Affinity tag nucleic acid and protein compositions, and processes for using same

This invention relates to affinity tag compositions including affinity tag nucleic acids and proteins, and processes useful for isolating and detecting or quantifying species of a nucleic acid of interest, and other processes for modifying, isolating, detecting or quantifying proteins and analytes of interest.

All patents, patent applications, patent publications, scientific articles and the like, cited or identified in this application are hereby incorporated by reference in their entirety in order to describe more fully the state of the art to which the present invention pertains.

For many purposes of manipulating or analyzing nucleic acids, the first important step is isolation of the nucleic acids from other cellular material. In this regard, the earliest methods were relatively crude methods using ethanol precipitation followed by phase partitioning with organic reagents. For instance, phenol has been widely used to separate DNA from cellular material while RNA is more commonly isolated using a guanidinium isothiocyanate/phenol/chloroform mixture. These methods do not depend on the particular sequences of the nucleic acids for their isolation, i.e., they are sequence independent and the basis of separation is strictly derived from general chemical properties of DNA and RNA.

More sophisticated methods were later developed that employed the particular sequences of the nucleic acids as an identifying feature for separation, thereby enabling the isolation of nucleic acids with selected sequences apart from other nucleic acids as well as from other cellular material. A notable example of this method is “hybrid capture” where a nucleic acid complementary to the sequence or sequences of interest is used to specifically hybridize to one or more target nucleic acids. At a later step, a tag on the capture probe is used to separate material that has hybridized to the capture probe from material that remained unhybridized. Examples of formats that exploit this methodology include beads with oligo T segments for isolation of polyA RNA, and strepavidin-coated microtitre plates that can bind biotinylated primers after amplification reactions. In either case, a moiety capable of binding the tag is fixed to a solid support, thus enabling a series of simple washing steps to remove nucleic acids lacking the sequences of interest. Thus, in one case, a nucleic acid sequence is added to the capture probe, and in the other case, one of the nucleotides is modified by the addition of a ligand. Unfortunately, these methods are disadvantaged by the slower kinetics of mixed phase hybridization in the first case and the low capacity engendered by the attachment of large bulky proteins to a solid matrix in the aforementioned biotin/strepavidin method.

While conceptually simple, the isolation and purification of proteins has been at the same time both easier and more problematic. Unlike nucleic acids that have similar chemical properties regardless of sequence differences, the variety of different amino acids and the existence of secondary and tertiary structures have allowed the application of various criteria to be used for isolation of a single species of protein. These criteria include differences in molecular weight, shape, salt solubility, net charge and polar versus nonpolar characteristics. Thus, for purification of any given protein, a series of separation steps can be carried out that will be unique to that particular protein. However, these standard methods of protein purification lack the advantages described earlier for isolation of unique nucleic acid sequences where essentially a single methodology can be applied to purification of any species of interest. Although this has remained true for most native proteins, the burgeoning field of recombinant DNA has allowed more flexibility in modifying desirable proteins such that they carry additional amino acid sequences that can be helpful during purification procedures. The most notable example of such methods is the histidine tag which has been added to either the carboxy or amino end of the coding sequence (Dobeli et al., U.S. Pat. No. 5,284,933). The important feature of this oligopeptide sequence is that it has an affinity for chelated metals, such that a matrix with immobilized metal can be used to bind any protein that has such a histidine tag (Dobeli et al., U.S. Pat. No. 4,877,830), a method commonly referred to as IMAC (Immobilized Metal Affinity Chromatography). Thus, a single isolation procedure can be used for a wide variety of proteins after the proteins have been suitably modified. Although oligohistidine is the best known example of an oligo peptide that can bind to an immobilized metal, other petides have been described as well, including one that has the amino acid sequence HGGHHG (Cheng et al. 2004 Bio-organic & Medicinal Chemistry Letters 14; 1987-1990)

The use of non-nucleic acid affinity tags has also been used in conjunction with nucleic acids. For instance, Min and Verdine (1996 Nucleic Acids Research 24:3806-3810) have described a nucleic acid primer with modified bases at the 5′ end with histidine moieties attached to the bases. As such, their primer does not contain an oligopeptide tag as described above, but rather the 5′ end has been modified with a series of histaminyl purine residues. Extension of these primers in a PCR reaction allows collection of the PCR products by means of a chelated resin. No application is described in this publication, however, for using these constructs for either signal detection or analyte isolation.

Stanley et al. (U.S. Pat. No. 5,843,663) describe the use of affinity agents attached to peptide nucleic acids (PNAs). As described previously in Min and Verdine (1996), cited supra., the individual amino acids are attached to each nucleotide analog as opposed to a true oligohistidine capture agent. It also should be pointed out that this is not an example of a chimeric molecule consisting of a nucleic acid and an affinity tag because the peptide nucleic acid is actually a synthetic substitute for a nucleic acid. The backbones of the constructs described by Stanley et al. have an essentially homogeneous nature because both the subunits of the amino acid segment and the pleptide nucleic acid analogue segment are joined together by a succession of peptide bonds to form a single polymeric molecule. The method described in this patent has drawbacks that are intrinsic to the use of peptide nucleic acids. Specifically, efficient synthesis is limited to only short PNA sequences and there is a high cost associated with the reagents used in PNA synthesis.

