CAP-BINDING DOMAIN OF HUMAN EUKARYOTIC PROTEIN SYNTHESIS INITIATION FACTOR eIF-4E AND THE USE THEREOF

Abstract: Binding of eIF-4E to the 5'm<7>G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In the present invention, the binding site cap-binding domain of eIF-4E was identified as the region containing the sequence Trp<113> to Arg<122>. Thus, in a first preferred embodiment the present invention provides a truncated peptide of the cap binding protein consisting essentially of the mRNA cap-binding domain. Specifically, the invention concerns eIF-4E cap binding peptide and its variants. In additional preferred embodiments, the invention provides an isolated nucleic acid molecule which encodes a truncated peptide of the eIF-4E protein consisting essentially of the mRNA cap-binding domain of eIF-4E; a recombinant vector and host cell comprising the above-described nucleic acid molecule; and a recombinant peptide produced thereby. The invention further provides the truncated or recombinant peptides immobilized on a solid support, a resin comprising the truncated or recombinant peptides immobilized on a solid support, methods and kits for isolating a capped mRNA molecule, and methods and kits for producing a full-length cDNA molecule.

Description: Cap-Binding Domain of Human Eukaryotic Protein Synthesis Initiation Factor eIF-4E and the Use Thereof STATEMENT AS TO U S GOVERNMENT RIGHTS The present invention was made in part under grants from the National Science Foundation (GER-9023681, MCB 9303661 and DGE-9553549) and the National Institutes of Health (NIH-CA63640) The U S government has certain rights in the present invention FIELD OF THE INVENTION The present invention is in the field of molecular biology Specifically, the invention provides truncated peptides of the eukaryotic protein synthesis initiation factor eIF-4E comprising the region of eIF-4E that binds to the cap regions of mRNA molecules The invention also provides methods and kits using these peptides suitable for use in the isolation of capped mRNA molecules and in the production of full-length cDNA molecules RELATED ART Overview The initiation phase of eukaryotic protein synthesis is characterized by recognition of the m 7 (5 ')Gppp(5 ')N (where "N" is any nucleotide) cap structure at the 5' terminus of eukaryotic mRNA by initiation factors (elF's) (reviewed in Rhoads, R.E , Trends Biochem. Sci. (Pers. Ed.) 13:52-56 (1988); Sonenberg, N., Prog. Nucleic Acid Res. Mol. Biol. 55:173-207 (1988)) Of the elF's which interact at or near the cap, only eIF-4E (alone or as part of the eIF-4F complex) has been shown to interact directly with the cap (Sonenberg, N., Nucleic Acids Res. 9: 1643-1656 (1981), Sonenberg, N , et al., Cell 27 563-572 (1981), Tahara, S , e/ al., J. Biol.. Chem. 256:7691-694 (1981); Hell ann, G.M., et al.→ J. Biol.. Chem. 257:4056-4062 (1982), Grifo, H.A , et al., J. BwL Chem. 255.5804-5810 (1981), Webb, N R , et al, Biochemistry 23 177 '-181 (1984)). This initial recognition of the cap structure represents the first committed step in the initiation phase of protein synthesis. Evidence that cap recognition by eEF-4E is a limiting step in protein synthesis is supported by the observation that it is the least abundant of all the initiation factors and thus may serve a regulatory function (Duncan, R.F., et al., J. Biol.. Chem. 262:380-388 (1987); Hire ath, L.S., et al., J. Biol.. Chem. 260:7843-7849 (1985)). In addition, there are a number of other observations indicating that this protein is a key point for post-transcriptional control of gene expression. For example, EGF and other growth regulatory peptides rapidly stimulate the phosphorylation of eIF-4E phosphorylation in intact cells (Donaldson, R.W., et al., J. Biol.. Chem. 266:3162-3166 (1991); Sonenberg, N„ Biochemie 76:839-846 (1994); Rhoads, R E., J. Biol. Chem. 268:3011-3020 (1993); Proud, C.G., Curr. Top. Cell. Regul. 32:243-369 (1992)). Furthermore, evidence has been obtained from two different cellular systems that hyperphosphorylation of elF-4E promotes the recruitment of other eIF-4E subunits to the m 7 G mRNA cap (Bu, X., et al., J. Biol.. Chem. 265:4975-4978 (1993); Morley, S.J., et al, Eur. J. Biochem. 218:39-4% (1993)) and a three- to four-fold increase in the affinity of eIF-4E binding to mRNA caps (Minich, W.B., et al, Proc. Natl. Acad. Sci. U.S.A. 97:7668-7672 (1994)). The regulation of eEF-4E function has been shown to be even more complicated by the recent identification of eIF-4E binding proteins that are regulated physiologically by insulin (Lin, T.A., et al., Science 266:653-656 (1994)). When bound to eIF-4E these proteins prevent its recognition of, and binding to, m 7 G caps of mRNA. To understand fully the mechanisms by which these covaient modifications and translational repressor 4E binding proteins exert their effects a more detailed understanding of eIF-4E structure and binding site sequence will be required. Structure of the eIF-4E Cap-binding Domain The amino acid sequence for the cap-binding protein has been previously reported for several species including yeast (Altmann, M., et α/., M?/. Cell. Biol.. 7:998-1003 (1987)), wheat (Metz, A M , et al, Nucl. Acids Res. 20:4096 (1992)), human (Rychlik, W., et al., Proc. Natl. Acad. Sci. U.S.A. 5-^:945-949 (1987)), and mouse (Alt ann, M, et al., . Biol. Chem. 26*-/: 12145-12147 (1989)) While there is some interspecies divergence in the amino acid sequences of the cap-binding proteins, the numbers and positions of eight tryptophan residues are highly conserved, with there being one additional tryptophan residue in p26 of wheat germ eIF-4F (Metz, A M , et al., Nucl. Acids Res. 20:4096 (1992)) Although no x-ray crystallographic or structural NMR information on the eEF-4E«cap complex have yet been reported, photoaffinity labeling studies of mammalian eIF-4E have suggested that the cap-binding domain is within amino acid residues 47-182 of the native protein (Chavan, A.J., et al, Biochemistry 29: 5521-5529) Furthermore, mutational analysis suggested that a glutamic acid residue, which is located three amino acid residues to the carboxy side of tryptophan 5 (numbering of tryptophan residues proceeds from the amino terminus to the carboxy terminus), participates in the binding of the mRNA cap to human eIF-4E via direct hydrogen bond formation with the 2-amino group of the m 7 G base (Ueda, H , et al, FEBSLett. 280 207-210 ( 199 ), Ueda, H , et al. , Biochem. Biophys. Ada. 1075' 181 - 186 ( 1991 )). Interestingly, however, previous work from this same group had proposed that a glutamic acid residue two amino acid residues to the carboxy side of tryptophan 7 participates in this hydrogen bond interaction (Ueda, H., et al., Biochem. Biophys. Res. Commun. 154. 199-204 ( 1988)) Regardless of which conclusion is correct, however, the cap-binding subunit of wheat germ eIF-4F lacks any such acidic residue in these regions, containing instead a β-turn-inducing proline residue in the region of tryptophan 5 and a basic histidine residue in the region of tryptophan 7 (Metz, A M , et al., Nucleic Acids Res. 20:4096 (1992)) These conflicting results and the lack of structural information indicate the need for a more precise localization of the eIF-4E binding site for the 5' m 7 G cap of mRNAs Photoaffinity Labeling Photoaffinity labeling of nucleotide binding sites has provided a highly selective method for identifying the participating amino acids of many proteins (Salvucci, M E , et al., Biochemistry 37:4479-4487 (1992); Shoemaker, M.T., and Haley, B E , Biochemistry 52: 1883-1890 (1993), Jayaram, B., and Haley, B E., J. Biol. Chem. 269:3233-3242 (1994)). Photoactivation of the azido moiety with ultra-violet light results in the generation of a nitrene and the subsequent covalent attachment of the probe to the binding domain through the remaining nitrogen (Potter, RL., and Haley, B E., Meth. Enzymol. 