Bifunctional protein for the isolation of capped mRNA

Details Bifunctional protein for the isolation of capped mRNA Bifunctional protein for the isolation of capped mRNA

1992-02-24

1993-06-15

Hill, Jr., Robert J.

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

530413, 4351721, C07K 1300, C12J 122

Patent

active

052199894

ABSTRACT:
A protein comprising at least a first functional site having the ability to bind the cap structure of mRNA and a second functional site having the ability to bind a solid support matrix in such a manner as to allow the first functional site to be immobilized and still remain functionally accessible to interact with the cap structure of mRNA. Also within the scope of the present invention is a method for generating a cDNA library mostly containing full-length cDNAs. The method comprises the incubation of a mixture comprising mRNA:cDNA hybrids with 1) a single strand RNA specific nuclease and 2) the above-mentioned protein. The resulting mixture is then passed through a column comprising a support matrix having the ability to bind the second functional site of the above-mentioned protein in order to selectively bind complete mRNA:cDNA hybrids. The mRNA:cDNA hybrids are then competitively eluted with a cap analog and full-length cDNA strands are separated and recovered. The present invention also includes a method for purifying capped mRNA using the above-mentioned protein. The process comprises the incubation of a mixture containing mRNA with the above-mentioned protein, passing the resulting mixture through a column comprising the support matrix having the ability to bind to the second functional site of the above-mentioned protein in order to selectively bind capped mRNAs, and competitively eluting the capped mRNAs with a cap analog.

REFERENCES:
Sonenbey et al. Proc. Natl Acad. Sci. USA 76(9), pp. 4345-4349 (1979).
Creighton, "Proteins; Structures and Molecular Properties," Freeman & Co.: 1984, pp. 347-348.
Maniatis et al., "Molecular Cloning; A Laboratory Manual" Cold Spring Harbor Lab., New York: 1982, pp. 197-198.

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