[0089] In selected embodiments of the invention, the transcription products generated from the transcription conditions in Table 2 can then be used for input into the SELEX^™ process in order to identify aptamers and/or to determine a conserved sequence that has binding specificity to a given target. The resulting sequences are already stabilized, thus eliminating the need for post-SELEX^™ modification and giving a more highly stabilized aptamer. [0090] The mutant T3 polymerases of the invention are also tested for their ability to promote a variety of other transcription yields by using transcription mixtures, such as, for example, a dCmD, dTmV, rTmV, rUmV or dUmV reaction mixture. Aptamers produced using these transcription mixtures are referred to as dCmD, dTmV, rTmV, rUmV or dUmV aptamers. The ability of the mutant T3 polymerases of the invention to promote the production of any of these aptamers is tested by varying the components used within the transcription mixture and/or by varying the concentration of components with the transcription mixture. For example, the mutant T3 polymerase is used in conjunction with one or more of the following components: modified and/or unmodified nucleotide triphosphates, a nucleic acid transcription template, T- OH guanosine, magnesium ions, manganese ions, a non 2'-OMe guanosine non-triphosphate residue, polyalkylene glycol (e.g., polyethylene glycol), inorganic pyrophosphatase, guanosine monophosphate, guanosine diphosphate, 2'-fluoro guanosine monophosphate, 2'-fluoro guanosine diphosphate, 2 '-amino guanosine monophosphate, 2 '-amino guanosine diphosphate, 2'-deoxy guanosine monophosphate, 2'-deoxy guanosine diphosphate, buffer, detergent (e.g., Triton X-IOO), polyamine (e.g., spermine or spermidine), reducing agent (e.g., DTT or βME), or any combination thereof. The concentrations of these components are varied, for example, using the concentrations described below.

[0091] As described below, useful yields of transcripts fully incorporating T- substituted nucleotides can be obtained under conditions other than the conditions described in Table 2 above. For example, variations to the above transcription conditions may include the following.

[0092] In some embodiments, it is contemplated that the HEPES buffer concentration can range from 0 to 1 M. The invention also contemplates the use of other buffering agents having a pKa between 5 and 10 including, for example, Tris-hydroxymethyl-aminomethane. [0093] In some embodiments, it is contemplated that the DTT concentration can range from 0 to 400 mM. It is also contemplated that other reducing agents could be used, including, for example, mercaptoethanol.

[0094] In some embodiments, it is contemplated that the spermidine and/or spermine concentration can range from 0 to 20 mM.

[0095] In some embodiments, it is contemplated that the PEG-8000 concentration can range from 0 to 50% (w/v). It is also contemplated that other hydrophilic polymers including, for example, other molecular weight PEGs or other polyalkylene glycols could be used.

[0096] In some embodiments, it is contemplated that the Triton X-100 concentration can range from 0 to 0.1% (w/v). It is also contemplated that other non-ionic detergents including, for example, other detergents, such as other Triton-X detergents could be used.

[0097] In some embodiments, it is contemplated that the MgCl₂ concentration can range from 0.5 mM to 50 mM. In some embodiments, it is contemplated that the MnCl₂ concentration can range from 0.15 mM to 15 mM.

[0098] In some embodiments, it is contemplated that the 2'-OMe NTP concentration

(each NTP) can range from 5 μM to 5 mM.

[0099] In some embodiments, it is contemplated that the 2'-OH GTP concentration can range from 0 μM to 300 μM.

[00100] In some embodiments, it is contemplated that the concentration of 2'-OH GMP, guanosine or other 2'-OH G substituted at a position other than the 2 '-sugar position can range from 0 to 5 mM, where 2'-OH GTP is not included in the reaction, 2'-OH GMP is required and may range from 5μM to 5 mM.

[00101] In some embodiments, it is contemplated that the DNA template concentration can range from 5 nM to 5 μM.

[00102] In some embodiments, it is contemplated that the mutant polymerase concentration can range from 2nM to 20 μM.

[00103] In some embodiments, it is contemplated that the inorganic pyrophosphatase can range from 0 to 100 units/ml. [00104] In some embodiments, it is contemplated that the pH can range from pH 6 to pH

9. The methods of the invention can be practiced within the pH range of activity of most polymerases that incorporate modified nucleotides.

[00105] In some embodiments, it is contemplated that the transcription reaction may be allowed to occur from one hour to weeks, preferably from 1 to 24 hours.

[00106] In some embodiments, the optional use of other chelating agents in the transcription reaction mixture are contemplated, for example, EDTA, EGTA and DTT.

Aptamer Medicinal Chemistry

[00107] Once aptamers that bind to a desired target are identified, several techniques may be optionally performed in order to further increase binding and/or functional characteristics of the identified aptamer sequences.

[00108] Aptamers that bind to a desired target may be truncated in order to obtain the minimal aptamer sequence (also referred to herein as "minimized construct" or "minimized aptamer") having the desired binding and/or functional characteristics. One method of accomplishing this is by using folding programs and sequence analysis, e.g., aligning clone sequences resulting from a selection to look for conserved motifs and/or covariation to inform the design of minimized constructs. Biochemical probing experiments can also be performed to determine the 5' and 3' boundaries of an aptamer sequence to inform the design of minimized constructs. Minimized constructs can then be chemically synthesized and tested for binding and functional characteristics as compared to the non-minimized sequence from which they were derived. Variants of an aptamer sequence containing a series of 5', 3' and/or internal deletions may also be directly chemically synthesized and tested for binding and/or functional characteristics as compared to the non-minimized aptamer sequence from which they were derived.

[00109] Additionally, doped reselections may be used to explore the sequence requirements within a single active aptamer sequence or a single minimized aptamer sequence. Doped reselections are performed using a synthetic, degenerate pool that has been designed based on the single sequence of interest. The level of degeneracy usually varies 70% to 85% from the wild type nucleotide, i.e., the single sequence of interest. In general, sequences with neutral mutations are identified through the doped reselection process, but in some cases sequence changes can result in improvements in affinity. The composite sequence information from clones identified using doped reselections can then be used to identify the minimal binding motif and aid in Medicinal Chemistry efforts.

[00110] Aptamer sequences and/or minimized aptamer sequences may also be modified post-SELEX^™ selection using Aptamer Medicinal Chemistry to perform random or directed mutagenesis of the sequence in order to increase binding affinity and/or functional characteristics, or alternatively to determine which positions in the sequence are essential for binding activity and/or functional characteristics.