97:613-633 (1983)). Immobilized aluminum(III)-chelate chromatography has facilitated the isolation of binding site peptides due to the presence of highly charged phosphates on the photoinserted azidonucleotide which interact with the Al 3+ (Salvucci, M.E., et al, Biochemistry 57:4479-4487 (1992); Shoemaker, M.T., and Haley, B E., Biochemistry 52:1883-1890 (1993); Jayaram, B., and Haley, B E., J. Biol. Chem. 269:3233-3242 (1994); Anderssen, L. J., Chromatography 539: 327-334 (1991)). Uses of eIF-4E Protein Isolated cap-binding eIF-4E proteins have several uses. For example, U.S. Patent No. 5,219,989 to Sonenberg et al. discloses the preparation of multifunctional fusion proteins comprising the eIF-4E protein and at least a second protein having the ability to bind to a solid support matrix. The exemplified second protein is protein A which is capable of binding to a resin having an IgG antibody attached to it. Such fusion proteins may be used for the preparation of a cDNA library mostly containing full-length cDNA and for the purification of capped mRNA. A preparation consisting essentially of the cap-binding region of eIF-4E (or fragments thereof), however, has not been reported. Such a preparation would provide a less expensive and more specific means for binding of capped mRNA molecules, and for production of full-length cDNA molecules, than the fusion proteins of U.S. Patent No. 5,219,989. In addition, such a preparation could be produced more efficiently in recombinant systems such as mammalian host cells, since the nucleic acid sequences directing the synthesis of such a preparation would be smaller and therefore more efficiently incorporated and transcribed than those for fusion proteins. It is therefore an object of the present invention to provide the amino acid sequence of the cap-binding region of eIF-4E, to provide peptides and fragments thereof comprising this region, and to provide methods for the use of these peptides and fragments in the isolation of capped mRNA molecules and in the preparation of full-length cDNA molecules. BRIEF SUMMARY OF THE INVENTION The present invention provides a truncated peptide of the eIF-4E protein consisting essentially of the mRNA cap-binding domain of eIF-4E, wherein the peptide has an amino acid sequence corresponding to that set forth in SEQ ED NO' 1. The invention further encompasses variant peptides comprising the cap-binding domain of eIF-4E within a β-sheet structural motif In preferred embodiments, the invention provides the peptides immobilized on a solid support, a resin comprising the peptides immobilized on a solid support, a method for isolating a capped mRNA molecule, a method for producing a full-length cDNA molecule, a kit for isolating a capped mRNA molecule and a kit for producing a full-length cDNA molecule Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a graph and an autoradiogram of a gel demonstrating saturation of [γ- 32 P]8- N 3 GTP photoincorporation into r eIF-4E r eIF-4E (4 micrograms) was incubated with the indicated concentrations of [γ- 32 P]8-N 3 GTP in 50 μl of photolysis buffer The reaction mixture was irradiated with UN light for 90 seconds and analyzed by SDS- AGE 32 P incorporation was determined by radioisotopic imaging and quantification FIG. IB is a graph and an autoradiogram of a gel demonstrating competitive Inhibition of [γ- 32 P]8-Ν 3 GTP photoincorporation into r eIF-4E by m 7 GTP. r eIF-4E (4 micrograms) was incubated with 40 μM [γ- 32 P]8-N 3 GTP in the presence of increasing concentrations of m 7 GTP under the conditions described for Fig 1A 32 P incorporation was determined by radioisotopic imaging and quantitation FIG. 2 is a bar graph representing a chromatogram (radioactivity profile) of immobilized Al 3+ chromatography of tryptic peptides .eIF-4E (5 mg) was photolabeled with 50 μM [γ- 32 P]8- N 3 GTP followed by a second photolabeling with 50 μM 8-N 3 GTP Following digestion with modified trypsin, Al τ chelate chromatography proceeded as described in the Example Fractions 1-5 represent flow through, 6-25 represent various buffer washes, and 26-31 represent fractions eluted with 10 mM K 2 HPO 4 32 P levels were determined by liquid scintillation counting. FIG. 3A is a chromatogram of microbore, C 8 reverse phase HPLC of tryptic peptide fractions from Al 3 ' chelate chromatography, demonstrating a typical UV profile of the radioactive fractions resulting from K 2 HPO 4 elution of the Al 3< chelate column FIG. 3B is a chromatogram demonstrating the corresponding 32 P cpm profile of the fractions resulting from the HPLC in Fig. 3A Radioactivity levels were determined by liquid scintillation counting. DETAILED DESCRIPTION OF THE INVENTION Identification of the Cap-binding Domain of eIF-4E Binding studies of m 7 GTP, m 7 GpppG, and various capped mRNAs with eIF-4E have provided insight into the nature of the eIF-4E*»cap interaction (Carberry, S E., et al, Biochemistry 25:8078-8083 (1989); Carberry, S.E., et al., Biochemistry 57:1427-1432 (1992), Goss, D.J., et al., Biochem. Biophys. Ada. 76*50:163-166 (1990)) Tryptophan stacking and hydrogen bonding with glutamic acid residues have been suggested to be important factors in binding of the mRNA cap structure by native eEF-4E ( Kamiichi, K , et al., J. Chem. Soc. Perkin Trans. II, 1739-1745 (1987); Ishida, T., et al, J. Am. Chem. Soc. 110:22X6-2294 (1988), Ueda, H., et a , Biochem. Biophys. Ada. 7075: 181-186 (1991)). Additionally, the involvement of a protonated histidine in cap binding by eIF-4E has been proposed (Carberry, S.E., et al. Biochemistry 25:8078-8083 (1989)). These results have allowed for much speculation regarding the specific amino acid residues that constitute the m 7 G cap binding site. In the examples of the present invention provided below, the photoaffinity probe [γ- 32 P]8- N 3 GTP was used as a photoactivable analogue of the m 7 G cap structure of mRNA to selectively label the m 7 GTP cap-binding domain of human recombinant eIF-4E ( r eEF-4E) The photolabeled peptide resulting from proteolysis was isolated using a!uminum(III)-chelate chromatography followed by reversed-phase high performance liquid chromatography The amino acid sequence of this peptide has been identified in the present invention as consisting essentially of residues 113- 122 of the amino acid sequence of human eIF-4E described previously (Rychlik, W., et al., Proc. Natl. Acad Sci. U.S.A. 5^:945-949 (1987)) This peptide thus contains tryptophan 6 (Trp 113 in full-length eIF-4E) Amino Acid sequence of the Cap-binding Domain of eIF-4E The present invention is thus related to a truncated cap binding protein (e.g eIF-4E protein) which is capable of binding to the 5' terminus cap structure of mRNA produced in eukaryotic cells Such truncated proteins comprise at least amino acids Trp 113 to Arg I22 corresponding to the human eIF-4E sequence as disclosed by Rychlik, W.. et al, Proc. Natl. Acad. Sci. U.S.A. 5*^:945-949 (1987), or suitable variants thereof Examples of such truncated proteins include Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg (SEQ ID NO 1) Variants of the truncated eIF-4E protein include any change or changes (deletions, substitutions or additions) in or to the amino acid sequence, provided that the variant retains the ability to bind to the cap region of mRNA. The ability to retain cap binding function can be determined by the assays described herein Preferably, changes are made so as to conserve the hydrophobicity and charge of the peptide. Examples of such variants include but are not limited to one or more substitutions within the peptide as follows Leu changed to Ala, Val, He or Phe, Asn changed to Gin, Gin changed to Asn, Thr changed to Ser or Gly; Lys changed to Arg or His, and Arg changed to Lys or His Previous fluorescence quenching studies have indicated the involvement of a tryptophan residue, which apparently resides in a somewhat hydrophobic environment, in eIF-4E binding of the mRNA cap (Carberry, S.E , et al., Biochemistry 25:8078-8083 ( 1989)) Computer-predicted secondary structure obtained using Chou and Fasman sequence analysis (Chou, P Y , and Fasman, G D , Biochemistry 13 222-245 (1974)) indicate that tryptophan 6 comes after a putative turn region in the protein and would be at least partially buried within a hydrophobic core with only partial exposure to the