ISOLATED RNA MOLECULES WHICH BIND TO ARGININE AND USES THEREOF

Abstract: The invention relates to isolated RNA molecules with high affinity for particular amino acids, processes for isolating these, and uses thereof. Via a process which includes various washing and heating steps, one isolates these RNA molecules.

Description: ISOLATED RNA MOLECULES WHICH BIND TO ARGININE AND USES THEREOF FIELD OF THE INVENTION This invention relates to isolated RNA molecules with high or strong affinity for amino acids, as well as uses thereof. Also, a part of the invention are processes lor securing these RNA molecules. BACKGROUND AND PRIOR ART Interactions between amino acids and RNA play substantial roles in a number of biological systems See, e.g., Draper, Ann. Rev. Biochem 64: 593-620 (1995). For example, arginine inhibits the self- splicing reaction of the grou jinrron of Tetrahymena by substituting for two H-donor sites ot the guanosine cofactor which contact the G 2M -C m base pair in the ribozvme ' s guanosine binding site. Yarus, Science 240:1751-1758 (1988); Michel, et al. Nature 342:391-395 (1989). Recently, the editing reactions of aminoacyl tRNA synthetases have been viewed as an example of RNA dependent amino acid recognition. Schimmel, et al., Trends Biochem. Sci 20:1-2 (1995). These editing reactions involve RNA dependent steps which eliminate errors of amino acid activation and aminoacvlation. Jakubowski, et al., Microbiol. Rev 56:412-429 (1992). A third example is the interaction of the HΓV-1 TAT protein with a stem loop structure of TAR RNA, located at the 5' -end of HIV-1 mRNA. Critical for the recognition of TAT and TAR is a single arginine within a basic region of TAT. Calnan, et al., Science 252:1167-1171 ( 1991). Short oligopeptides resembling the basic region as well as tree arginine bind specifically to TAR, although weaker than within the context of the whole protein Weeks, et al., Science 249:1281-1285 ( 1990). The TAT-TAR interaction provided the first example to show that in protein-RNA recognition RNA structures are involved which interact with individual amino acid side chains in the protein. It seems likelv that other, yet undiscovered RNA-protein interactions exist in which single amino acid side chains within a protein or peptide torm specific contacts to structural elements provided by RNA to largely determine specificity, functionality, and strength of binding Draper, supra. The isolation and characterization of RNA sequences which specifically recognize individual amino acids might facilitate a better understanding of biologically relevant protein-RNA or RNA-amino acid interactions. A powerful tool to obtain amino acid binding RNAs is in vitro selection as taught by, e.g. Famulok, et al., Agnew. Chem. Int. Ed. Engl. 31:979-988 (1992). Klug, et al., Mol. Biol. Rep 20:97-107 (1994); Gold, et al. Ann. Rev. Biochem. 64:763-797 (1995); Joyce, Curr. Opin. Struct. Biol 4:331-336 (1994), all of which are incorporated by reference. RNA aptamers which specifically recognize amino acids, such as ύrrimobilized tryptophan, arginine, cirxulline, and valine have been extracted from pools of up to 10 15 different RNA sequences. See Connell, et al., Biochemistry 32:5497-5502 (1993); Famulok, J. Am. Chem. Soc. 116:1698-1706 (1994); Majesfeld, et al., Natur. Struct. Biol. 1:287-292 (1994), all of which are incorporated by reference. The reported affinities ranged from 60μM (Famulok) to 12mM (Majesfeld) with a high level of discriinination against other amino acids being obtained in each case. Among these amino acid binding RNAs, the arginine specific aptamers might be especially relevant to protein-RNA recognition because arginine side chains carrying a positive charge at neutral pH seem to be particularly suited to form specific contacts with a negatively charged nucleic acid. See Aboul-ela et al., J. Mol. Biol 253:313-332 (1995). For example, the HIV-1 Rev protein contains a basic region in which ten of seventeen amino acids between positions 34-50 are arginines. A corresponding 17-mer peptide binds to the Rev responsive element (RRE) RNA IIB hairpin in the same way as within the context of the lull-length protein with four arginines being important for specificity. See, respectively, Kjems, et al., EMBO J 11:1119-1129 (1992); Tan, et al. CeU 73:1031-1040 (1993). In the HIV- TAR/ Tat complex an -irginine and an isoleucine residue were found to be critical for binding and specificity. Frankel, in Nagai, et al., ed, RNA-Protein Interactions IRL Press, New