[00111] Aptamer Medicinal Chemistry is an aptamer improvement technique in which sets of variant aptamers are chemically synthesized. These sets of variants typically differ from the parent aptamer by the introduction of a single substituent, and differ from each other by the location of this substituent. These variants are then compared to each other and to the parent. Improvements in characteristics may be profound enough that the inclusion of a single substituent may be all that is necessary to achieve a particular therapeutic criterion. [00112] Alternatively, the information gleaned from the set of single variants may be used to design further sets of variants in which more than one substituent is introduced simultaneously. In one design strategy, all of the single substituent variants are ranked, the top 4 are chosen and all possible double (6), triple (4) and quadruple (1) combinations of these 4 single substituent variants are synthesized and assayed. In a second design strategy, the best single substituent variant is considered to be the new parent and all possible double substituent variants that include this highest-ranked single substituent variant are synthesized and assayed. Other strategies may be used, and these strategies may be applied repeatedly such that the number of substituents is gradually increased while continuing to identify further-improved variants.

[00113] Aptamer Medicinal Chemistry may be used particularly as a method to explore the local, rather than the global, introduction of substituents. Because aptamers are discovered within libraries that are generated by transcription, any substituents that are introduced during the SELEX process must be introduced globally. For example, if it is desired to introduce phosphorothioate linkages between nucleotides then they can only be introduced at every A (or every G, C, T, U, etc.) if globally substituted. Aptamers that require phosphorothioates at some As (or some G, C, T, U, etc.) (locally substituted) but can not tolerate it at other As (or some G, C, T, U, etc.) can not be readily discovered by this process.

[00114] The kinds of substituents that can be utilized by the Aptamer Medicinal

Chemistry process are only limited by the ability to generate them as solid-phase synthesis reagents and introduce them into an oligomer synthesis scheme. The process is certainly not limited to nucleotides alone. Aptamer Medicinal Chemistry schemes may include substituents that introduce steric bulk, hydrophobicity, hydrophilicity, lipophilicity, lipophobicity, positive charge, negative charge, neutral charge, zwitterions, polarizability, nuclease-resistance, conformational rigidity, conformational flexibility, protein-binding characteristics, mass, etc. Aptamer Medicinal Chemistry schemes may include base-modifications, sugar-modifications or phosphodiester linkage-modifications.

[00115] When considering the kinds of substituents that are likely to be beneficial within the context of a therapeutic aptamer, it may be desirable to introduce substitutions that fall into one or more of the following categories:

(1) Substituents already present in the body, e.g., 2'-deoxy, 2'-ribo, 2'-O-methyl purines or pyrimidines, or 5-methyl cytosine.

(2) Substituents already part of an approved therapeutic, e.g., phosphorothioate-linked oligonucleotides.

(3) Substituents that hydro lyze or degrade to one of the above two categories, e.g., methylphosphonate-linked oligonucleotides.

The aptamers of the invention include aptamers developed through Aptamer Medicinal Chemistry as described herein.

[00116] Target binding affinity of the aptamers can be assessed through a series of binding reactions between the aptamer and the target (e.g., a protein) in which trace³²P-labeled aptamer is incubated with a dilution series of the target in a buffered medium and then analyzed by nitrocellulose filtration using a vacuum filtration manifold. Referred to herein as the dot blot binding assay, this method uses a three layer filtration medium consisting (from top to bottom) of nitrocellulose, nylon filter and gel blot paper. RNA that is bound to the target is captured on the nitrocellulose filter whereas the non-target bound RNA is captured on the nylon filter. The gel blot paper is included as a supporting medium for the other filters. Following filtration, the filter layers are separated, dried and exposed on a phosphor screen and quantified using a phosphorimaging system. The quantified results can be used to generate aptamer binding curves from which dissociation constants (K_D) can be calculated. In a preferred embodiment, the buffered medium used to perform the binding reactions is IX Dulbecco's PBS (with Ca⁺⁺ and Mg⁺⁺) plus 0.1 mg/mL BSA.

[00117] Generally, the ability of an aptamer to modulate the functional activity of a target, i.e., the functional activity of the aptamer, can be assessed using in vitro and in vivo models, which will vary depending on the biological function of the target. In some embodiments, the aptamers of the invention may inhibit a known biological function of the target. In other embodiments, the aptamers of the invention may stimulate a known biological function of the target. The functional activity of aptamers can be assessed using in vitro and in vivo models designed to measure a known function of the aptamer target. [00118] The aptamers of the invention may be routinely adapted for diagnostic purposes according to any number of techniques employed by those skilled in the art. Diagnostic utilization may include both in vivo or in vitro diagnostic applications. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. The ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would also be able to adapt any aptamer by procedures known in the art to incorporate a labeling tag in order to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures.

Aptamers Having Immunostimulatory Motifs

[00119] Recognition of bacterial DNA by the vertebrate immune system is based upon the recognition of unmethylated CG dinucleotides in particular sequence contexts ("CpG motifs"). One receptor that recognizes such a motif is Toll-like receptor 9 ("TLR 9"), a member of a family of Toll-like receptors (-10 members) that participate in the innate immune response by recognizing distinct microbial components. TLR 9 binds unmethylated oligodeoxynucleotide ("ODN") CpG sequences in a sequence-specific manner. The recognition of CpG motifs triggers defense mechanisms leading to innate and ultimately acquired immune responses. For example, activation of TLR 9 in mice induces activation of antigen presenting cells, up regulation of MHC class I and II molecules, and expression of important co-stimulatory molecules and cytokines, including IL- 12 and IL-23. This activation both directly and indirectly enhances B and T cell responses, including robust up regulation of the THl cytokine IFN-gamma. Collectively, the response to CpG sequences leads to: protection against infectious diseases, improved immune response to vaccines, an effective response against asthma, and improved antibody-dependent cell-mediated cytotoxicity. Thus, CpG ODNs can provide protection against infectious diseases, function as immuno-adjuvants or cancer therapeutics (monotherapy or in combination with a mAb or other therapies), and can decrease asthma and allergic responses.