aqueous environment (McCubbin, W D , et al., J. Biol. Chem. 263: 17663 - 17671 (1988)) The calculations also suggest that tryptophan 6 lies within a region of β-sheet structure Circular dichroism data obtained in studies related to those disclosed here reveal that a large conformational transition occurs in wheat eEF-(iso)4F upon its binding of mRNA cap analogues Taken together, these data suggest that the interaction between eIF-4E and the mRNA cap may involve a structural β-sheet motif, this conformational transition may thus play a regulatory role, as has been proposed for other proteins containing similar motifs (Wang, Y , et al, in Spe roscopy of Biological Molecules, Merlin, J C , et al , eds ), pp 335-336 (1995)), by determining the interaction of the eIF-4E binding domain with the m 7 G cap structure of mRNA It is well-known in the art that β-strands, which are organized into higher secondary structures such as β-sheets and α/β barrels in a variety of proteins, share certain general properties with respect to their preferred amino acid sequences (Sternberg, M J E , et al, Phil. Trans. R. Soc. Lond B 293 177- 189 ( 1981 ), Wodak, S J , et al. , Biochem. Soc. Symp. 57 99- 121 ( 1990), Siezen, R J , et al., Prot. Eng. 4(7) 719-737 (1991), Chothia, C , et al., Ciba Found. Symp 162 36-57 (1991), Bork, P , et al., J. Mol. Biol. 242 309-320 (1994), Yoshida, M , and Amano, T., FEBS Letts. 359 1-5 (1995)) In general, β-strands comprising β-sheet structural motifs in most proteins are composed of stretches of nonpolar or hydrophobic amino acid residues and are typically 3-10 residues in length While any hydrophobic residue may be found in these regions, including alanine (Ala), valine (Val), leucine (Leu), isoleucine (He), phenylalanine (Phe), methionine (Met), tryptophan (Trp), proline (Pro) or cysteine (Cys), the branched aliphatic residues (Leu, He and Val) are particularly preferred (Stemberg, M J E , et al, Phil. Trans. R. Soc. Lond. B 295.177-189 (1981)) In addition, β-sheet motifs typically have one or more charged (especially aspartic acid (Asp) or glutamic acid (Glu)), aromatic (especially tyrosine (Tyr) or threonine (Thr)) or β-turn-inducing (especially Pro or glycine (Gly)) residues immediately prior to and/or following the stretch of hydrophobic amino acids (Stemberg, M.J.E., et al, Phil. Trans. R Soc. Lond B 293:177-189 (1981); Wodak, S.J., etal, Biochem. Soc. Symp. 57:99-121 (1990); Siezen, R.J., et al., Prot. En 4(7):1\9-131 (1991); Chothia, C, et al, Ciba Found. Symp. 762:36-57 (1991); Bork, P., et al., J. Mol. Biol. 242:309-320 (1994); Yoshida, M., and Amano, T., FEBS Letts. 559:1-5 (1995)). The amino acid residues thus arranged in the primary structure of the protein or peptide interact via aromatic ring stacking, hydrogen bonding and hydrophobic interactions to form β-strands and the resulting higher order secondary structures such as β- sheets. Accordingly, the present invention also encompasses variants of the eIF-4E peptide described above (for SEQ ID NO: l) in which Trpl 13 (tryptophan 6) is maintained in the same β-sheet structural motif. Included in these variants are peptides having an amino acid sequence corresponding to the general pattern Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Xl (1 . 2) -X2 (4 . 10) -Xl (1 . 2) (SEQ ID NO:2) wherein XI is a charged, aromatic or β-turn-inducing amino acid, most preferably Asp, Glu, Tyr, Thr, Gly or Pro; X2 is a hydrophobic amino acid, most preferably Leu, He, Val or Trp, and the subscripts for XI and X2 indicate the number of such residues in consecutive sequence at the indicated position. Other preferred variant peptides of the invention include those having an amino acid sequence corresponding to the general pattern X^-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-X,, (SEQ ID NO:3) wherein X is any amino acid, and n is a whole number ranging in value from 0 to 70, preferably 0 to 50, 0 to 25 or 0 to 10. Other preferred variant peptides of the invention include those having an amino acid sequence corresponding to the general pattern X n -Tφ-Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-X n (SEQ ID NO 4) wherein X is any amino acid, and n is a whole number ranging in value from 0 to 70, preferably 0 to 50, 0 to 25 or 0 to 10. Other preferred variant peptides of the invention include those having an amino acid sequence corresponding to the general pattern X n -Trp-Glu-Asp-Glu-Lys-Asn-Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln- Arg-X,, (SEQ ID NO:5) wherein X is any amino acid, and n is a whole number ranging in value from 0 to 70, preferably 0 to 50, 0 to 25 or 0 to 10. Other preferred variant peptides of the invention include those having an amino acid sequence corresponding to the general pattern Xl (0 . 10) -X2 (0 . 2) -Xl (0 . 10) -Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg- X1 (I . 2) -X2 (4 _ 10) -X1 (I.2) (SEQ ID NO:6) wherein XI is generally a charged, aromatic or β-turn-inducing amino acid (such as Asp, Glu, Tyr, Thr, Gly or Pro), X2 is a hydrophobic amino acid (such as Leu, He, Val or Trp) and the subscripts for XI and X2 indicate the number of such residues in consecutive sequence at the indicated position. Examples of such variant peptides include, but are not limited to: Tφ-Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg (SEQ ID NO: 7); Tφ-Glu-Asp-Glu-Lys-Asn-Lys-Arg-Gly-Gly-Arg-Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Lys- Lys (SEQ ID NO:8); Tφ-Leu-He-Thr-Leu-Asn-Lys-Gln-Gln-Arg-GIy-Asp-Ile-Val-Val-Leu-Val-Pro (SEQ ID NO:9); Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Asp-Gly-Val-Val-He-Ile-Val-Leu-Leu-Asp-Gly (SEQ ID NO 10), Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Asp-Leu-Leu-Val-Ile-Val-Glu-Gly (SEQ ID NO 11), Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Glu-Asp-Val-IIe-Val-Leu-Leu-Ile-Tyr (SEQ ID NO 12), Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Pro-Ile-Val-Val-Ile-Val-Leu-Val-Ile-Ile-Asp-Gly (SEQ ID NO 13), Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Asp-Thr-Val-Leu-Val-Val-Ile-Gly-Pro (SEQ ID NO 14), Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Gly-Asp-Ile-He-Val-Val-Val-Ile-Val-Asp-Pro (SEQ ID NO 15), Tφ-Leu-He-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Gly-Val-Val-Val-Ile-Ile-Val-Leu-Leu-Asp (SEQ ID NO 16), Tφ-Leu-He-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Pro-Thr-Val-Ile-Val-Val-Ile-Asp (SEQ ID NO 17), Trp-Lys-Arg-Gly-Gly-Arg-Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg (SEQ ID NO 18), Tφ-Glu- j Asp-Glu-Lys-Asn-Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Lys- Lys (SEQ ID NO 19), Lys-Leu-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Val-Leu-Gln (SEQ ID NO 20), Ile-Gln-Leu-Val-Tφ-Leu-Ue-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Leu-Lys-Ala-Gly (SEQ ID NO.21); Val-Ala-Tφ-Lys-Arg-Gly-Gly-Arg-Tφ-Leu-Ile-Thr-Leu-Ue-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Gln- Ala-Lys-Thr (SEQ ID NO:22); Ue-Thr-Val-Gln-Ala-Tφ-Lys-Arg-Gly-Gly-Arg-Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Lys- Thr-Leu-Phe (SEQ ID NO:23); Leu-Arg-Val-Tφ-Glu-Asp-Glu-Lys-Asn-Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr-Leu-Asn-Lys- Gln-Gln-Arg-Lys-Tyr (SEQ ID NO:24); Asn-Ue-Ser-Tφ-Lys-Arg-Gly-Gly-Arg-Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Phe-Lys-Ala- Val-Gln-Gln (SEQ ID NO:25); Glu-Tyr-Glu-Gly-Val-Val-Pro-Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gly-Glu-Arg-Asp-Leu-Ile-Leu-Val- Ile-Gly (SEQ ID NO:26); and Asp-Gly-Pro-Gly-Leu-Tφ-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Tyr-Thr-Trp-Val-Val-Ile-Leu- Val-Thr-Gly (SEQ ID NO:27). Other amino acid sequences for peptides that may be used in the present invention will be apparent to one of ordinary skill in the art, in light of the above descriptions of preferred sequences. Based on structural and thermodynamic considerations as described above, this general sequence of amino acids should localize Trpl 13 within the β-strand or β-sheet structural motif that is optimal for maintenance of mRNA cap recognition and binding by eIF-4E. Synthesis of eIF-4E Peptides Truncated eIF-4E