York, NY 1994) pp. 221-247. Furthermore, the Rex-protein of HTLV-I might interact with its natural RNA binding element XBE through arginine residues. Baskerville et al., J. Virol 69:7559-7569 ( 1995). Further work has now been carried out, resulting in the development of techniques which permit, and have permitted, the isolation of RNA molecules which have strong or high affinities for particular amino acid molecules. The present invention is a development of the work described by Famulok, J. Am. Chem. Soc. 116:1698-1706 (1994), incorporated by reference. Via modifications in the protocols described therein, it has been possible to isolate RNA molecules of extremely high affinity, as is shown via the examples which follow. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a representative elution protocol obtained in accordance with the invention. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1 Two pools of RNA were prepared for screening for high affinity binding to amino acids. The first pool ("pool 1" hereafter) was prepared according to Famulok, J. Am Chem. Soc. 116(5) :1698-1706 (1994), incorporated by reference. The Famulok paper teaches the production of a pool of about 10 l3 RNA molecules which were 111 nucleotides in length. These lllmers were prepared using defined 5' and 3' sequences, together with a randomized 74mer insert. -D- Famulok teaches, inter alia, a particular RNA molecule ("clone 16"), which has high L-citrulline affinity. This clone was used as the basis for preparing a second, degenerate pool. This pool was prepared, also following Famulok except that in preparing the 74mer insert, the reservoirs of phospharamidite were each doped with 10% of each of the other phospharamidites, to provide reservoirs of, e.g., 70%A, and 10% of each of G, C and T, and so forth. (The 74mer insert is placed in between an 18-mer and a 19-mer primer binding site.) The entirety of pool 2 was then subjected to PCR amplification. The PCR amplification was carrier out as described by Famulok, supra, resin the primers described therein, i.e., TCTAATACGA CTC ACTA TAG GAG CTC AGC CTT CAC TGC (SEQ ID NO.:l) and GTGGATCCGACCGTG GTGCC (SEQ ID NO: 2), respectively, as the 5' and 3' primers. Six cycles of amplification were carried out (94 °C 4min; 42 °C, 7min; 72 °C 7min), and then the amplification product was extracted with phenol/ CHCL y precipitated with ethanol, dissolved in 1.3 ml of TE (lOmM Tris-HCl, pH 7.6; ImM EDTA. pH 80), followed by gel filtration on Sephadex G-50. RNA was transcribed from the PCR product and used in the s election protocol s wh ich fo l l ow . EXAMPLE 2 The RNA produced in Example 1, supra, was selected for high affinitv amino acid binding. Prior to this, however, it was treated to purifv it. This procedure is now set forth. Samples ot the RNA containing solutions of example 1 (10-15 μg, or 0.4nmoles of RNA), were diluted with water to final volumes of 80 μl. Then, the solutions were heated to 90 °C for 10 minutes, followed by cooling to 23 °C for 5 additional minutes, after which they were added to a preselection column. The preselection column consisted of 0.5 ml of agarose which had derivatized glycine bound thereto (following manufacturer's instructions), and which had been equilibrated with several column volumes of binding buffer prior to the addition of the samples. The RNA containing samples were added to the preselection column, and then washed with 2-3 volumes of buffer. The effect of this wash is to start a flow-through from the preselection column, which was placed directly on top of the selection column (discussed infra). Positioned direct lv below the preselection column was the selection column, which was agarose with derivatized arginine bound thereto, the arginine having been derivatized and attached to the agarose, in accordance with the manufacturer's instructions. The selection column had been rinsed, previously, with binding buffer. Once the RNA had flowed onto the selection column, a selection scheme was employed to find onlv tightly binding aptamers. First, the column was washed with five volumes of binding buffer. Then, counter selection was carrier out, by washing the column with six column volumes of bmding buffer which contained 20mM of citruJLline. Next, in order to increase stringency of competition between free citrulline and bound arginine. the agarose, and hence the RNA bound to it, was heated to 90°C tor 10 minutes then cooled to 23°C for 15 minutes in the presence of the 20mM citrulline