[00120] Aptamers of the invention may comprise one or more CpG or other immunostimulatory sequences. Such aptamers can be identified or generated by a variety of strategies using, e.g., the SELEX^™ process described herein. In general, the strategies can be divided into two groups. In group one, the strategies are directed to identifying or generating aptamers comprising both a CpG motif (or other immunostimulatory sequence) as well as a binding site for a target, where the target (hereinafter "non-CpG target") is a target other than one known to recognize CpG motifs (or other immunostimulatory sequences) and known to stimulates an immune response upon binding to a CpG motif. The first strategy of this group comprises the steps of performing SELEX to obtain an aptamer to a specific non-CpG target using an oligonucleotide pool wherein a CpG motif has been incorporated into each member of the pool as, or as part of, a fixed region, e.g., in some embodiments the randomized region of the pool members comprises a fixed region having a CpG motif incorporated therein, and identifying an aptamer comprising a CpG motif. The second strategy of this group comprises the steps of performing SELEX^™ to obtain an aptamer to a specific non-CpG target, and appending a CpG motif to the 5' and/or 3' end or engineering a CpG motif into a region, preferably a non-essential region, of the aptamer. The third strategy of this group comprises the steps of performing SELEX^™ to obtain an aptamer to a specific non-CpG target, wherein during synthesis of the pool the molar ratio of the various nucleotides is biased in one or more nucleotide addition steps so that the randomized region of each member of the pool is enriched in CpG motifs, and identifying an aptamer comprising a CpG motif. The fourth strategy of this group comprises the steps of performing SELEX to obtain an aptamer to a specific non-CpG target, and identifying an aptamer comprising a CpG motif. The fifth strategy of this group comprises the steps of performing SELEX to obtain an aptamer to a specific non-CpG target, and identifying an aptamer which, upon binding, stimulates an immune response but that does not comprise a CpG motif.

[00121] In group two, the strategies are directed to identifying or generating aptamers comprising a CpG motif and/or other sequences that are bound by the receptors for the CpG motifs (e.g., TLR9 or the other Toll-like receptors) and upon binding stimulate an immune response. The first strategy of this group comprises the steps of performing SELEX^™ to obtain an aptamer to a target known to bind to CpG motifs (or other immunostimulatory sequences) and upon binding stimulate an immune response, using an oligonucleotide pool wherein a CpG motif has been incorporated into each member of the pool as, or as part of, a fixed region, e.g., in some embodiments the randomized region of the pool members comprise a fixed region having a CpG motif incorporated therein, and identifying an aptamer comprising a CpG motif. The second strategy of this group comprises the steps of performing SELEX to obtain an aptamer to a target known to bind to CpG motifs (or other immunostimulatory sequences) and upon binding stimulate an immune response, and then appending a CpG motif to the 5' and/or 3' end or engineering a CpG motif into a region, preferably a non-essential region, of the aptamer. The third strategy of this group comprises the steps of performing SELEX to obtain an aptamer to a target known to bind to CpG motifs (or other immunostimulatory sequences) and upon binding stimulate an immune response, wherein during synthesis of the pool, the molar ratio of the various nucleotides is biased in one or more nucleotide addition steps so that the randomized region of each member of the pool is enriched in CpG motifs, and identifying an aptamer comprising a CpG motif. The fourth strategy of this group comprises the steps of performing SELEX^™ to obtain an aptamer to a target known to bind to CpG motifs (or other immunostimulatory sequences) and upon binding stimulate an immune response, and identifying an aptamer comprising a CpG motif. The fifth strategy of this group comprises the steps of performing SELEX to obtain an aptamer to a target known to bind to CpG motifs (or other immunostimulatory sequences), and identifying an aptamer that upon binding stimulates an immune response but that does not comprise a CpG motif.

[00122] A variety of different classes of CpG motifs have been identified, each resulting in a different cascade of events, release of cytokines and other molecules, and activation of certain cell types. See, e.g., CpG Motifs in Bacterial DNA and Their Immune Effects, Annu.

Rev. Immunol. 2002, 20:709-760, which is incorporated herein by reference. Additional immunostimulatory motifs are disclosed in the following U.S. Patents, each of which is incorporated herein by reference: U.S. Patent No. 6,207,646; U.S. Patent No. 6,239,116; U.S. Patent No. 6,429,199; U.S. Patent No. 6,214,806; U.S. Patent No. 6,653,292; U.S. Patent No. 6,426,434; U.S. Patent No. 6,514,948 and U.S. Patent No. 6,498,148. Any of these CpG or other immunostimulatory motifs can be incorporated into an aptamer. The choice of aptamers is dependent upon the disease or disorder to be treated. Preferred immunostimulatory motifs are as follows (shown 5' to 3' left to right) wherein "r" designates a purine, "y" designates a pyrimidine, and "X" designates any nucleotide: AACGTTCGAG (SEQ ID NO: 8); AACGTT; ACGT, rCGy; rrCGyy, XCGX, XXCGXX, and XiX₂CGYiY₂ wherein Xi is G or A, X₂ is not C, Yi is not G and Y₂ is preferably T.

[00123] In those instances where a CpG motif is incorporated into an aptamer that binds to a specific target other than a target known to bind to CpG motifs and upon binding stimulate an immune response (a "non-CpG target"), the CpG is preferably located in a non-essential region of the aptamer. Non-essential regions of aptamers can be identified by site-directed mutagenesis, deletion analyses and/or substitution analyses. However, any location that does not significantly interfere with the ability of the aptamer to bind to the non-CpG target may be used. In addition to being embedded within the aptamer sequence, the CpG motif may be appended to either or both of the 5' and 3' ends or otherwise attached to the aptamer. Any location or means of attachment may be used so long as the ability of the aptamer to bind to the non-CpG target is not significantly interfered with.

[00124] As used herein, "stimulation of an immune response" can mean either (1) the induction of a specific response (e.g., induction of a ThI response) or the production of certain molecules or (2) the inhibition or suppression of a specific response (e.g., inhibition or suppression of the Th2 response) or of certain molecules.

Modulation of Pharmacokinetics and Biodistribution of Aptamer Therapeutics [00125] It is important that the pharmacokinetic properties for all oligonucleotide-based therapeutics, including aptamers, be tailored to match the desired pharmaceutical application. While aptamers directed against extracellular targets do not suffer from difficulties associated with intracellular delivery (as is the case with antisense and RNAi-based therapeutics), such aptamers must still be able to be distributed to target organs and tissues, and remain in the body (unmodified) for a period of time consistent with the desired dosing regimen. [00126] Thus, the invention provides materials and methods to affect the pharmacokinetics of aptamer compositions, and, in particular, the ability to tune aptamer pharmacokinetics. The tunability of (i.e., the ability to modulate) aptamer pharmacokinetics is achieved through conjugation of modifying moieties (e.g., PEG polymers) to the aptamer and/or the incorporation of modified nucleotides (e.g., 2'-fluoro or 2'-O-methyl) to alter the chemical composition of the nucleic acid. The ability to tune aptamer pharmacokinetics is used in the improvement of existing therapeutic applications, or alternatively, in the development of new therapeutic applications. For example, in some therapeutic applications, e.g., in antineoplastic or acute care settings where rapid drug clearance or turn-off may be desired, it is desirable to decrease the residence times of aptamers in the circulation. Alternatively, in other therapeutic applications, e.g., maintenance therapies where systemic circulation of a therapeutic is desired, it may be desirable to increase the residence times of aptamers in circulation. [00127] In addition, the tunability of aptamer pharmacokinetics is used to modify the biodistribution of an aptamer therapeutic in a subject. For example, in some therapeutic applications, it may be desirable to alter the biodistribution of an aptamer therapeutic in an effort to target a particular type of tissue or a specific organ (or set of organs). In these applications, the aptamer therapeutic preferentially accumulates in a specific tissue or organ(s). In other therapeutic applications, it may be desirable to target tissues displaying a cellular marker or a symptom associated with a given disease, cellular injury or other abnormal pathology, such that the aptamer therapeutic preferentially accumulates in the affected tissue. For example, PEGylation of an aptamer therapeutic (e.g. , PEGylation with a 20 kDa PEG polymer) is used to target inflamed tissues, such that the PEGylated aptamer therapeutic preferentially accumulates in inflamed tissue.