peptides such as those described above may be prepared according to well-known methods of solid phase peptide synthesis, using routine methods of organic and inorganic chemistry Alternatively, the peptides may be produced by recombinant DNA techniques, which would yield a recombinant peptide comprising the above-described truncated eIF-4E peptides Production of such recombinant peptides is preferably accomplished by expression of the peptide in a host cell, including a bacterial, yeast or mammalian cell, in which the peptide coding sequence is operably linked to a promoter sequence Typically, the promotor- peptide encoding sequence is contained by a vector Techniques for isolation of nucleic acids, insertion into a vector, transformation of a host cell and isolation of the recombinant protein from the host cell are well-known in the art (see, for example, Sambrook, J , et al, eds , Molectdar Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1992)) Use of eIF-4E Peptides Isolation of Capped mRNA Molecules In one preferred embodiment of the invention, the peptides produced as described above may be covalently or non-covalently immobilized on a solid phase support By "solid phase support" is intended any solid support to which a peptide can be immobilized Such solid phase supports include, but are not limited to nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, beads and microtitre plates Preferred are beads made of glass, latex or a magnetic material Linkage of the peptide of the invention to a solid support can be accomplished by attaching one or both ends of the peptide to the support Attachment may also be made at one or more internal sites in the peptide. Multiple attachments (both internal and at the ends of the peptide) may also be used according to the invention Attachment can be via an amino acid linkage group such as a primary amino group, a carboxyl group, or a sulfhydryl (SH) group or by chemical linkage groups such as with cyanogen bromide (CNBr) linkage through a spacer For non-covalent attachments, addition of an affinity tag sequence to the peptide can be used such as GST (Smith, D B and Johnson, K.S. Gene 67:31 (1988); polyhistidines (Hochuli, E et al., J. Chromatog. 411 11 (1987)); or biotin. Such affinity tags may be used for the reversible attachment of the peptide to the support Such immobilized peptides are useful in chromatographic isolation of mRNA molecules by binding to the cap region of the mRNAs. In this scenario, a solution containing the capped mRNA molecules would be contacted, en masse or on a column, with the resin containing the immobilized cap-binding peptides of the present invention. After allowing binding of the mRNA molecules to the resin, unbound materials may be simply washed away. The mRNA molecules may then be rapidly removed from the resin or other solid phase support by contacting the resin or support with an elution buffer which may contain, for example, a competitive analog of the mRNA cap such as m 7 GDP (see U.S. Patent No. 5,219,989), a buffer that causes a change in the pH, and/or ionic strength of the solution. Immobilized capped mRNA molecules may instead be used directly in an RT-PCR method according to methods that are well known. Production of Full-length cDNA Molecules The mRNA cap-binding peptides of the present invention can be used to obtain full-length cDNAs, as has been described for eIF-4E protein A fusion proteins (U.S. Patent No. 5,219,989). Production of cDNA molecules is effected by reverse transcription of a desired mRNA with a protein having reverse transcriptase activity, for example, according to U.S. Patent No. 5,244,797 with reverse transcriptase, or with a DNA polymerase having reverse transcriptase activity. Preferably, the reverse transcriptase is a truncated or mutated reverse transcriptase that lacks RNase H activities that gives a much higher proportion of full-length cDNA compared to the wild type reverse transcriptase. Such reverse transcriptases (Superscript™ and Superscript II ™ respectively) may be obtained from Life Technologies, Inc. (Gaithersburg, MD) Preferred DNA polymerases having reverse transcriptase activity are thermostable enzymes. Examples of DNA polymerases having reverse transcriptase activity include the wild type and mutant Tne enzymes (see WO96/ 10640 and copending application entitled "Cloned DNA Polymerases from Thermotoga and Mutants thereof by A. John Hughes and Deb K. Chatterjee, filed August 14, 1996); Tth enzyme (U.S. Patent Nos. 5,192,674, 5,413,926, 5,242,818 and WO91/09950); and Taq (U.S. Patent Nos. 5,474,920, 5,079,3524,889,818 and 5,352,600). See also U.S. Patent Nos. 5,407,800, 5,322,770 and 5,310,652. An intermediate in the production of cDNA molecules via this process is a hybrid mRNA cDNA molecule Once a mixture containing mRNA cDNA hybrids has been obtained after reverse transcription of mRNA, it is incubated with a single-stranded RNA specific nuclease, such as a T. or T 2 nuclease, and an endonuclease that specifically attacks the 3' adjacent phosphodiester-bound GpN The naturally modified m 7 G part of the cap structure will not be recognized by this enzyme T, does not attack RNA that is hybridized to DNA If the reverse transcriptase copies the entire length of the mRNA, or if it falls short by a few nucleotides such that there is no unhybridized GpN residue in the corresponding mRNA, T, will not degrade the mRNA and, as a result, the cap structure will remain covalently bound to the mRNA.cDNA hybrid If, however, cDNA synthesis was not complete, the single-stranded RNA specific nuclease will degrade unpaired RNA and remove the cap structure from the mRNA cDNA hybrid Following nuclease treatment, the mRNA cDNA hybrids are incubated with the truncated eIF-4E protein of the present invention, which may be immobilized on a solid phase support as described above As a result of this incubation, only those mRNA cDNA hybrids that have a covalently attached cap structure will bind to the protein of the present invention By immobilizing the truncated eIF-4E to a solid phase support, all of the non-capped containing hybrids, or incomplete cDNAs, will wash away The bound full-length capped mRNA cDNA hybrids may then be released competitively by treatment with a cap analog such as m 7 GDP as described in U S 5,219,989 The resulting purified fraction contains only full length or near full length first strand cDNAs which then act as templates for second strand synthesis The steps for completing the cDNA library are the same as those normally used by those of ordinary skill in the art By use of a reverse transcriptase lacking RNase H activity to reverse transcribe the mRNA together with the purification of the capped mRNA cDNA hybrids after nuclease digestion, it is possible to obtain an unexpectedly high yield of full length cDNA, even when the DNA is long Thus, the present invention is a major advance in the art The present invention is also directed to a kit for isolation of capped mRNA molecules Kits according to this aspect of the invention comprise a carrier means having in close confinement therein one or more container means, each of which contain the reagents described -1*6- herein. For example, a first container means may contain a truncated eIF-4E peptide of the present invention, which may be bound to a solid phase as described above. A second container means may contain a competitive analogue of the cap region of mRNA molecules, which is preferably m 7 GDP. Also encompassed within the present invention is a kit for the production of full-length cDNA molecules. Kits according to this aspect of the invention comprise a carrier means having in close confinement therein one or more container means, each of which contain the reagents described herein. For example, a first container means may contain the truncated eIF-4E protein of the present invention, which may be bound to a solid phase as described above. A second container means may contain a nuclease, which is preferably T, nuclease. A third container means may contain a protein having reverse transcriptase activity, which is preferably a