Creation of variable length and sequence linker regions for dual-domain or multi-domain molecules
View Patent ↗Disclosed are methods and compositions for creating a DNA, RNA or protein molecule with two or more nucleic acid or polypeptide domains, respectively, joined by a linker region. These methods are used to generate random linker libraries of nucleic acids that encode dual-domain or multi-domain polypeptides. The linker regions are characterized by both length and sequence variability.
1. A library of linker nucleic acid molecules or sequences, each of which joins two nucleic acid domains or two nucleic acid sequences encoding two polypeptide domains, each of which has a pattern of degenerate repeated triplet nucleotides with the following properties:
(i) position 1 of each repeated triplet cannot be the same nucleotide as position 2 of the repeated triplet; or
(ii) position 2 of each repeated triplet cannot be the same nucleotide as position 3 of the repeated triplet; or
(iii) position 1 of each repeated triplet cannot be the same nucleotide as position 3 of the repeated triplet; and
(iv) wherein each of said molecules or sequences that joins said domains does not encode Gly 4 Ser (SEQ ID NO:52) or a repeat thereof.
2. A method for making the library of linker nucleic acid molecules or sequences of claim 1 , comprising:
(a) obtaining two template DNA sequences that comprise the first and the second domains;
(b) preparing amplification primer pairs which amplify the first and second domains where each primer pair comprises an upstream primer and a downstream primer, each primer having a 5′ end and a 3′ end, wherein the downstream primer for the first domain or the upstream primer for the second domain comprises a nontemplated sequence,
said nontemplated sequence comprising said repeated pattern of degenerate repeated triplet nucleotides, wherein at least two of the 5′ terminal triplets of said repeated pattern of degenerate repeated triplet nucleotides have the same degenerate sequence;
(c) amplifying the domains with the amplification primers to generate at least one population of nucleic acid domains having different lengths and sequences in the nontemplated sequence;
(d) ligating the nucleic acid domains generated in step (c) to generate said population of dual-domain molecules; and
(e) excising or amplifying said linker nucleic acid molecules or sequences from said population of dual domain molecules.