[00128] To determine the pharmacokinetic and biodistribution profiles of aptamer therapeutics (e.g., aptamer conjugates or aptamers having altered chemistries, such as modified nucleotides) a variety of parameters are monitored. Such parameters include, for example, the half- life (ti/2), the plasma clearance (Cl), the volume of distribution (Vss), the area under the concentration-time curve (AUC), maximum observed serum or plasma concentration (C_max), and the mean residence time (MRT) of an aptamer composition. As used herein, the term "AUC" refers to the area under the plot of the plasma concentration of an aptamer therapeutic versus the time after aptamer administration. The AUC value is used to estimate the bioavailability (i.e., the percentage of administered aptamer therapeutic in the circulation after aptamer administration) and/or total clearance (Cl) (i.e., the rate at which the aptamer therapeutic is removed from circulation) of a given aptamer therapeutic. The volume of distribution relates the plasma concentration of an aptamer therapeutic to the amount of aptamer present in the body. The larger the Vss, the more an aptamer is found outside of the plasma (i.e., the more extravasation).

[00129] The invention provides materials and methods to modulate, in a controlled manner, the pharmacokinetics and biodistribution of stabilized aptamer compositions in vivo by conjugating an aptamer to a modulating moiety, such as a small molecule, peptide, or polymer terminal group, or by incorporating modified nucleotides into an aptamer. As described herein, conjugation of a modifying moiety and/or altering nucleotide(s) chemical composition alters fundamental aspects of aptamer residence time in the circulation and distribution to tissues. [00130] In addition to clearance by nucleases, oligonucleotide therapeutics are subject to elimination via renal filtration. As such, a nuclease-resistant oligonucleotide administered intravenously typically exhibits an in vivo half-life of <10 min, unless filtration can be blocked. This can be accomplished by either facilitating rapid distribution out of the blood and into tissues, or by increasing the apparent molecular weight of the oligonucleotide above the effective size cut-off for the glomerulus. Conjugation of small therapeutics to a PEG polymer (PEGylation) can dramatically lengthen residence times of aptamers in the circulation, thereby decreasing dosing frequency and enhancing effectiveness against vascular targets. [00131] Aptamers can be conjugated to a variety of modifying moieties, such as high molecular weight polymers, e.g., PEG; peptides, e.g., Tat (a 13-amino acid fragment of the HIV Tat protein (Vives, et al. (1997), J. Biol. Chem. 272(25): 16010-7)), Ant (a 16-amino acid sequence derived from the third helix of the Drosophila antennapedia homeotic protein (Pietersz, et al. (2001), Vaccine 19(11-12): 1397-405)) and Arg₇ (a short, positively charged cell-permeating peptides composed of polyarginine (Arg₇) (Rothbard, et al. (2000), Nat. Med. 6(11): 1253-7; Rothbard, J et al. (2002), J. Med. Chem. 45(17): 3612-8)); and small molecules, e.g., lipophilic compounds, such as cholesterol. Among the various conjugates described herein, in vivo properties of aptamers are altered most profoundly by complexation with PEG groups. For example, complexation of a mixed 2'-F and 2'-0Me modified aptamer therapeutic with a 20 kDa PEG polymer hinders renal filtration and promotes aptamer distribution to both healthy and inflamed tissues. Furthermore, the 20 kDa PEG polymer-aptamer conjugate proves nearly as effective as a 40 kDa PEG polymer in preventing renal filtration of aptamers. While one effect of PEGylation is on aptamer clearance, the prolonged systemic exposure afforded by presence of the 20 kDa moiety also facilitates distribution of aptamer to tissues, particularly those of highly perfused organs and those at the site of inflammation. The aptamer-20 kDa PEG polymer conjugate directs aptamer distribution to the site of inflammation, such that the PEGylated aptamer preferentially accumulates in inflamed tissue. In some instances, the 20 kDa PEGylated aptamer conjugate is able to access the interior of cells, such as, for example, kidney cells.

[00132] Modified nucleotides can also be used to modulate the plasma clearance of aptamers. For example, an unconjugated aptamer that incorporates both 2'-F and 2'-0Me stabilizing chemistries, which exhibits a high degree of nuclease stability in vitro and in vivo, displays rapid loss from plasma {i.e., rapid plasma clearance) and a rapid distribution into tissues, primarily into the kidney, when compared to unmodified aptamer.

PEG-Derivatized Nucleic Acids

[00133] As described above, derivatization of nucleic acids with high molecular weight non-immunogenic polymers has the potential to alter the pharmacokinetic and pharmacodynamic properties of nucleic acids and make them more effective therapeutic agents. Favorable changes in activity can include increased resistance to degradation by nucleases, decreased filtration through the kidneys, decreased exposure to the immune system, and altered distribution of the therapeutic throughout the body.