truncated or mutant reverse transcriptase lacking RNAse H activity. A fourth container means may contain an elution buffer which may contain a competitive analogue of the cap region of mRNA molecules, which is preferably m 7 GDP. A fifth container means may contain one or more nucleoside triphosphates and at least one buffer salt. Additional container means may contain an oligo(dT) primer or a control RNA. Two or more of the enzymes described above may alternatively be combined into a single container means The enzymes may be present in a buffered solution or in lyophilized form. Alternatively, the enzymes may be stabilized by drying in the presence of a sugar such as trehalose (U.S. patent nos 5,098,893 and 4,824,938) or acacia gum, pectin, carboxymethyl cellulose, carboxymethylhydroxyethyl cellulose, guar, carboxy guar, carboxymethylhydroxypropyl guar, laminaran, chitin, alginates or carrageenan. It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following example, which is included herewith for purposes of illustration only and is not intended to be limiting of the invention. Examples Example 1: Identification of Cap-binding Domain of eIF-4E Materials and Methods Materials. Sequencing grade modified trypsin was from Boehringer Mannheim Biochemica (Indianapolis, IN). Electrophoresis reagents were from Bio-Rad (Melville, NY). HPLC reagents were from E M. Science (Gibbstown, NJ). All other reagents were obtained from Sigma Chemical Company (St Louis, MO) and were molecular biology grade unless otherwise noted. Recombinant eIF-4E. Human recombinant eIF-4E was purified from Escherichia coli containing a T7 polymerase-driven vector which was described in detail elsewhere (Baker et al., J. Biol. Chem. 267: 1 1495-1 1499 (1992). See also Rychlik et al, Proc. Natl. Acad. Sci USA 5^:945-949 (1987)). Recombinant eIF-4E was purified from bacterial lysates by m 7 GTP Sepharose affinity chromatography followed by Mono Q FPLC (Pharmacia, Piscataway, NJ) (Haas D W. & Hagedom C.H., Arch. Biochem. Biophys. 254:84-89 (1991); Baker B.F., et al, J. Biol. Chem. 267:11495-9 (1992); Bu, X. & Hagedom, CH. FEBS Lett. 507:15-18 (1992)) The FPLC purification step was performed in 50 mM HEPES (pH 8.0), 1 mM MgCl 2 , 10% glycerol, 1 mM DTT and used a 0-500 mM gradient of NaCl. Fractions that contained greater than 95% pure recombinant eIF-4E were identified by measuring absorbance at 280 nm and SDS- PAGE analysis. The isoelectric point of the recombinant eIF-4E was identical to that of the dephosphorylated iso-species of eEF-4E isolated from cultured human cells (Bu, X. & Hagedom, CH. FEBS Lett. 507:15-18 (1992)). In addition to its ability to bind the m 7 GTP cap structure of mRNA, the recombinant eIF-4E prepared using these methods is phosphorylated in vitro by protein kinase C at the same sites as native eIF-4E (Haas D.W. & Hagedorn C.H., Second Messengers and Phosphoproteins 14:55-63 (1992)). Synthesis of Photoaffinity Probe. The radioactive photo probe [γ- 32 P]8-N 3 GTP (specific activity, 32 mCi/μmol) was synthesized and purified as previously reported (Potter, R.L. & Haley, B.EMethods Enzymol. 97:613-633 (1983)). Photoaffinity Labeling of r eIF-4E. To demonstrate saturation effects, samples containing 4 μg of r eIF-4E in photolysis buffer (10 mM Tris»Cl, no salt, no DTT, pH 7.6), were incubated at 4°C in 1.5 ml Eppendorf tubes with the appropriate concentration of [γ- 32 P]8- N 3 GTP for 60 seconds. The reaction mixture was then irradiated for 90 s from a distance of 1 cm with a hand held 254 nm UVB UV lamp (intensity, 1400 μW/cm 2 ). The total reaction volume was 50 μl. The reaction was quenched by the addition of 250 μl of cold acetone. The sample was kept at 4°C for 3 hours and then centrifuged in a Savant HSC10K high speed centrifuge at 10,000 φm for 20 minutes. The supernatant was drawn off and the pellet was resuspended in a protein solubilizing mixture (PSM) consisting of 10% SDS, 3.6 M urea, 2.5% (w/v) DTT, 2% (w/v) pyronin Y (tracking dye), and 20 mM Tris'Cl (pH 8.0). For studies of protection against photoinsertion, 4 μg of r eIF-4E were incubated in photolysis buffer for 60 s at 4°C with the required competitor. At 60 s 40 μM [γ- 32 P]8-N 3 GTP was added and at 60 s the samples were irradiated, precipitated, and solubilized as described above. SDS-PAGE, Scintillation, and Radioisotopic Imaging and Quantitation. Following solubilization, photolabeled samples were analyzed by SDS-PAGE according to Laemmli (Laemmli, U.K. Nature 227:680-685 (1970)). The gels were fixed in 25% isopropanol 10% acetic acid for one hour with frequent changes of fixing solution and dried at 80 °C on a Bio-Rad Model 583 slab gel dryer. 32 P incorporation into r eIF-4E was determined with an Ambis 4000 radioisotopic imaging and quantitation system (Scanalytics/CSPI, Billerica, MA). Photoaffinity Labeling and Enzymatic Digestion of ,eIF-4E for Binding Domain Peptide Isolation. r eIF-4E (5 mg) was photolabeled with 50 μM [γ- 32 P]8-N 3 GTP in a 4 ml total volume of photolysis buffer. The reaction mixture was incubated at 4°C for 5 minutes in a plastic weighing boat followed by irradiation for 2 minutes. This was followed by a second incubation and irradiation with non-radioactive 8-N 3 GTP under identical conditions. The reaction was quenched and the protein precipitated by the addition of 4 ml of cold acetone followed by incubation at 4 °C for 3 hours. The sample was collected by centrifugation for 20 minutes at 10,000 φ . The pellet was resuspended in 2 ml of 2 M urea in 75 M NH 4 HCO 3 and the pH adjusted to 8.5-9.0 by the addition of concentrated NH 4 OH. r eIF-4E was proteolyzed by the addition of modified trypsin (10% w/v), with shaking overnight at 20 C C Immobilized Aluminum(III)-Chelate Chromatography. Iminodiacetic acid-epoxy- activated Sepharose (2 ml) was placed into a 15 ml centrifuge tube and washed as follows 3 x 10 ml distilled water, 4 x 10 ml 50 mM A1C1 3 , 3 x 10 ml distilled water, 3 x 10 ml buffer A (50 mM NH 4 OAC, pH 6 0), 3 x 10 ml buffer B (100 mM NH 4 OAC and 0 5 M NaCl, pH 7.0), 3 x 10 ml buffer A The resin was then transferred to a 10 ml disposable column (Bio-Rad) The labeled r eIF-4E digestion mixture was brought to a total volume of 3 ml with buffer A, the pH was adjusted to 6 0 with concentrated acetic acid, and the sample was loaded onto the column To increase the recovery of the labeled peptide the sample tube was washed with 2 ml buffer A The column was successively eluted with 2 x 10 ml buffer A, 1 x 10 ml buffer B, 1 x 10 ml buffer A, and the radiolabeled peptide was eluted with 6 ml buffer C (10 mM K 2 HPO 4 , pH 8 0) Chromatography was conducted at room temperature with a flow rate of 0 5 ml/min All fractions were assayed for radioactivity on a Wallac Model 1409 Liquid Scintillation Counter (99% counting efficiency for 32 P). Reverse Phase High Performance Liquid Chromatography and Amino Acid Sequencing of Photolabeled Peptide. The radioactive fractions resulting from buffer C elution from the aluminum(III)-chelate chromatography were pooled and concentrated in a Savant SVC100H speed vac concentrator until a volume of 1 5 ml was reached 250 μl samples were then analyzed by reversed-phase HPLC using a microbore C column (Brownlee Lab) and an Applied Biosystems 130A separation system The mobile system consisted of a 25 μM Al yo 1 % TFA solution (X) and a 0.1% TFA 70% acetonitrile (Y) solvent system The gradient was as follows; 100% X at a flow rate of 50 μl/min for 5 minutes, 0- 100%Y at a flow rate of 125 μl/min for 45 minutes, and 100% Y at a flow rate of 125 μl/min for 5 minutes Photolabeled peptides were detected by exact correlation of absorbance at 220 nm and 32 P cpm Fractions containing photolabeled peptides were concentrated to 20 μl and subjected to amino acid sequence analysis at the Hunter College Sequencing and Synthesis Facility using an Applied Biosystems 477A protein sequencer and an Applied Biosystems 120A analyzer with on-line PTH derivative identification. Results Photoaffinity Labeling of→eIF-4E. The specificity of [γ- 32 P]8-N 3 GTP was demonstrated by saturation effects with the probe