Patent ID: 8632972

Claim:
An in vitro method of identifying a protein that binds to a predetermined RNA sequence in a cell, said method comprising the steps of: a) providing a membrane permeable construct to a cell under conditions suitable for transport of said construct across the membrane of said cell, wherein said construct comprises: i) a nucleic acid analog which hybridizes with an untranslated region of an intracellular polynucleotide comprising a predetermined target mRNA sequence, said nucleic acid analog further comprising at least one photoreactive moiety; ii) a peptide moiety comprising R 1 -CPP-R 2 , wherein CPP is a cell-penetrating peptide, further wherein each of R 1 and R 2 are independently selected from the group consisting of a peptide, an amino acid, NH 2 , H, or OH, further wherein said nucleic acid analog is covalently attached to one of the members selected from the group consisting of R 1 , R 2 , a cysteine residue within said peptide moiety, or a lysine (K) residue within said peptide moiety; and iii) a chemical bond linking said nucleic acid analog and said peptide moiety; b) incubating said construct with said cell under conditions suitable to allow said construct to bind with an untranslated region of an intracellular polynucleotide comprising a predetermined target mRNA sequence through a Watson/Crick base-pair binding between said nucleic acid analog and said untranslated region of said target mRNA sequence to form a construct-polynucleotide complex in said cell; c) activating said photoreactive moiety of said construct, thereby covalently cross-linking said nucleic acid analog with a nearby RNA binding protein that binds to said predetermined target mRNA sequence; d) isolating said crosslinked nucleic acid analog-protein from said cell following step c); and e) identifying said protein crosslinked to said nucleic acid analog; thereby identifying a protein that binds to a predetermined target RNA sequence.