[00134] The aptamer compositions may be derivatized with polyalkylene glycol

("PAG") moieties. Examples of PAG-derivatized nucleic acids are found in United States Patent Application Serial No. 10/718,833, filed on November 21, 2003, which is herein incorporated by reference in its entirety. Typical polymers include polyethylene glycol ("PEG"), also known as polyethylene oxide ("PEO"), and polypropylene glycol (including poly isopropylene glycol). Additionally, random or block copolymers of different alkylene oxides {e.g., ethylene oxide and propylene oxide) can be used in many applications. In its most common form, a polyalkylene glycol, such as PEG, is a linear polymer terminated at each end with hydroxyl groups: HO-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-OH. This polymer, alpha-, omega-dihydroxylpolyethylene glycol, can also be represented as HO-PEG-OH, where it is understood that the -PEG- symbol represents the following structural unit: -CH₂CH₂O- (CH₂CH₂O)_n-CH₂CH₂-, where n typically ranges from about 4 to about 10,000. [00135] As shown, the PEG molecule is di-functional and is sometimes referred to as

"PEG diol". The terminal portions of the PEG molecule are relatively non-reactive hydroxyl moieties, the -OH groups, that can be activated or converted to functional moieties for attachment of the PEG to other compounds at reactive sites on the compound. Such activated PEG diols are referred to herein as bi-activated PEGs. For example, the terminal moieties of PEG diol have been functionalized as active carbonate ester for selective reaction with amino moieties by substitution of the relatively non-reactive hydroxyl moieties, with succinimidyl active ester moieties from N-hydroxy succinimide.

[00136] In many applications, it is desirable to cap the PEG molecule on one end with an essentially non-reactive moiety so that the PEG molecule is mono-functional (or mono- activated). In the case of protein therapeutics, which generally display multiple reaction sites for activated PEGs, bi-functional activated PEGs lead to extensive cross-linking, yielding poorly functional aggregates. To generate mono-activated PEGs, one hydroxyl moiety on the terminus of the PEG diol molecule is typically substituted with a non-reactive methoxy end moiety, -OCH3. The other, un-capped terminus of the PEG molecule is typically converted to a reactive end moiety that can be activated for attachment at a reactive site on a surface or a molecule, such as a protein.

[00137] PAGs are polymers that typically have the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PAGs is to covalently attach the polymer to insoluble molecules to make the resulting PAG-molecule "conjugate" soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al, J. Org. Chem., 60:331-336 (1995). PAG conjugates are often used not only to enhance solubility and stability, but also to prolong the blood circulation half-life of molecules. [00138] Polyalkylated compounds of the invention are typically between 5 and 80 kDa in size, however any size can be used, the choice dependent on the aptamer and application. Other PAG compounds of the invention are between 10 and 80 kDa in size. Still other PAG compounds of the invention are between 10 and 60 kDa in size. For example, a PAG polymer may be at least 10, 20, 30, 40, 50, 60, 70 or 80 kDa in size. Such polymers can be linear or branched. In some embodiments the polymers are PEG.

[00139] In contrast to biologically-expressed protein therapeutics, nucleic acid therapeutics are typically chemically synthesized from activated monomer nucleotides. PEG- nucleic acid conjugates may be prepared by incorporating the PEG using the same iterative monomer synthesis. For example, PEGs activated by conversion to a phosphoramidite form can be incorporated into solid-phase oligonucleotide synthesis. Alternatively, oligonucleotide synthesis can be completed with site-specific incorporation of a reactive PEG attachment site. Most commonly this has been accomplished by addition of a free primary amine at the 5'- terminus (incorporated using a modifier phosphoramidite in the last coupling step of solid phase synthesis). Using this approach, a reactive PEG (e.g., one which is activated so that it will react and form a bond with an amine) is combined with the purified oligonucleotide and the coupling reaction is carried out in solution.

[00140] The ability of PEG conjugation to alter the biodistribution of a therapeutic is related to a number of factors, including the apparent size (e.g., as measured in terms of hydrodynamic radius) of the conjugate. Larger conjugates (>1 OkDa) are known to more effectively block filtration via the kidney and to consequently increase the serum half-life of small macromolecules (e.g., peptides, antisense oligonucleotides). The ability of PEG conjugates to block filtration has been shown to increase with PEG size, up to approximately 50 kDa (further increases have minimal beneficial effect as half life becomes defined by macrophage-mediated metabolism rather than elimination via the kidneys). [00141] Production of high molecular weight PEGs (>10 kDa) can be difficult, inefficient and expensive. As a route toward the synthesis of high molecular weight PEG- nucleic acid conjugates, previous work has been focused on the generation of higher molecular weight activated PEGs. One method for generating such molecules involves the formation of a branched, activated PEG in which two or more PEGs are attached to a central core carrying the activated group. The terminal portions of these higher molecular weight PEG molecules, i.e., the relatively non-reactive hydroxyl (-OH) moieties, can be activated or converted to functional moieties for attachment of one or more of the PEGs to other compounds at reactive sites on the compound. Branched activated PEGs will have more than two termini, and in cases where two or more termini have been activated, such activated higher molecular weight PEG molecules are referred to herein as, multi-activated PEGs. In some cases, not all termini in a branched PEG molecule are activated. In cases where any two termini of a branched PEG molecule are activated, such PEG molecules are referred to as bi-activated PEGs. In some cases where only one terminus in a branched PEG molecule is activated, such PEG molecules are referred to as mono-activated. As an example of this approach, activated PEG prepared by the attachment of two monomethoxy PEGs to a lysine core, which is subsequently activated for reaction, has been described (Harris et ah, Nature, vol.2: 214-221, 2003). [00142] The invention provides another cost effective route to the synthesis of high molecular weight PEG-nucleic acid (preferably, aptamer) conjugates, including multiply PEGylated nucleic acids. The invention also encompasses PEG-linked multimeric oligonucleotides, e.g., dimerized aptamers. The invention also relates to high molecular weight compositions where a PEG stabilizing moiety is a linker that separates different portions of an aptamer, e.g. , the PEG is conjugated within a single aptamer sequence such that the linear arrangement of the high molecular weight aptamer composition is, e.g., nucleic acid-PEG- nucleic acid (-PEG-nucleic acid)_n, where n is greater than or equal to 1. [00143] High molecular weight compositions of the invention include those having a molecular weight of at least 10 kDa. Compositions typically have a molecular weight between 10 and 80 kDa in size. High molecular weight compositions of the invention are at least 10, 20, 30, 40, 50, 60, 70 or 80 kDa in size.

[00144] A stabilizing moiety is a molecule or portion of a molecule that improves pharmacokinetic and pharmacodynamic properties of the high molecular weight aptamer compositions of the invention. In some cases, a stabilizing moiety is a molecule or portion of a molecule that brings two or more aptamers or aptamer domains into proximity, or provides decreased overall rotational freedom of the high molecular weight aptamer compositions of the invention. A stabilizing moiety can be a polyalkylene glycol, such a polyethylene glycol, which can be linear or branched, a homopolymer or a heteropolymer. Other stabilizing moieties include polymers, such as peptide nucleic acids (PNA). Oligonucleotides can also be stabilizing moieties, such oligonucleotides can include modified nucleotides, and/or modified linkages, such as phosphorothioates. A stabilizing moiety can be an integral part of an aptamer composition, i.e., it is covalently bonded to the aptamer.