and by protection studies with m 7 GTP, unmethylated nucleotides, and a capped mRNA. Photoinsertion was saturated at 70 μM with an apparent K d of about 19 μM (Fig. 1A). Increasing concentrations of m GTP resulted in decreased photoinsertion by [γ 32 P]8-N 3 GTP with more than 75% maximal protection observed at 15 μM m 7 GTP with an apparent K d of 7 μM (Fig. IB). The percent maximal protection of 12% is determined by dividing the [γ 32 P]8-N 3 GTP concentration (40 uM) by the total maximum nucleotide plus probe concentration (340 uM). This result provides evidence that photoinsertion of [γ- 32 P]8-N 3 GTP was indeed occurring at the m 7 G cap binding site of eIF-4E. To further study the specificity of photoinsertion of the probe into the m 7 G mRNA cap binding site the effect of other nucleotides and a capped mRNA was studied (Table I). Table 1. Percent of photolabeling of r eIF-4E by 40 μM [γ- 32 P]8N 3 GTP in the presence of various nucleotides and capped mRNA Nucleotide nucleotide concentration % of control none O μM 100 GTP 300 μM 21 GDP 300 μM 26 GMP 300 μM 73 CTP 300 μM 43 ATP 300 μM 35 ADP 300 μM 51 AMP 300 μM 89 UTP 300 μM 58 UDP 300 μM 93 UMP 300 μM 93 m 7 GTP 300 μM 16 capped mRNA" 300 μM 5 "capped mRNA, 34 base long oligoribonucleotide Among the unmethylated nucleotides, GTP (21% of control), GDP (26% of control), and ATP (36% of control) proved to be the best inhibitors to photoinsertion. Although other nucleotides were also able to act as inhibitors they were much less effective. Capped mRNA was able to dramatically reduce photoinsertion (5% of control) as compared to equimolar concentrations of m 7 GTP (18% of control) and unmethylated nucleotides (maximum of 21% of control). Isolation and Identification offγ- 32 P]8-N s GTP Photolabeled Peptides. r eIF-4E was photolabeled as described above to determine the amino acid sequence that was covalently modified by [γ- 32 P]8-N 3 GTP within the m 7 G cap binding site. The second round of photolysis was performed with nonradioactive 8-N 3 GTP to increase the amount of binding domain peptide while reducing nonspecific radiolabeling. TCA was initially used to precipitate photolabeled r eEF- 4E, however this approach resulted in low yields of peptide for sequencing probably due to the disruption of the acid sensitive probe-peptide bond However, acetone precipitation of photolabeled r eIF-4E resulted in higher yields of labeled peptide. Photolabeled r eIF-4E was digested overnight with modified trypsin The labeled peptide was isolated by affinity chromatography and further purified by reversed-phase HPLC, using the methods described above. To locate the photolabeled peptide in the various buffer solutions the flow through fractions (fractions 1-5), washes (fractions 6-25), and phosphate elutions (fractions 26-31) (Fig 2) were separately pooled and subjected to reversed-phase HPLC The flow through and wash fractions contained no radioactive peaks as determined by liquid scintillation counting thus no labeled peptides However, these fractions did contain many peptide fragments, demonstrating that most of the peptides are unmodified and not retained on the Al 3 ' resin The HPLC absorbance profile at 220 nm and the corresponding 32 P cpm profile for the phosphate elution (fractions 16-31) is shown in figure 3. Two major radioactive peaks were observed which also gave a UN absorbance. The first peak at 3 minutes represents the flow through and injection disturbance and contained no peptides as revealed by sequencing data The radioactivity detected with this peak was free photolyzed [γ- 32 P]8-Ν 3 GTP, or probe hydrolyzed during HPLC due to the lability of the N-glycosyl bond to acidic conditions (King, S , Kim, H , & Haley, B., Methods Enzymol. 196:449-466 (1991)) The second major radioactive peak at 23 minutes was concentrated for amino acid sequence analysis. Photolabeling, peptide isolation, and sequencing were repeated in three separate experiments to ensure that only one binding domain was covalently modified by [γ- 32 P]8-N 3 GTP In all experiments the radioactivity coeluted with fraction 23. The sequenced peptide consisted of residues 1 13-122 of human eIF-4E which includes tryptophan 6 (see Table 2). Table 2. Sequence analysis of (γ- 32 P|8N 3 GTP the photolabeled tryptic peptide from the radioactive peak at 23 minutes in the HPLC chromatogram cycle number identified residue picomoles observed 1 W 5.6 2 L 17.5 3 I 14.1 4 T 10.6 5 L 11.7 6 N 7.0 7 K NF" 8 Q 6.4 9 Q 4.9 10 R 2.1 "NF, not found In addition the lysine residue at position 1 19 remained uncleaved by trypsin. This information combined with the lack of identification of lysine 1 19 by sequence analysis indicates that the residue is the likely site of photoinsertion. No minor peptides were detected in the sequencing data. Example 2: Binding of Capped mRNA to Synthetic Peptides To more closely examine the eEF-4E cap-binding domain, synthetic peptides were produced and the binding of m 7 GTP to these peptides were measured. With one peptide (WKRGGRWLITLNKQQR; SEQ ID NO 7), direct fluorescence titration experiments were used to measure the equilibrium binding constant for the formation of the 4E/m 7 GTP complex. Measurements were made in 20 mM HEPES.KOH/lmM DTT, pH 7.6, at 25 °C on a SPEX fluorolog-T2 spectrofluorometer equipped with a high-intensity (450 W) xenon arc lamp. Changes in fluorescence (ΔF) over time were plotted on an Eadie-Hofstee plot In these experiments, an equilibrium binding constant of 8 5 x 107 was calculated from the plotted results Analogous experiments were conducted with a second peptide (WEDEKNKRGGRWLITLNKQQKK, SEQ ID NO: 8), using capped mRNA at a ratio of 1.20 (RNA:peptide). Binding of capped mRNA to the peptide was determined in gel shift assays under various ionic and buffer conditions In a binding solution of 20 mM HEPES, pH 7.6, 1 mM DTT, 50mM NaCl and 5% glycerol, there was a shift of the peptide/mRNA complex towards the negative pole of the gel When binding was conducted in 10 mM potassium phosphate, pH 8.0, 0.25 M KC1, 2 mM EDTA, 5% glycerol, however, an electrophoretic shift was observed towards the positive pole The net charge of the peptide in the presence and absence of phosphate in these experiments was 4 " and 3\ respectively. Discussion The present experiments demonstrate the ability of [γ- 32 P]8-N 3 GTP to serve as a substitute for the m 7 (5')Gppp(5')N cap structure of mRNA in binding to eIF-4E Selectivity of the probe for the active site was determined in saturation studies with [γ- 32 P]8-N-,GTP and in protection studies utilizing m 7 GTP, various nucleotides, and capped mRNA as inhibitors to photoinsertion. Photoinsertion into eIF-4E was saturated with 70 μM [γ- 32 P]8-N 3 GTP and the apparent K d was 19 μM. The observation that photoinsertion of [γ- 32 P]8-N,GTP was inhibited by 90% in the presence of 90 μM m 7 GTP demonstrates the specificity of this binding site. The ability of the purine nucleotides GTP, GDP, and ATP to reduce photoinsertion is not surprising because the cap binding site is a dinucleotide site and the probe used in these studies was a mononucleotide Additional evidence for the site specific insertion of [γ- 32 P]8-N 3 GTP into the cap binding domain is that of capped mRNA (m 7 G(5')'ppp(5')G, 34 nucleotides) as the inhibitor which drastically reduced photoinsertion of the probe to 5% of the control (no inhibitor). The peptide resulting from photoinsertion of [γ- 32 P]8-N 3 GTP and subsequent isolation has been identified as the tryptic peptide consisting of residues 1 13-122 and thus contains tryptophan 6 Detection of the actual site of nitrene insertion is often difficult due to the lability of the N-glycosyl bond to the acidic conditions of HPLC (King, S., Kim, H., & Haley, B., Meth. Enzymol. 