containing solution. The column was then eluted with another 5 column volumes of binding buffer containing 20mM citrulline so as to remove any non- bound RNA. The column was then washed with 15 column volumes of binding buffer plus 20mM arginine, and was then heat denatured, in the presence of this solution, using the parameters set forth supra. This was done because it was assumed that molecules which have a slow dissociation rate and hence elute slowly are tight binders when heat denatured, these tight binding RNA molecules should separate from the column and renarure in the presence of excess ligand in solution, thereby avoiding undesired enrichment of those molecules which require functional groups in the matrix plus the ligand for binding. The column was then eluted with 5 column volumes of binding buffer plus 20mM arginine. Eluted RNA was collected by (a) removing amino acids with phenol (b) precipitating the RNA, and then (c) amplifying the RNA. This cycle was repeated, twenty times, and was carried out on both pool 1 and pool 2, as described in Example 1. A representative elution profile from cycle 20, is shown in Figure 1. EXAMPLE 3 The RNA aptamers which eluted after cycle 10 and cycle 20 were subjected to PCR amplification in accordance with Famulok. supra. and then sequenced. Of 36 clones sequenced following cycle 10, 35% showed the arginine binding motif taught by Famulok, supra and 65% did not. When materials obtained after twenty cycles were sequenced, however, none of those in cycle 10 were found, indicating that the prior art sequences do not have the same level of affinity for their partner amino acid as do those left after 20 cycles. The sequences found after 20 cycles are presented in SEQ ID NOS: X through Y. Of the 31 clones found, one sequence appeared 8 times, four sequences each appeared twice, and eleven sequences, once only. When the sequences found in pool 1 and pool 2 were compared, only one, Le., Ag. 06 (SEQ ID NO: 3 ) was found in both pools. This molecule was also the most prevalent one. EXAMPLE 4 Experiments were then carried out to determine the strength of binding of the isolated aptamers. This is determined by calculating the dissociation constant, using the well known methods of e.g., Lorsch, et al. Biochemistry 33:973-982 (1994); Fersht, Enzyme Structure and Mechanism, 2 nd Ed. (W.H. Freeman and Co., New York, 1985), p 186; Connors, Binding Constants (Wiley, New York 1987), p. 314, incorporated by reference. This is an equilibrium dialysis method using dialvsis chambers of 500 ul volume. The membrane used had a molecular weight cutoff of 5000 daltons. A sample of RNA, typically at a concentration of l.O^M was used. Measurements were generally taken at three different concentrations of L-[2, 3, 4, 5- 3 H] - arginine - HQ (Le., 250, 25 and 2.5 lvl). Sample volumes of 200 μl were used on each side of the membrane. The samples were equilibrated at 23 °C overnight, after which the volume on both sides of the membrane was determined and adjusted to ensure the amounts were the same. Aliquots were withdrawn, and subjected to scintillation counting. Three RNA molecules were used, i.e., Ag. 06 (SEQ ID NO: ), as well as two other molecules representing point mutations , SEQ ID NOS 4 + 5. Using the specific activity of arginine, concentrations of bound ligand [ESJeg, free amino acid [S]eq, and free RNA [E]eg at equiUbrium were calculated. Kd is defined by the equation: Kd = ([E]eq X[S]eq) x [ESJeq 1 The value for Ag. 06 was about 330nM, i.e., nearly 200 times greater than the tightest binders ot Famulok supra. Note that a strong affinity is represented by a low numerical value, and a weaker affinity by a higher numerical value. The specific values ranged from 4mM to 330nM, and are on a par with affinity values for other, non-amino acid substances, such as theophylline (320nM) cyanco balamine (88nM), and the aminoglycoside antibiotics tobramycin, kanamycin, lividomycin, and neomycin (20nM - 220nM). See Jenison et al., Science 263:1425- 1429 (1996) Lorch , et al., Biochemistry 33:973-982 (1994); Wang et al., Chem & Biol 2:281-290 (1995); Lato, et al., Chem & Biol 2;291-303 (1995); Walks, et al., Chem & Biol 2:543-552 (1995). EXAMPLE 5 Experiments were carried out to determine if the RNA molecules could distinguish between L-arginine and D-arginine. Using the analvtical affinity chroma tography method of Arnold, et al. J. Chromatog 355:1-2 ( 1986), and when applied to a 4.0mM D-arginine agarose column, the RNA eluted in the void volume. This