[00145] Compositions of the invention include high molecular weight aptamer compositions in which two or more nucleic acid moieties are covalently conjugated to at least one polyalkylene glycol moiety. The polyalkylene glycol moieties serve as stabilizing moieties. In compositions where a polyalkylene glycol moiety is covalently bound at either end to an aptamer, such that the polyalkylene glycol joins the nucleic acid moieties together in one molecule, the polyalkylene glycol is said to be a linking moiety. In such compositions, the primary structure of the covalent molecule includes the linear arrangement nucleic acid-PAG- nucleic acid. One example is a composition having the primary structure nucleic acid-PEG- nucleic acid. Another example is a linear arrangement of: nucleic acid-PEG-nucleic acid-PEG- nucleic acid.

[00146] To produce the nucleic acid-PEG-nucleic acid conjugate, the nucleic acid is originally synthesized such that it bears a single reactive site (e.g., it is mono-activated). In a preferred embodiment, this reactive site is an amino group introduced at the 5 '-terminus by the addition of a modifier phosphoramidite as the last step in solid phase synthesis of the oligonucleotide. Following deprotection and purification of the modified oligonucleotide, it is reconstituted at high concentration in a solution that minimizes spontaneous hydrolysis of the activated PEG. In a preferred embodiment, the concentration of oligonucleotide is 1 mM and the reconstituted solution contains 200 mM NaHCOs buffer, pH 8.3. Synthesis of the conjugate is initiated by slow, step-wise addition of highly purified bi-functional PEG. In a preferred embodiment, the PEG diol is activated at both ends (bi-activated) by derivatization with succinimidyl propionate. Following reaction, the PEG-nucleic acid conjugate is purified by gel electrophoresis or liquid chromatography to separate fully-, partially- and un-conjugated species. Multiple PAG molecules concatenated as random or block copolymers or smaller PAG chains can be linked to achieve various lengths (or molecular weights). Non-PAG linkers can be used between PAG chains of varying lengths.

[00147] The 2'-O-methyl, 2'-fluoro and other modified nucleotide modifications stabilize the aptamer against nucleases and increase its half life in vivo. The 3'-dT cap also increases exonuclease resistance. See, e.g., U.S. Patents Nos. 5,674,685; 5,668,264; 6,207,816 and 6,229,002; each of which is incorporated by reference herein in its entirety. PAG-Derivatization of a Reactive Nucleic Acid

[00148] High molecular weight PAG-nucleic acid-PAG conjugates can be prepared by reaction of a mono-functional activated PEG with a nucleic acid containing more than one reactive site. In one embodiment, the nucleic acid is bi-reactive, or bi-activated, and contains two reactive sites: a 5 '-amino group and a 3 '-amino group introduced into the oligonucleotide through conventional phosphoramidite synthesis, for example: 3'-5'-di-PEGylation, as illustrated in Figure 2. In alternative embodiments, reactive sites can be introduced at internal positions, using for example, the 5-position of pyrimidines, the 8-position of purines, or the T- position of ribose as sites for attachment of primary amines. In such embodiments, the nucleic acid can have several activated or reactive sites and is said to be multiply activated. Following synthesis and purification, the modified oligonucleotide is combined with the mono-activated PEG under conditions that promote selective reaction with the oligonucleotide reactive sites while minimizing spontaneous hydrolysis. In the preferred embodiment, monomethoxy-PEG is activated with succinimidyl propionate and the coupled reaction is carried out at pH 8.3. To drive synthesis of the bi-substituted PEG, stoichiometric excess PEG is provided relative to the oligonucleotide. Following reaction, the PEG-nucleic acid conjugate is purified by gel electrophoresis or liquid chromatography to separate fully, partially and un-conjugated species. [00149] The linking domains can also have one or more polyalkylene glycol moieties attached thereto. Such PAGs can be of varying lengths and may be used in appropriate combinations to achieve the desired molecular weight of the composition. [00150] The effect of a particular linker can be influenced by both its chemical composition and length. A linker that is too long, too short, or forms unfavorable steric and/or ionic interactions with the target will preclude the formation of a complex between the aptamer and the target. A linker, which is longer than necessary to span the distance between nucleic acids, may reduce binding stability by diminishing the effective concentration of the ligand. Thus, it is often necessary to optimize linker compositions and lengths in order to maximize the affinity of an aptamer to a target.

[00151] Each publication and patent document cited herein is incorporated herein by reference in its entirety as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same.

EXAMPLES Example 1: Mutant T3 Polymerase Expression and Purification

[00152] Mutant T3 RNA polymerases for use in the methods of the invention were prepared as follows. i) Preparation and Amplification of T3

[00153] One pill of T3 bacteriophage (ATCC #BAA-1O25-B1) was dissolved in 200 μL dH2O at 37⁰C. T3 was then amplified via polymerase chain reaction ("PCR") using the following T3-5 'primer: CW339.122.A (BamHI site underline) TCA CCA TCA CGG ATC CAT GAA CAT CAT CGA AAA CAT CGA AAA G (SEQ ID NO: 9) and T3-3 'primer: CW339.122.B (Hindlll site underlined) TCA GCT AAT TAA GCT TGT TAT GCA AAG GCA AAG TCA GAC TTG (SEQ ID NO: 10). The resulting PCR product was cloned into TOPO pCR4.1. A Hindlll and BamHI cut product was swapped into a T3 polymerase expression vector. A multi site-directed mutagenesis reaction was then performed using the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA). ii) Introduction of Mutation into T3 LA Mutant Polymerase Expression Vector [00154] A Y640L primer: GGT CAT GAC GCT GGC TCT CGG TTC CAA GGA GTT

CG (TAC->CTC mutation) (SEQ ID NO: 11) and H785A primer: CGC TCC TAA CTT TGT TGC CTC ACA GGA CGG TAG CC (CAC->GCC mutation) (SEQ ID NO: 12) were used to translate the T3 LA Mutant Polymerase. T3 LA Mutant Polymerase was screened by sequencing and the product was then subcloned into T3 RNAP expression vector (via BamHl/Hindlll site). The clones produced from this reaction were transformed into BL21(DE3) competent cells (Stratagene, CA). iii) Introduction of Mutation into T3 Single Mutant Polymerase Expression Vector [00155] In order to produce the T3 Single Mutant from T3 produced according to the teaching in Section i) above, a mutation was introduced using: i) the forward primer: GGTCATGACGCTGGCTTTCGGTTCCAAGGAGTTCG (SEQ ID NO: 13) and ii) the reverse primer: CGAACTCCTTGGAACCGAAAGCCAGCGTCATGACC (SEQ ID NO: 14). The clones produced from this reaction were transformed into BL21 (DE3) competent cells (Stratagene, CA). iv) Expression and Production of Mutant T3 Polymerases