196:449-466 (1991)). However these data suggest that lysine 119 is the modified residue for two reasons: the photolabeled binding site peptide is the product of tryptic cleavage at Arg-112 and Arg-122 yet Lys-119 remained uncleaved; and amino acid sequence analysis was unable to identify Lys-119 despite the continuation of sequencing for three residues beyond this position. Such a gap in sequencing usually indicates reaction of the probe with the missing amino acid (Shoemaker, M.T. & Haley, B.E., Biochemistry 32: 1883-1890 (1993)). The amino acid is "missing" because it does not elute in the expected position when the probe has been covalently attached to it. Fluorescence and circular dichroism studies have been performed with Trp==»Lys mutants of tryptophan 6 of yeast eIF-4E which indicate that this residue may be responsible for imparting specificity to cap recognition (McCubbin, W.D., et al., J. Biol. Chem. 265:17663-17671 (1988)). It was found that 7-methylated analogs of GDP and G(5')ppp(5')G bind strongly to the mutant, but unmethylated analogs also have binding affinity. The presence of the phosphate groups results in a stacking interaction with the indole ring regardless of the state of methylation. This has been attributed to electrostatic or hydrogen-bonding between the tryptophan side chain and the highly charged phosphates (Kamiichi, K., et al., J. Chem. Soc. Perkin Trans. II, 1739-1745 (1987)). The substitution of glycine 1 1 1 by aspartic acid in yeast eIF-4E reduced cap binding activity as measured by binding of eEF-4E to m 7 GDP agarose affinity columns (Altmann, M. & Trachsel, H., Nucleic Acids Res. 9: 1643-1656 ( 1989)). This glycine is two residues to the N-terminal side of tryptophan 6. The identification of residues 113-122 as playing a role in m 7 GpppG cap recognition by eIF-4E is consistent with previous studies. The eight tryptophans in yeast eEF-4E have been classified into three separate groups on the basis of the mRNA cross-linking activity of Trp=»Phe mutants: 1) tryptophans that are strongly required for eIF-4E cross-linking to the mRNA cap which include tryptophans 1, 2, 5 and 8; 2) tryptophans that reduce the cross-linking ability of eIF-4E which include 3, 6 and 7; 3) tryptophans that are not required for cap recognition which includes 4. Furthermore, the mutation of tryptophans 1 and 8 completely obliterated cap binding activity (Altmann, M., et al., J. Biol. Chem. 265:17229-17232 (1988)). The large number of residues affecting binding may indicate that the overall folding of eIF-4E plays a crucial role in cap recognition. Identification of amino acid residues that participate in the m 7 G(5')ppp(5)N binding site of eIF-4E will aid in further mutational studies designed to understand the molecular details of eIF-4E*-cap recognition. In addition, further knowledge of the structural motif required for eH E'mRNA cap interactions will aid our understanding of precisely how translational repressor 4E-binding proteins function (Lin, T.A., et al. Science 266, 653-656 (1994); Mader, S , et al, Mol. Cell Biol. 15:4990-4991 ( 1995)). Having now fully described the present invention in some detail by way of illustration and example for puφoses of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Life Technologies, Inc. (B) STREET: 9800 Medical Center Drive (C) CITY: Rockville (D) STATE: Maryland (E) COUNTRY: USA (F) POSTAL CODE (ZIP) : 20850 (ii) TITLE OF INVENTION: Cap-Binding Domain of Human Eukaryotic Protein Synthesis Initiation Factor eIF-4E and the Use Thereof (iii) NUMBER OF SEQUENCES: 27 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US (to be assigned) (B) FILING DATE: 29-AUG-1997 (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/024,881 (B) FILING DATE: 30-AUG-1996 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : Trp Leu lie Thr Leu Asn Lys Gin Gin Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 11..13 (D) OTHER INFORMATION: /note= "residues at positions 11 and 13 may be charged, aromatic or beta- turn- inducing amino acids (such as D, E, Y, T, G or P) ; residue at position 12 may be a hydrophobic amino acid (such as L, I or V) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Xaa Xaa Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "residue at position 1 may be any amino acid" (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 12 (D) OTHER INFORMATION: /note= "residue at position 12 may be any amino acid" (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : Xaa Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "residue at position 1 may be any amino acid" (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 18 (D) OTHER INFORMATION: /note= "residue at position 18 may be any amino acid" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Xaa Trp Lys Arg Gly Gly Arg Trp Leu lie Thr Leu Asn Lys Gin Gin 1 5 10 15 Arg Xaa (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "residue at position 1 may be any amino acid" (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 23 (D) OTHER INFORMATION: /note= "residue at position 23 may be any amino acid" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Xaa Trp Glu Asp Glu Lys Asn Lys Arg Gly Gly Arg Trp Leu lie Thr 1 5 10 15 Leu Asn Lys Gin Gin Arg Xaa 20 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 1..3 (D) OTHER INFORMATION: /note-- "residues at positions 1 and 3 may be charged, aromatic or beta- turn- inducing residues (such as D, E, Y, T, G or P) ; residue at position 2 may be a hydrophobic amino acid (such as L, I, V or W) " (ix) FEATURE: (A) NAME/KEY: Modified- site (B) LOCATION: 14..16 (D) OTHER INFORMATION: /note= "residues at positions 14 and 16 may be charged, aromatic or beta-turn-inducing amino acids (such as D, E, Y, T, G or P) ; residue at position 15 may be a hydrophobic amino acid (such as L, I, V or W) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Xaa Xaa Xaa Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Xaa Xaa Xaa 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : Trp Lys Arg Gly Gly Arg Trp Leu lie Thr Leu Asn Lys Gin Gin Arg 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Trp Glu Asp Glu Lys Asn Lys Arg Gly Gly Arg Trp Leu lie Thr Leu Asn Lys 1 5 10 15 Gin Gin Lys Lys 20 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Gly Asp lie Val Val Leu 1 5 10 15 Val Pro (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Asp Gly Val Val lie lie 1 5 10 15 val Leu Leu Asp Gly 20 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Asp Leu Leu Val lie Val 1 5 10 15 Glu Gly (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Glu Asp Val lie Val Leu 1 5 10 15 Leu lie Tyr (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Pro lie Val Val lie Val 1 5 10 15 Leu Val lie lie Asp Gly 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Asp Thr Val Leu Val Val 1 5 10 15 lie Gly Pro (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Trp Leu He Thr Leu Asn Lys Gin Gin Arg Gly Asp He He Val Val 1 5 10 15 Val He Val Asp Pro 20 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Trp Leu He Thr Leu Asn Lys Gin Gin Arg Gly Val Val Val He He 1 5 10 15 Val Leu Leu Asp 20 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 17 : Trp Leu He Thr Leu Asn Lys Gin Gin Arg Pro Thr Val He Val Val 1 5 10 15 He Asp (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Trp Lys Arg Gly Gly Arg Trp Leu He Thr Leu Asn Lys Gin Gin Arg 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Trp Glu Asp Glu Lys Asn Lys Arg Gly Gly Arg Trp Leu He Thr Leu 1 5 10 15 Asn Lys Gin Gin Lys Lys 20 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: Lys Leu Trp Leu He Thr Leu Asn Lys Gin Gin Arg Val Leu Gin 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: He Gin Leu Val Trp Leu He Thr Leu Asn Lys Gin Gin Arg Leu Lys 1 5 10 15 Ala Gly (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Val Ala Trp Lys Arg Gly Gly Arg Trp Leu He Thr Leu He Thr Leu 1 5 10 15 Asn Lys Gin Gin Arg Gin Ala Lys Thr 20 25 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: He Thr Val Gin Ala Trp Lys Arg Gly Gly Arg Trp Leu He Thr Leu 1 5 10 15 Asn Lys Gin Gin Arg Lys Thr Leu Phe 20 25 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Leu Arg Val Trp Glu Asp Glu Lys Asn Lys Arg Gly Gly Arg Trp Leu 1 5 10 15 He Thr Leu Asn Lys Gin Gin Arg Lys Tyr 20 25 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Asn He Ser Trp Lys Arg Gly Gly Arg Trp Leu He Thr Leu Asn Lys 1 5 10 15 Gin Gin Arg Phe Lys Ala Val Gin Gin 20 25 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE : peptide (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 26 : Glu Tyr Glu Gly Val Val Pro Trp Leu He Thr Leu Asn Lys Gly Glu 1 5 10 15 Arg Asp Leu He Leu Val He Gly 20 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Asp Gly Pro Gly Leu Trp Leu He Thr Leu Asn Lys Gin Gin Arg Tyr 1 5 10 15 Thr Trp Val Val He Leu Val Thr Gly 20 25

