The University of Chicago

Krystyna Maria Kazmierczak

Elena K Davydova

Lucia B Rothman-Denes

The present invention comprises novel methods, compositions and kits that use N4 vRNAP deletion mutants to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide or non-nucleic acid analytes, by detecting a target sequence tag that is joined to an analyte-binding substance. The method consists of an annealing process, a DNA ligation process, an optional DNA polymerase extension process, a transcription process, and, optionally, a detection process.

Target-dependent transcription using deletion mutants of N4 RNA polymerase

A histidine-tagged, deletion mutant of bacteriophage N4-coded, virion RNA polymerase (mini-vRNAP) which is active has been developed. The his-tagged mini-vRNAP has been cloned under the control of the pBAD promoter, is stable and is purified in a single step yielding large amounts (10 mg/liter ofexpressing cells). This RNA polymerase uses single-stranded DNA containing 17 bases (the promoter) upstream of the transcribed regions as a template. In the presence ofSSB protein, it transcribes this template efficiently, providing a unique system to synthesize RNAs of the desired sequence using single-stranded DNA templates. The enzyme incorporates derivatized nucleoside triphosphates with high efficiency. A mutant of mini-vRNAP has been generated that incorporates deoxynucleoside triphosphates. In addition, the inventors have developed an in vivo system to express RNAs and proteins under mini vRNA polymerase promoter control.

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Bloomington, IN, United States of America

Krystyna Maria Kazmierczak