[00156] After the expression vector comprising the mutant T3 polymerase nucleic acid sequences (e.g., the T3 LA Mutant or the T3 Single Mutant) was transformed into BL21 (DE3) competent cells as described above, these cells were incubated on ice for 20 minutes. Heat shock was then performed by putting the cell containing tubes in a 42⁰C water bath for 2 minutes. After performing heat shock, these tubes were returned to ice for 1 minute. 1 ml of L broth ("LB") was added to each tube and the tubes were then incubated at 37⁰C, with shaking, for 45 minutes. 100 μl of culture liquid from each tube was plated onto LB+Amp agar plates and incubated at 37⁰C overnight.

[00157] A single colony from the overnight cultured plate was inoculated into 100 ml

LB-Amp+ (150ug/ml) and then maintained at 37⁰C overnight. On the second day, two 4-liter flasks containing 2 liters of pre-warmed LB+Amp were inoculated with 50 ml of overnight culture and grown at 37⁰C until the OD600 reached between 0.6-0.8. 200 μl of IM IPTG was then added to each 2L cell culture with a final concentration of 100 μM and grown for another 3 hours at 37°C. The cells were then pelleted by spinning at 5000 rpm for 10 minutes. Cells were then resuspended in a 200 ml lysis buffer (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% Glycerol, 1 mM imidazole, betamercaptoethanol ("BME") 5mM) and divided into 6 conical 50 ml tubes. The cells were sonicated and then bacterial debris was spun down at 11,000 rpm for 60 minutes.

[00158] The resulting supernatant was then filtered through a 0.22 μM filter. Imidazole was then added to the filtrate to a final concentration of 10 mM. The filtrate was then loaded onto a 5 ml Ni-NTA column (GE Healthcare Bio-Sciences, NJ) with sample pump. The column was washed with 10 column volumes ("CVs") of Buffer A (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% Glycerol, 10 mM imidazole, BME 10 mM) containing 20 mM imidazole. The column was then washed with 10 CVs of buffer with a linear gradient of imidazole concentration from 40 mM to 70 mM in buffer A. The protein was then eluted with 6 CVs of Buffer B (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% Glycerol, 250 mM imidazole, BMElO mM). [00159] After checking the collection fractions with 5 μl of sample on 4-12% SDS-

PAGE, all of the fractions of interest were combined and dialyzed (dialysis tubing: Spectrum Spectra/por Molecular porous membrane (VWR) MWCO: 12-14000) in IL of dialysis buffer (dialysis buffer: 50 mM Tris-Cl, pH 7.9, 100 mM NaCl, 50% Glycerol, 0.ImM EDTA, 0.1% Triton X-100, BME 20 mM) overnight. The dialysis buffer was changed after 12 hours and dialysis was carried out for an additional 4 hours. The concentration of T3 RNA polymerase is measured using the Bradford assay as described in Bradford, M. M. (1976) Anal. Biochem. 72, and confirmed by SDS-PAGE.

Example 2: MNA, rGmH, mRfY, dCmD, rRfY and dRmY Transcription using Mutant

T3 RNA Polymerases

[00160] The following DNA templates and primers were used to program a polymerase chain reaction in order to generate a double-stranded transcription template. "N" indicates a degenerate position with an approximately equal probability of being any one of: adenine (A), thymidine (T), guanine (G) or cytosine (C). All sequences are listed in the 5' to 3' direction.

PCR Template (ARC6002): TGAATTAACCCTCACTAAAGGAAATGCCCAAGATCAGCNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNCTTATTTGATGTCATCCGAGATCG (SEQ ID NO: 6)

5'-primer (ARC6005): GACTGAATTAACCCTCACTAAAGGAAATGCCCAAGATCAGC (SEQ ID NO: 15)

3'-primer (ARC6006): CGATCTCGGATGACATCAAATAAG (SEQ ID NO: 16)

PCR Template (ARCl 590):

GGGAGAGGAGAGAACGTTCACTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNG GTCGATCGATCGATCATCGATG (SEQ ID NO: 7) 5 ' -primer (KMT .172.141.K) : TAATACGACTCACTATAGGGAGAGGAGAGAACGTTCACT (SEQ ID NO: 17)

3 '-primer (KMT.172.14 LL): CATCGATGATCGATCGATCGAC (SEQ ID NO: 18)

[00161] The resultant double-stranded transcription template was then used to program transcription mixtures for each of the transcription mixtures set out in Table 2. The transcription mixture was incubated at 37°C overnight for 16 hours.

[00162] After incubation, the mixtures were precipitated with isopropanol. The resultant pellet was dissolved and quantitated using denaturing PAGE ( 10% acrylamide) for 60 minutes at 25W. The samples were visualized and quantitated by UV shadow (at 260nm) with Alpha Imager quantitation software relative to a known standard. The transcriptional yield was documented in Table 3. Table 3: Transcription Yield

Example 3: rRfY SELEX for Ap tamers to Integrins Using Mutant T3 Polymerases for

Transcription

[00163] SELEX experiments using an rRfY pool composition and the Y640F mutant T3 polymerase construct were carried out against the purified integrin human αvβ3 (Chemicon). [00164] For SELEX experiments with αvβ3, previously selected aptamer 17.16 was used as a base for the generation of a randomized pool for selection. This aptamer is described in PCT Patent Publication No. WO 01/09159. The following DNA templates and primers were used to program a polymerase chain reaction in order to generate a double-stranded transcription template. In these sequences, "A" indicates adenine, "C" cytosine, "G" guanine and "T" thymidine. "%" indicates a partially degenerate position with an approximately 85% probability of this position being the nucleotide indicated to the right of the "%" and approximately 5% probability of this position being each of the remaining three possible nucleotides (ex., %A ≡ 85% A, 5% C, 5% G, 5% T). AU sequences are listed in the 5' to 3' direction.