Claims: WHAT IS CLAIMED IS 1 A truncated peptide of the cap-binding protein consisting essentially of the mRNA cap-binding domain 2 The peptide of claim 1, wherein said cap-binding protein is eIF-4E 3 The peptide of claim 1, wherein said peptide has an amino acid sequence corresponding to SEQ ID NO 1 4 The peptide of claim 3, wherein said peptide contains one or more substitutions, deletions and/or deletions and wherein said peptide maintains mRNA cap binding function 5 The peptide of claim 1, wherein said peptide has an amino acid sequence corresponding to a general pattern selected from the group consisting of Trp-Leu-Ile-Thr-Leu- Asn-Lys-Gln-Gln-Arg-Xl (1 . 2) -X2 (4 . 10) -Xl (1.2) (SEQ ID NO 2), and X l (0 . 10) -X2 (0 . 2) -Xl (0 . 10) -Trp-Leu- Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-Xl (] . 2) -X2 (4 . )) -X 1 (1 . H)) (SEQ ID NO 6), wherein XI is a charged, aromatic or β-turn-inducing amino acid, X2 is a hydrophobic amino acid, and wherein the subscripts for XI and X2 indicate the number of such residues in consecutive sequence at the indicated position 6 The peptide of claim 1, wherein said peptide has an amino acid sequence corresponding to the group consisting of (a) X^-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-X,, (SEQ ID NO 3), (b) X n -T -Lys-Arg-Gly-Gly-Arg-T -Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg- X„ (SEQ ID NO 4); and (c) X n -Trp-Glu-Asp-Glu-Lys-Asn-Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr- Leu-Asn-Lys-Gln-Gln-Arg-X,, (SEQ ID NO:5), wherein X is any amino acid, and n is a whole number ranging in value from 0 to 70 7 The peptide of claim 5, wherein XI is Asp, Glu, Tyr, Thr, Gly or Pro 8 The peptide of claim 5, wherein X2 is Leu, He, Val or Trp. 9 The peptide of claim 6, wherein n is a whole number ranging in value from 0 to 25 10 The peptide of claim 6, wherein n is a whole number ranging in value from 0 to 10 1 1 The peptide of claim 1, wherein said peptide has an amino acid sequence corresponding to SEQ ID NO 7 12 The peptide of claim 1, wherein said peptide has an amino acid sequence corresponding to SEQ ID NO 8 13 The peptide of any one of claims 1-6, 1 1 or 12, wherein said peptide is immobilized on a solid support 14 The peptide of claim 13, wherein said peptide is immobilized by an amino acid linkage group or a chemical linkage 15 The peptide of claim 14, wherein one end of the peptide is immobilized to the solid support 16 The peptide of claim 13, wherein said solid support is selected from the group consisting of nitrocellulose, diazocellulose, glass, latex, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, silica gel, a bead and a microtitre plate 17. The peptide of claim 13 wherein said solid support is a glass bead, a latex bead or a magnetic bead. 18. A resin comprising the peptide of claim 1 covalently or non-covalently attached to a solid support matrix. 19. The resin according to claim 18, wherein said attachment is accomplished by an amino acid linking group or a chemical linking group. 20. The resin of according to claim 18, wherein one end of the peptide is covalently or non-covalently attached to the resin. 21. The resin of claim 18, which is selected from the group consisting of nitrocellulose, diazocellulose, glass, latex, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, silica gel and a bead. 22. The resin of claim 18, which is a glass bead, a latex bead or a magnetic bead. 23. A method for the isolation of a capped mRNA molecule, said method comprising the steps of (a) contacting a solution containing said capped mRNA molecule with the peptide of any one of claims 1-6, 11 or 12 under conditions favoring the noncovalent binding of said capped mRNA molecule to said peptide; and (b) removing materials not bound to said peptide. 24. The method of claim 23, further comprising an elution step (c), wherein said capped mRNA molecule is eluted from said peptide using an elution buffer comprising a competitive analog of the cap region of a mRNA molecule, a changed the pH, or changed ionic strength. 25 The method of claim 23, wherein said peptide is immobilized on a solid support 26 The method of claim 25, wherein said solid support is selected from the group consisting of nitrocellulose, diazocellulose, glass, latex, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, silica gel, a bead and a microtitre plate 27 The method of claim 26, wherein said bead is a glass bead, a latex bead or a magnetic bead 28 A method for the production of a full-length cDNA molecule, said method comprising the steps of (a) contacting a solution containing a capped mRNA molecule with a protein having reverse transcriptase activity under conditions favoring the production of a plurality of cDNA molecules from said mRNA and the formation of a mixture of mRNA cDNA hybrids, (b) contacting said mixture of mRNA cDNA hybrids with a single-stranded RNA-specific nuclease under conditions favoring the digestion of RNA not hybridized to cDNA, (c) contacting said mixture ofmRNA cDNA hybrids with an endonuclease that specifically attacks the 3' adjacent phosphodiester-bound GpN, (d) contacting said mixture of mRNA cDNA hybrids with the peptide of any one of claims 1-6, 1 1 or 12, under conditions favoring the noncovalent binding to said peptide of said mRNA cDNA hybrids, (e) removing materials not bound to said peptide, and (f) eluting said bound mRNA.cDNA hybrids from said peptide 29 The method of claim 28, wherein said protein having reverse transcriptase activity is a truncated or mutated reverse transcriptase enzyme which lacks RNAse H activity 30 The method of claim 28, wherein said protein having reverse transcriptase activity is a DNA polymerase having reverse transcriptase activity 31. The method of claim 30, wherein said DNA polymerase is a thermostable DNA polymerase. 32. The method of claim 31, wherein said DNA polymerase is selected from the group consisting of Tne, Tth, and Taq or a mutant thereof. 33. The method of claim 28, wherein said single-stranded RNA-specific nuclease is T j nuclease. 34. The method of claim 28, wherein said elution step (f) is accomplished using a competitive analog of the cap region of a mRNA molecule, changing the pH, or the ionic strength of the solution 35. The method of claim 28, wherein said peptide is immobilized on a solid support. 36. A kit for the isolation of a capped mRNA molecule, said kit comprising one or more containers, wherein a first container contains the peptide of any one of claims 1-6, 1 1 or 12, and wherein a second container contains an elution buffer for recovering the capped mRNA from said peptide. 37. A kit for the production of a full-length cDNA molecule, said kit comprising one or more containers, wherein a first container contains the peptide of any one of claims 1-6, 1 1 or 12, a second container contains a protein having reverse transcriptase activity, and a third container contains a nuclease enzyme. 38. The kit of claim 36, wherein said peptide is immobilized on a solid support. 39. The kit of claim 37, wherein said peptide is immobilized on a solid support. 40 The kit of claim 37, wherein said protein having reverse transcriptase activity is a truncated or mutated reverse transcriptase enzyme lacking RNAse H activity. 41. The kit of claim 37, wherein said protein having reverse transcriptase activity is a DNA polymerase having reverse transcriptase activity. 42. The kit of claim 37, wherein said nuclease enzyme is T, nuclease.

Inventors: FRIEDLAND DIANA, GOSS DIXIE J, FRIEDLAND, DIANA, GOSS, DIXIE, J.
Applicants: FRIEDLAND DIANA, GOSS DIXIE J, LIFE TECHNOLOGIES INC, FRIEDLAND, DIANA, GOSS, DIXIE, J., LIFE TECHNOLOGIES, INC.
Application year: 1997
Main classification: C07H21
IPC codes: C07K14