leads to the conclusion that the Kd is 4mM, i.e., the affinity was very weak. Thus, the mole ules such as Ag. 06 (SEQ ID NO: 3), show great discrirnination. In this particular case, it is on the order of 12,000 fold better. The foregoing examples set forth particular embodiments of the invention which includes, inter alia, isolated ribonucleic acid (RNA) molecules which have high affinity for a particular amino acid. Especially preferred are isolated ribonucleic acid molecules having affinities of around 300nM, more preferably greater than about 60μM. These RNA molecules can have the strong affinity for any L- or D- amino acid. Of particular interest are RNA molecules with high affinity for L-arginine. and which may permit discrimination between L- and D-arginine, and /or L-citrulline and L-arginine. Exemplary of such RNA molecules are those set forth in SEQ ID NOS: 3 through 20 , inclusive. These RNA molecules have various uses, which will be clear to the skilled artisan. For example, the RNA molecules may be used to remove particular amino acids from a solution, or determine the presence or amount ol a particular amino acid, by exploiting this high affinitv to form complexes of RNA and amino acid which can be removed from a solution, followed by identification and/ or quantization of the amino acid via elution, or other standard analytical techniques. Enantiomers of amino acids may be identified and/ or separated, such as L and D arginine; different, structurally related amino acids, such as L-arginine and L-άtrulline may be separated, and so forth. Of particular interest as a feature of the invention is a process for isolating an RNA molecule with high affinity for an amino acid. The essential steps of this process involve the denaturing of an RNA molecule or sample, followed by binding the denatured RNA to a solid phase, using art recognized techniques which need not be repeated here. Next, the bound RNA is contacted with a solution of an amino acid which is not the amino acid for which a high affinity binder is sought. Then, any RNA which remains bound to a solid phase is heated, followed by contact, a second time, with the amino acid solution referred to supra. These three steps can be repeated for any number of cycles, using the same, or different amino acids. For example, were one looking tor high affinity L-arginine binders, one might contact the solid phase bound RNA with D-arginine, heat it, and contact it a second time with D-arginine, followed by a repeat of these steps using L-citrulline. Preferably, one carries out the cycling for anywhere from 5 to 20 cycles, most preferably, 10-20 cycles. Following these steps, any remaining RNA is heated and then contacted with a solution ot the amino acid for which a strong or high affinity binder is sought. This serves to elute the desired RNA from the solid phase. In these processes, it is preferred that the RNA be bound to the solid phase by amino acid molecules for which strong affinity binders are sought. A strong or high affinity binder, as was noted, supra, is an RNA molecule which has a low Kd value. For the invention as described herein, strong affinitv (or high affinity) binders are those with a Kd value of 60μm or less. More preferably, these molecules have a Kd value that is an order of magnitude lower, i.e., about 6.0μm or less. More preferably, this value is about 500 nM or less, and most preferably, about 300 nM or less. Other features ot the invention will be clear to the skilled artisan, and need not be elaborated upon herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention. UAAACCGAUGCUGGGCGAUUCUCCUGAAGUAGGGGAAGAGUUGUCAUGUAUGGG ag 04 r- AUGAUAAACCGAUGCUGGGCGAUUCUCCUGAAGCAGGGGAAGAGUUGUCAUGUAUGGG ag.16 > AUGAUAAACCGAUGCUGGGCGAUUCCCCUGAAGUAGGGGAAGAGUUGUCAUGUAUGGG ag.02* 0 CCAGCCUAUGGACGUGCCUUUACGGCUGUCCGGAAAAGGUGGUGCCGUCCGGCAUGGC — ag 10* / CCAGCCUAUGGACGUGCCUUUACGGCUGUCCGGAAAAGGUAGUGCCGUCCGGCAUGGC a Ol* 23 " GCGGCGGGUGUACUUCGCAAGAGGCCCGUGCGGUGGGAGUGGUAGCCUUGGAAACCAUAGA agll* GGCGGGUGUACUUCGCAAGAGGCCCGUGCGGUGGGAGUGGUAGCCUUGGAAACCAUAGA . ag 13 AGAUCAGGCACGCCGGGUACGAAAUAGCACCAAUGUCAACAUGACUUUUAAAGAGC M ae 17 AGAUCAGGCACGCCGGGUACGAAAUAGCACCAGUGUCAACAUGACUUUUAAAGAGC ag 03* 1 2 ACAGCCAACGGUUCGGUCACGGGACUGCUCUUCAUCGUAGGGAGUGGUGGCCUUGGUACGU GCGGA ag.12 1 3 AAUCUCGAACUCUGCGGUAGUCAAUACCGGCUUUCAGAGAACGGUCGAGGUCGCUUGUCGG UCUGC ag.05 CGCUGAGAGAGAGUCCUCUGUUUAGGAAGUGUAGCUUAACUUGCGACAGGCAGGCUUCA ag.07 I S " ACAGCAACGCGUGUAAGUCGAUAAACUUCUCUUUAGCAGAAGUGGUAAGCUGUGUGACGU AAGGC I <p ag 08 AGCUGCGGUGUGGCGGAAGGAGUGUAGCAUAAGUUAGAGUGAGCCUUCUGCCUGUG _ ag.09 ' / ACGGACUACCAGGUAGGUCGCUGGACGAAUCGGAAGGAGGGUAACCUUGCGGUAGGACUGU G I $f ag.14 GGAUCAGGGACGGCGGGUAGAAAUAGCACCAAUGUUCAAUCAGUGGGCCCAAAGAGC I ύ ag.15 1 A ' CAGCAACGCGUGUAAGUCGAUAAACUUCUCUCUAGCAGAAGUGGUAGCUGUGCGACGUGA 2^0 AG 18* AAUGCUAACCGCAUUGGCCGCGGGACUUCUCGUCUGACCAGUUAGCAUGGCGUGGU ag.19 AUAGCGCAAAACGGUUCGGCUACGGGCUGCCCGGCGAAGGGUGGUAGCCUUGCUGAUGUUU GGAC 2.2- ag.20 ACGCGUGGUCCUCCUCUCCAUGACUCUCUGGACGGCGUGUAGUCUUGAAACGUCUGAU ag-21 2.? ACACCGAUGCGGGCGAUGGACAUCUCUUCCCUAAUGACUGGAAGGGAUGGUAAGGUGGCUG UGUGGC 7 <■ ag.24 AACAGGUAGGUCGCUGCACUGUCCUUUGGGACUGUCCGGAAGGAGCGUUGUCAGGUAUCGC

