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OwnerGert-Jan Arts, Ellen Vera Langemeijer +3inventors
InventorsGert-Jan Arts, Ellen Vera Langemeijer, Ivo Piest, Helmuth Hendrikus Gerardus Van Es, Godefridus Augustinus Maria Michiels
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Isolated polynucleotides, and vectors including the same, are disclosed as useful for down-regulation of specific RNA in cells, including a first sequence of about 17 to about 23 nucleotides, complementary to said RNA, and linked to a second sequence capable of forming a loop when said second sequence is RNA. The polynucleotides include self-complementing single-stranded polynucleotides, including a third sequence linked by said second sequence where all nucleotides in said first and said third sequences are complementary. Functional genomic, diagnostic and therapeutic methods are disclosed that involve reducing the amount of a unique RNA sequence in cells using a vector encoding the self-complementing polynucleotide including a first sequence complementary to said RNA sequence. Methods are also disclosed for preparing the polynucleotides, vectors, libraries of vectors, and the temporary knock-down of proteins, such as lethal proteins, during virus or recombinant protein production.
An isolated polynucleotide useful for the down regulation or degradation of a specific RNA molecule in a host cell, consisting essentially of a first polynucleotide sequence consisting of about 17 to about 23 nucleotides and complementary to about 17 to about 23 nucleotides of said RNA sequence in said host cell, said first sequence covalently linked to a second sequence capable of forming a stem-loop structure when said second sequence is an RNA sequence, wherein said first sequence consists essentially of a RNA sequence, or a single stranded DNA equivalent thereof.
A self complementing single-stranded polynucleotide useful for the down regulation or degradation of RNA in a host cell, consisting essentially of a first polynucleotide sequence consisting of about 17 to about 23 nucleotides and complementary to about 17 to about 23 nucleotides of said RNA sequence in said host cell, and a third nucleotide sequence covalently linked by a second nucleotide sequence capable of forming a stem-loop structure when said second sequence is an RNA sequence, and such that all nucleotides in said first sequence and said third sequence are capable of base pairing with each other, wherein said second nucleotide sequence comprises a stem-loop forming region that comprises a sequence derived from naturally occurring RNA sequences and that does not functionally target a specific RNA molecule in a host cell.
A method for preparing a self-complementing single stranded polynucleotide including complementary sequences covalently linked by a polynucleotide sequence forming a stem loop structure, comprising treating a single stranded polynucleotide consisting essentially of a first polynucleotide sequence covalently linked to a second polynucleotide sequence that includes two nucleotide sequences capable of complementary base pairing and thereby forming a stem-loop structure and that has a 3′ OH terminus, under conditions such that said first sequence serves as a template starting at the 3′ OH terminus for the synthesis of a complementary sequence thereto.
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