Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-03-16
2001-12-04
Martinell, James (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S455000, C435S325000, C435S091530, C800S008000, C514S04400A, C536S023100
Reexamination Certificate
active
06326166
ABSTRACT:
BACKGROUND OF THE INVENTION
DNA-binding proteins, such as transcription factors, are critical regulators of gene expression. For example, transcriptional regulatory proteins are known to play a key role in cellular signal transduction pathways which convert extracellular signals into altered gene expression (Curran and Franza,
Cell
55:395-397 (1988)). DNA-binding proteins also play critical roles in the control of cell growth and in the expression of viral and bacterial genes. A large number of biological and clinical protocols, including among others, gene therapy, production of biological materials, and biological research, depend on the ability to elicit specific and high-level expression of genes encoding RNAs or proteins of therapeutic, commercial, or experimental value. Such gene expression is dependent on protein-DNA interactions.
Attempts have been made to change the specificity of DNA-binding proteins. Those attempts rely primarily on strategies involving mutagenesis of these proteins at sites important for DNA-recognition (Rebar and Pabo,
Science
263:671-673 (1994), Jamieson et al.,
Biochemistry
33:5689-5695 (1994), Suckow et al.,
Nucleic Acids Research
22(12):2198-2208 (1994)). This strategy may not be efficient or possible with some DNA-binding domains because of limitations imposed by their three-dimensional structure and mode of docking to DNA. In other cases it may not be sufficient to achieve important objectives discussed below. Therefore, it is desirable to have a strategy which can utilize many different DNA-binding domains and can combine them as required for DNA recognition and gene regulation.
SUMMARY OF THE INVENTION
This, invention pertains to chimeric proteins which contain at least one composite DNA-binding region and possess novel nucleic acid binding specificities. The chimeric proteins recognize nucleotide sequences (DNA or RNA) sparning at least 10 bases and bind with high affinity to oligonucleotides or polynucleotides containing such sequences. (It should be understood that the nucleotide sequences recognized by the chimeric proteins may be RNA or DNA, although for the sake of simplicity, the proteins of this invention are typically referred to as “DNA-binding”, and RNA too is understood, if not necessarily mentioned.)
The terms “chimeric” protein and “composite” domain are used to denote a protein or domain containing at least two component portions which are mutually heterologous in the sense that they do not occur together in the same arrangement in nature. More specifically, the component portions are not found in the same continuous polypeptide sequence or molecule in nature, at least not in the same order or orientation or with the same spacing present in the chimeric protein or composite domain.
As discussed in detail below, a variety of component DNA-binding polypeptides known in the art are suitable for adaptation to the practice of this invention. The chimeric proteins contain a composite region comprising two or more component DNA-binding domains, joined together, either directly or through one amino acid or through a short polypeptide (two or more amino acids) to form a continuous polypeptide. Additional domains with desired properties can optionally be included in the chimeric proteins. For example, a chimeric protein of this invention can contain a composite DNA-binding region comprising at least one homeodomain, such as the Oct-1homeodomain, together with a second polypeptide domain which does not occur in nature identically linked to that homeodomain. Alternatively, the composite DNA-binding domain can comprise one or more zinc finger domains such as zinc finger 1 and/or finger 2 of Zif268, together with a second polypeptide domain which does not occur in nature linked to that zinc finger domain(s).
A number of specific examples examined in greater detail below involve chimeric proteins containing a composite DNA-binding region comprising a homeodomain and one or two zinc finger domains. In one embodiment, the chimeric protein is a DNA-binding protein comprising at least one homeodomain, a polypeptide linker and at least one zinc finger domain. Such a chimeric protein is exemplified by a composite DNA-binding region containing zinc finger 1 or zinc finger 2 of Zif268, an amino acid or a short (2-5 amino acid residue) polypeptide, and the Oct-1 homeodomain. Another example is a chimeric protein containing a composite DNA-binding region comprising zinc fingers 1 and 2 of Zif268, a short linker, such as a glycine-glycine-arginine-arginine polypeptide, and the Oct-1 homeodomain. The latter chimeric protein, designated ZFHD1, is described in detail below. Other illustrative composite DNA-binding regions include those comprising the Oct1 POU specific domain (aa 268-343) and its own flexible linker (aa 344-366) fused to the amino terminus of ZFHD1 and ZFHD1 fused at its carboxy terminus to Zif268 fingers 1 and 2 (aa 333-390) via the Oct1 flexible linker.
In other embodiments, the chimeric protein comprises a composite DNA-binding region containing a chimeric zinc finger-basic-helix-loop-helix protein. One such chimeric protein comprises fingers 1 and 2 of Zif268 and the MyoD bHLH region, joined by a polypeptide linker which spans approximately 9.5 Å between the carboxyl-terminal region of finger 2 and the amino-terminal region of the basic region of the bHLH domain.
In another embodiment, the chimeric protein comprises a composite DNA-binding region containing a zinc finger-steroid receptor fusion. One such chimeric protein comprises fingers 1 and 2 of Zif268 and the DNA-binding domains of the glucocorticoid receptor, joined at the carboxyl-terminal region of finger 2 and the amino-terminal region of the DNA-binding domain of the glucocorticoid receptor by a polypeptide linker which spans approximately 7.4 Å.
As will be seen, one may demonstrate experimentally the selectivity of binding of a chimeric protein of this invention for a recognized DNA sequence. One aspect of that specificity is that the chimeric protein is capable of binding to its recognized nucleotide sequence preferentially over binding to constituent portions of that nucleotide sequence or binding to different nucleotide sequences. In that sense, the chimeric proteins display a DNA-binding specificity which is distinct from that of each of the component DNA-binding domains alone; that is, they prefer binding the entire recognized nucleotide sequence over binding to a DNA sequence containing only a portion thereof. That specificity and selectivity means that the practitioner can design composite DNA-binding regions incorporating DNA-binding domains of known nucleotide binding specificities with the knowledge that the composite protein will selectively bind to a corresponding composite nucleotide sequence and will do so preferentially over the constituent nucleotide sequences.
These chimeric proteins selectively bind a nucleotide sequence, which may be DNA or RNA, spanning at least 10 bases, preferably at least 11 bases, and more preferably 12 or more bases. By way of example, one can experimentally demonstrate selective binding for a 12-base pair nudeotide sequence using the illustrative ZFHD1 composite DNA-binding domain. Typically one will obtain binding to the selected DNA sequence with a Kd value of about 10
−8
or better, preferably 10
−9
or better and even more preferably 10
−10
or better. Kd values may be determined by any convenient method. In one such method one conducts a series of conventional DNA binding assays, e.g. gel shift assays, varying the concentration of DNA and determining the DNA concentration which correlates to half-maximal protein binding.
The nucleotide sequence specificity of binding by chimeric proteins of this invention, illustrated by proteins comprising the peptide sequence of ZFHD1, renders them useful in a number of important contexts because their DNA-binding properties are distinct from those of known proteins. Such uses include the selective transcription, repression or inhibition of transcripti
Pabo Carl O.
Pomerantz Joel L.
Sharp Phillip A.
Martinell James
Massachusetts Institute of Technology
Ropes & Gray LLP
Vincent Matthew P.
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