Soderlund et al. (U.S. Patent Appl. No. 20040053300) describe a method of determining the quantity of discrete polynucleotide analytes by the use of a pool of nucleic acid probes of various sizes. The probes hybridize to analytes that have been modified by the addition of an affinity tag (such as oligo histidine) to the base portion. After hybridization of the probes to analytes, complexes are isolated by virtue of the presence of the affinity agent in the analyte allowing binding to a matrix. In a subsequent step the bound probes are released and quantified, thus giving an indirect measurement of the amount of analytes present in a sample. In this particular instance, the analytes themselves have been covalently attached to an affinity agent.

Affinity binding pairs have also been used in conjunction with RNA molecules in Krause and Simmons (U.S. Patent Appl. No. 20060105341). In this application, the use of a so-called RNA “fusion” molecule with “RNA tags” is described. In this particular case, however, the “fusion” is not RNA linked to a non-nucleic acid but rather the molecule is a fusion of different nucleic acid sequences resulting in a homogenous nucleic acid where a first RNA segment with a protein binding sequences is appended to a second RNA segment with a selected nucleic acid sequence. This second RNA segment may bind, in turn, to a fusion protein with two domains where one domain binds the RNA tag and the other domain can be an affinity partner, such as an oligo-His tag, that can be used to bind the RNA protein complex to a matrix. This composition has been used for identification and purification of RNA protein complexes and it has not been used for signal generation or isolation of nucleic acid analytes.

Histidine has also been used for other purposes besides an affinity label. For example, Van Ness et al. (U.S. Pat. No. 7,247,434) describe methods for simultaneously determining a number of different nucleic acid sequences by the use of tagged nucleic acid fragments. Sequences are derived from the association of a different tag for each nucleotide base incorporated into nucleic acids synthesized from analyte templates. In one particular instance, a single histidine moiety is used as one of the base-specific tags where identification is carried out by mass spectrometry after the nucleic acids have been separated by length. In this particular instance the histidine is not being used as an affinity agent but only as an identifier tag.

Many of the drawback in the previous uses of affinity tags such as histidine tags are overcome by the present invention.

This invention provides a composition which comprises a nucleic acid and one member of an affinity binding pair, wherein the member is attached to one or more nucleotides of the nucleic acid through a phosphate or sugar of the nucleotide or nucleotides.

This invention also provides a composition just described wherein the affinity binding pair comprises: (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group.

This invention additionally provides a chimeric nucleic acid comprising at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising a metal binding peptide, wherein the metal binding peptide is attached to one or more nucleotides of the nucleic acid in the first portion through a sugar or phosphate of the nucleotide or nucleotides.

Also provided by this invention is a chimeric nucleic acid comprising at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising one member of a peptide affinity group, wherein the member is attached to one or more nucleotides of the nucleic acid in the first portion.

The present invention provides a process for isolating one or more species of a nucleic acid of interest. Various steps are used including the first step of providing a sample containing or suspected of containing the nucleic acid of interest, a composition which comprises a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid species of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group; and wherein the first member of the affinity binding pair is attached to one or more nucleotides in the nucleic acid portion; and a matrix comprising a second member of the affinity binding pair. The composition hybridizes with any nucleic acid of interest contained in the sample to form a first complex. The first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair. Bound material is separated from unbound material, thereby isolating the nucleic acid species of interest.

The present invention also provides a process for detecting the presence or quantity of a nucleic acid of interest. In an initial step, the following elements are provided: a sample containing labeled nucleic acids, a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, and a matrix comprising a second member of the affinity binding pair; wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group; and wherein the first member of the affinity binding pair is attached to one or more nucleotides of the nucleic acid portion. The composition is allowed to hybridize with any nucleic acid of interest contained in the sample to form a first complex. This first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair. The matrix is washed to remove unhybridized nucleic acids from the matrix. Detecting or quantifying the nucleic acid of interest is carried out by means of detecting or quantifying a signal from the labels.

The present invention provides yet another process for detecting the presence or quantity of a nucleic acid of interest. Various steps are performed including the initial step of providing the following elements: a sample containing or suspected of containing the nucleic acid of interest; a labeled probe complementary to the nucleic acid of interest; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein the first member of the affinity binding pair is attached to one or more nucleotides of the nucleic acid portion through a sugar, phosphate or base of the nucleotide or nucleotides; and a matrix comprising a second member of the affinity binding pair. Any nucleic acids of interest in the sample are allowed to hybridize with labeled probe and the composition to form a first complex. This first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair. The matrix is washed to remove unbound materials from the sample. The nucleic acid of interest is detected or quantified by means of detecting or quantifying a signal from the labels.

Yet another process provided by the present invention is one for detecting the presence or quantity of a nucleic acid of interest. Various steps are performed including the initial step of providing a sample containing or suspected of containing nucleic acid of interest; a probe complementary to the nucleic acid of interest and comprising two portions, wherein a first comprises sequences complementary to the nucleic acid of interest, and a second portion comprising a signal sequence; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein the first member of the affinity binding pair is attached to one or more nucleotides of the nucleic acid; and a matrix comprising a second member of the affinity binding pair. Any nucleic acids of interest in the sample are hybridized with labeled probe and the composition to form a first complex. The first complex is contacted with the matrix to form a second complex by means of a binding interaction between the one or more binding partners and the affinity peptide. The matrix is washed to remove unbound materials from the sample. The nucleic acid of interest is detected or quantified by hybridizing labeled oligonucleotides complementary to the signal sequence.

The present invention also provides a fusion protein comprising a biologically active polypeptide or protein and at least one affinity peptide attached to the amino-terminus or the carboxyl-terminus of the biologically active polypeptide or protein, wherein the affinity peptide comprises at least a portion of the amino acid sequence of kininogen, such portion comprising a metal binding peptide.