PCR Template (ARC14576):

GGGAGACAAGAATAAACGCTCAATTC%A%A%C%G%C%T%G%T%G%A%A%G%G %G%C%T%T%A%T%A%C%G%A%G%C%G%G%A%T%T%A%C%C%C%T%TCGACA GGAGGCTCACAACAGGC (SEQ ID NO: 19)

5'-primer (ARC14577):

GACTGAATTAACCCTCACTAAAGGGAGACAAGAATAAACGCTCAATTC (SEQ ID NO: 20)

3'-primer (ARCl 1761): GCCTGTTGTGAGCCTCCTGTCG (SEQ ID NO: 21)

[00165] The resultant double-stranded transcription template was then used to program a transcription mixture with the T3 polymerase construct Y640F (6 μg/mL) and the rRfY composition of NTPs to generate a SELEX pool. The transcription mixture was incubated at 37°C for -16 hours. After incubation, the mixtures were precipitated with ethanol. The resultant pellet was dissolved and purified using denaturing PAGE (10% acrylamide). The transcription sample was visualized using UV shadow and the gel pieces that contained the transcript were removed with a razor blade. The transcription was eluted from the gel, ethanol precipitated, and resuspended for use in SELEX experiments. [00166] SELEX experiments were carried out by first immobilizing the protein target

(αvβ3) on hydrophobic beads at various concentrations (1 μM - 50 nM), followed by blocking unbound sites on the beads with BSA. The SELEX pool (1.67 - 1 μM) was then incubated with the bead-bound target for 1 hour at 37°C. Unbound pool was removed from the beads, followed by washing with DPBS buffer. Bound sequences (aptamers) were then removed from the beads by denaturation with a hot urea-containing buffer and precipitated with ethanol. The pellet was resuspended in the presence of the appropriate 3 '-primer and used to program a reverse transcription reaction to generate a DNA strand that was complementary to the rRfY aptamer. The resulting DNA strand was then used to program a polymerase chain reaction, with the appropriate 5'- and 3 '-primers, to generate a double-stranded transcription template. The resultant double-stranded transcription template was then used to program a transcription mixture with the T3 polymerase construct Y640F (6 μg/mL) and the rRfY composition of NTPs to generate a SELEX pool enriched for target-binding aptamers for use in the next round of selection. The transcription reactions were carried out at either 37°C or 32°C and purified using denaturing PAGE, as above. Four selection rounds were carried out against αvβ3, with a selection round consisting of one cycle of binding reaction, reverse transcription, polymerase chain reaction, and transcription with T3 polymerase mutant Y640F.

[00167] The selection against αvβ3 resulted in a pool of aptamers that was enriched for binding to αvβ3 above the binding observed with the starting pool of sequences. The resulting sequences of aptamers contained aptamer 17.16 and several sequences that were highly related to aptamer 17.16.

[00168] The invention having now been described by way of examples, those of skill in the art will recognize that the invention can be practiced in a variety of ways and that the description and examples above are for purposes of illustration only and not for limiting the claims.

Claims

CLAIMSWhat is claimed is:

1. A mutant T3 RNA polymerase comprising altered amino acids at position 640 and position 785, wherein the altered amino acid at position 640 is a leucine and the altered amino acid at position 785 is an alanine.

2. The polymerase of claim 1 , wherein the polymerase comprises a mutation at position 640 from a tyrosine to a leucine, and a mutation at position 785 from a histidine to an alanine (Y640L/H785A).

3. The polymerase of claim 1, wherein the polymerase comprises the amino acid sequence of SEQ ID NO: 3.

4. The polymerase of claim 3, wherein the polymerase is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 5.

5. An expression vector comprising the polynucleotide of claim 4.

6. A host cell comprising the vector of claim 5.

7. A method for producing a mutant polymerase comprising culturing the host cell of claim 6 under conditions in which the polymerase is expressed, and recovering the polymerase.

8. The polymerase of claim 1, wherein the polymerase increases the incorporation of 2'- OMe modified nucleotides into an oligonucleotide relative to a wild-type T3 RNA polymerase that lacks the altered amino acids.

9. The polymerase of claim 1, wherein the polymerase exhibits decreased discrimination against 2'-0Me modified nucleotides relative to a wild-type T3 RNA polymerase that lacks the altered amino acids.

10. A mutant T3 RNA polymerase comprising an altered amino acid at position 640, wherein the altered amino acid at position 640 is a phenylalanine.

11. The polymerase of claim 10, wherein the polymerase comprises a mutation at position 640 from a tyrosine to a phenylalanine (Y640F).

12. The polymerase of claim 10, wherein the polymerase comprises the amino acid sequence of SEQ ID NO: 2.

13. The polymerase of claim 12, wherein the polymerase is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 4.

14. An expression vector comprising the polynucleotide of claim 13.

15. A host cell comprising the vector of claim 14.

16. A method for producing a mutant polymerase comprising culturing the host cell of claim 15 under conditions in which the polymerase is expressed, and recovering the polymerase.

17. The polymerase of claim 10, wherein the polymerase increases the incorporation of 2'- OMe modified nucleotides into an oligonucleotide relative to a wild-type T3 RNA polymerase that lacks the altered amino acids.

18. The polymerase of claim 10, wherein the polymerase exhibits decreased discrimination against 2'-OMe modified nucleotides relative to a wild-type T3 RNA polymerase that lacks the altered amino acids.

19. A method for identifying an aptamer that binds to a target, wherein the aptamer comprises a modified nucleotide, comprising the steps: a) preparing a transcription reaction mixture comprising: (i) the mutant T3 RNA polymerase of claim 1 or claim 10, (ii) one or more 2'-OMe modified nucleotide triphosphates (2'-0Me NTPs), (iii) magnesium ions, (iv) manganese ions and (v) one or more double- stranded oligonucleotide transcription templates; b) preparing a candidate mixture of single-stranded nucleic acids by transcribing the one or more oligonucleotide transcription templates under conditions whereby the mutant polymerase incorporates the 2'-0Me NTPs into each nucleic acid of the candidate mixture, wherein each nucleic acid of the candidate mixture comprises a 2'-0Me modified nucleotide; c) contacting the candidate mixture with the target; d) partitioning the nucleic acids having an increased affinity to the target from the remainder of the candidate mixture; and e) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby aptamers are identified.

20. A method for preparing a nucleic acid comprising one or more 2'-0Me modified nucleotides comprising the steps:

(a) preparing a transcription reaction mixture comprising: (i) the mutant T3 RNA polymerase of claim 1 or claim 10, (ii) one or more 2'-0Me modified nucleotide triphosphates (2'-0Me NTPs), (iii) magnesium ions, (iv) manganese ions and (v) one or more double- stranded oligonucleotide transcription templates; and

(b) contacting the one or more double-stranded oligonucleotide transcription templates with the mutant polymerase under conditions whereby the mutant polymerase incorporates the one or more 2'-0Me NTPs into a nucleic acid transcription product.