

Jacek Jemielity

Dieter Blaas

Timothy Skern

Wolfgang Sommergruber

Horst Johann Ahorn

Hans-Dieter Liebig

Debra Waters

Arrico De Benedetti

Barry J Lamphear

Regina Kirchweger

Elisabeth Ziegler

Janusz Stepinski

Ewa M Grudzien-Nogalska

Joanna Zuberek

Maciej Lukaszewicz

Dinucleotide cap analogs are disclosed, modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group. The analogs are useful as reagents in the preparation of capped mRNAs and have increased stability both in vitro and in vivo. They may be used as inhibitors of cap-dependent translation. Optionally, the boranophosphate or phosphoroselenoate group has a 2'-O or 3&#x2032;-O-alkyl group, preferably a methyl group, producing analogs called BH-ARCAs or Se-ARCAs. ARCAs may be modified with &#x3b1;-, &#x3b2;-, or &#x3b3;-boranophosphate or phosphoroselenoate groups.

mRNA cap analogs

New RNA cap analogs are disclosed containing one or more phosphorothioates groups. The analogs also contain modifications at the 2'-O position of 7-methylguanosine that prevent them from being incorporated in the reverse orientation during in vitro synthesis of mRNA and that hence are 'anti-reverse cap analogs' (ARCAs). The ARCA modification ensures that the S atom is precisely positioned within the active sites of cap-binding proteins in both the translational and decapping machinery. The new S-ARCA analogs are resistant to in vivo decapping enzymes. Some S-ARCAs have a higher affinity for eIF4E than the corresponding analogs not containing a phosphorothioate group. When mRNAs containing the various S-ARCAs are introduced into cultured cells, some are translated as much as five-fold more efficiently than mRNAs synthesized with the conventional analog mGpppG.

Synthesis and use of anti-reverse phosphorothioate analogs of the messenger RNA cap

The ability to synthesize capped RNA transcripts in vitro has been of considerable value in a variety of applications. However, one-third to one-half of the caps have, until now, been incorporated in the reverse orientation. Such reverse caps impair the translation of in vitro-synthesized mRNAs. Novel cap analogues, such as P-3'-deoxy-7-methylguanosine-5&#x2032;P-guanosine-5&#x2032;triphosphate and P-3&#x2032;-O,7-dimethylguanosine-5&#x2032;P-guanosine-5&#x2032;triphosphate, have been designed that are incapable of being incorporated into RNA in the reverse orientation. Transcripts produced with SP6 polymerase using 'anti-reverse' cap analogues were of the predicted length. Analysis of the transcripts indicated that reverse caps were not formed. The in vitro translational efficiency of transcripts with the novel 'anti-reverse' cap analogues was significantly higher than that of transcripts formed with conventional caps.

Synthesis and use of anti-reverse mRNA cap analogues

Methods of making and using Picornavirus L proteinase peptides (PLPPs) are provided, including, but not limited to, expression of DNA encoding all or a portion thereof, such as a Picornavirus L proteinase (PLP) and variants thereof, as well as methods for determining active sites and inhibitors of a PLP.

Picornavirus I proteinase and methods of making and using thereof

A hybrid vector carrying a first and second DNA segments operationally linked thereto, the first DNA segment encoding a protein capable of cross-linking to the cap structure of mRNA and mediating ribosome-binding, and the second DNA segment encoding a polypeptide or protein, the vector being capable of replication, transcription and translation to express the factor and the polypeptide or protein upon transformation of a eukaryotic host, and the polypeptide or protein being expressed at a level higher than the level of expression thereof in the absence of the first DNA segment. A eukaryotic host is transformed with this hybrid vector. Also disclosed is a method of increasing the synthesis of a polypeptide or protein in a eukaryotic host cell.

Hybrid vector and method resulting in protein overproduction by

Growing community of inventors

Shreveport, LA, United States of America

Robert E Rhoads