Claims: WHAT IS CLAIMED IS: 1. Isolated RNA molecule which has a high affinity for a spec i fic amino acid said affinity being less than about 6.0μm. 2. The isolated RNA molecule of claim 1, having an affinity less than about 500 nM. 3. The isolated RNA molecule of claim 1, having an affinity less than about 300 nM. 4. The isolated RNA molecule of claim 1, wherein said amino acid is arginine. 5. The isolated RNA molecule of claim 1, wherein said amino acid is L-arginine. 6. The isolated RNA molecule of claim 1, having the nucleotide sequence of Ag.06 (SEQ ID NO: 3). 7. Method for removing an amino acid from a solution comprising contacting said solution with the isolated RNA molecule of claim 1, bound to a solid phase, wherein said isolated RNA molecule has affinity for said arruno acid, and removing complexes of said amino acid and said RNA bound to a solid phase from said sample. 8. Method for separating enantiomers of amino acids from each other, comprising contacting a sample containing said enantiomers with a complex of a solid carrier and the isolated RNA molecule of claim 1, wherein said isolated RNA molecule has affinity for one of said enantiomers, and removing complexes of said enantimoer and sohd carrier bound RNA molecule from said sample. 9. The method of claim 8, wherein said enantiomers are L and D arg^Lnine, and said isolated RNA molecule has affinity for L-arginine. 7/33 10. Method for separating a first amino acid from a sample containing at least one additional amino acid, comprising contacting a sample containing at least two amino acids with the isolated RNA molecule of claim 1, bound to a solid carrier, wherein said RNA molecule has affinity tor said first amino acid, and removing complexes of said first amino acid and said complexes of solid phase carrier and said RNA molecule from said sample. 11. The method of claim 10, wherein said first amino acid is arginine and said additional amino acid is citrulline. 12. A process for purifying an RNA molecule which has affinity of at least about 100 nM for a first amino acid, comprising, in sequence: (a) denaturing an RNA sample; (b) binding said RNA sample to a solid phase; (c) contacting said RNA bound to a solid phase with a solution of a second am o acid which differs from said first arnino acid to elute RNA with low affinity for said second acid; (d) heating said RNA bound to a solid phase; (e) contacting said RNA bound to a solid phase with said solution of a second amino acid for a second time, to elute RNA with high affinity binding for said second amino acid; (f) contacting remaining solid phase bound RNA with a solution of said first amino acid, to elute RNA which has low affinity for said first amino acid; (g) heating any remaining, solid phase bound RNA; and (h) contacting said remaining solid phase bound RNA for a second time with a solution of said first amino acid, to elute RNA with high affinity for said amino acid. 13. The process of claim 12, comprising bmding said RNA sample to a sohd phase having said first amino acid immobilized thereon. 14. The method of claim 12, further comprising isolating said RNA from eluate formed in step (h). 15. The process of claims 12 - 14, wherein steps (d) and (e) are repeated at least once. 16. The process of claims 12 - 15 , further comprising contacting said solid phase bound RNA with a solution of at least one additional amino acid at least once, to elute RNA having affinity for said amino acids. 17. Isolated RNA molecule of claim 1, comprising a nucleotide sequence as set forth in Seq. ID. No: 3-20.

Inventors: FAMULOK MICHAEL, GEIGER ALBERT, FAMULOK, MICHAEL, GEIGER, ALBERT
Applicants: BOEHRINGER MANNHEIM GMBH, FAMULOK MICHAEL, GEIGER ALBERT, FAMULOK, MICHAEL, GEIGER, ALBERT
Application year: 1997
Main classification: C07H1
IPC codes: C07B57, C07C227, C07C273, C07C275, C07C277, C07C279, C07H21, C12N15, C12Q1, G01N33