JP2011504357A - Materials and methods for generating transcripts containing modified nucleotides - Google Patents

Abstract

Materials and methods are provided for generating aptamer therapeutics in which modified nucleotide triphosphates are introduced into the sequence. In one embodiment, the present invention provides a method of transcribing a nucleic acid comprising a) (i) a modified T7 RNA polymerase (ii) at least one nucleic acid transcription template and (iii) a nucleotide triphosphate. Preparing a transcription reaction mixture; and b) transferring the transcription reaction mixture under conditions resulting in a single-stranded nucleic acid.

Description

REFERENCE TO RELATED APPLICATIONS This patent application claims, under United States Patent Act §119 (e), the benefit of the following provisional application: US Provisional Patent Application No. 60 / filed on December 22, 2006. No. 876,780 and US Provisional Patent Application No. 60 / 879,830, filed Jan. 10, 2007. Each of these is hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION The present invention relates to materials and methods for transcription of nucleic acids, particularly modified enzymes and materials for increasing the introduction of modified nucleotides into nucleic acids, particularly aptamers, using modified enzymes in template-directed polymerization. Regarding the method.

  Aptamers, by definition, are isolated nucleic acid molecules that bind with high specificity and affinity to a target, such as a protein, through interactions other than Watson-Crick base pairing. Aptamers are nucleic acid based molecules, but there are fundamental differences between aptamers and other nucleic acid molecules such as genes and mRNAs. In the latter, the nucleic acid structure encodes information by its linear base sequence, and thus this sequence is an important sequence for information storage function. In stark contrast, aptamer function based on highly specific binding of a target molecule is dependent on a specific secondary / tertiary structure rather than a conserved linear base sequence. That is, aptamers are non-coding sequences. Any coding capability that an aptamer can have is entirely accidental and plays no role in binding of the aptamer to the relevant target. Thus, aptamers that bind to the same target, and even to the same site on the target, may have similar linear base sequences, but most are not.

  Aptamers must also be distinguished from natural nucleic acid sequences that bind to certain proteins. These latter sequences are natural sequences embedded in the genome of an organism that bind to a specific subgroup of proteins involved in the transcription, translation and transport of natural nucleic acids, ie nucleic acid binding proteins. On the other hand, aptamers are short, isolated, non-naturally occurring nucleic acid molecules. Aptamers that bind to nucleic acid binding proteins can be identified, but in most cases, such aptamers have little or no sequence identity to sequences that are recognized by nucleic acid binding proteins in nature. More importantly, aptamers can bind virtually any protein (not just nucleic acid binding proteins), as well as almost any target of interest, including small molecules, carbohydrates, peptides, and the like. For most targets, even a protein does not have a natural nucleic acid sequence to which it binds. For targets having such sequences, ie nucleic acid binding proteins, such sequences differ from aptamers due to their relatively low binding affinity compared to aptamers that bind strongly.

  Aptamers, like peptides produced by phage display or antibodies, can bind to selected targets with high specificity and modulate target activity or binding interactions. For example, aptamer binding can block their ability to function. Similar to antibodies, this functional property of high specificity binding to a target is an inherent property. Also, like antibodies, those skilled in the art cannot know the exact structural properties of an aptamer to a target, but know how to identify, create, and use such molecules in the absence of an accurate structural definition.

  Aptamers have the ability to dramatically (on the order of magnitude) affect aptamer binding and / or other activity (s), no matter how seemingly minor, a single structural change. It is similar to small molecule drugs. On the other hand, some structural changes have little or no effect. This arises from the importance of the secondary / tertiary structure of the aptamer. In other words, an aptamer is a three-dimensional structure maintained in a fixed conformation that provides chemical contact that binds its given target with high specificity. Consequently, (1) several regions or specific sequences are essential (a) as specific contact points with the target and / or (b) as sequences that contact the molecule with the target; (2) Several regions or specific sequences have various variability, for example, nucleotide X must be pyrimidine, nucleotide Y must be purine, or nucleotides X and Y must be complementary. (3) some regions or specific sequences can be arbitrary, i.e. they are basically spacing elements, e.g. arbitrary length sequences of arbitrary length, or PEG molecules, etc. It can also be a non-nucleotide spacer.

  Aptamers have been discovered by an in vitro selection process from a pool of random sequence oligonucleotides and have been generated for over 130 proteins, including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. Typical aptamers are 10-15 kDa in size (20-45 nucleotides), bind targets with nanomolar to sub-nanomolar affinity, and distinguish closely related targets (eg, aptamers are in the same gene family Normally does not bind other proteins). Through a series of structural experiments, aptamers can take advantage of the same types of binding interactions that promote affinity and specificity in antibody-antigen complexes (eg, hydrogen bonding, electrostatic complementarity, hydrophobic contact, steric exclusion) It is shown.

  Aptamers are used for therapeutics and in affinity purification, detection and diagnosis, including high specificity and affinity, biological efficacy, and superior pharmacokinetic properties, depending on the intended use It has a number of desirable characteristics for. In addition, they offer certain competitive advantages over antibodies and other protein biologics, such as:

  1) Speed and control. Aptamers are produced by a complete in vitro process, allowing rapid generation of initial lead compounds, including therapeutic lead compounds. In vitro selection allows tight control of aptamer specificity and affinity and enables the generation of lead compounds, including lead compounds for both toxin and non-immunogenic targets.

  2) Toxicity and immunogenicity. Aptamers, as a class, exhibit therapeutically acceptable toxicity or lack of immunogenicity. Although the efficacy of many monoclonal antibodies can be very limited by the immune response against the antibodies themselves, it is extremely difficult to induce antibodies against aptamers. It is most likely due to the inability of aptamers to be presented by T cells via MHC and that the immune response generally does not recognize nucleic acid fragments.

  3) Administration. Most currently approved antibody drugs are administered by intravenous infusion (typically 2-4 hours), but aptamers can be administered by subcutaneous injection (the bioavailability of aptamers by subcutaneous administration is > 80% in non-patent document 1)). This difference is mainly due to the relatively low solubility and therefore the large amount of most therapeutic mAbs required. Aptamers with good solubility (> 150 mg / mL) and relatively low molecular weight (aptamer: 10-15 kDa; antibody: 150 kDa) can be administered in weekly doses by injection in a volume of less than 0.5 mL. In addition, because aptamers are small in size, they can penetrate conformational stenotic areas where antibodies or antibody fragments cannot penetrate, which is an additional advantage of aptamer-based therapeutic or prophylactic agents.

  4) Scalability and cost. Therapeutic aptamers are chemically synthesized and can therefore be easily scaled as needed to meet production demand. Currently, the difficulty of scaling production limits the availability of some biologics, and the capital cost of large protein production plants is enormous, but a single large-scale oligonucleotide synthesizer is Can produce at least 100 kg / year and requires relatively little initial investment. The current cost of goods for aptamer synthesis at the kilogram scale is estimated at $ 500 / g, comparable to the cost of highly optimized antibodies. Continuing process development improvements are expected to reduce the cost of goods to <$ 100 / g in five years.

  5) Stability. Therapeutic aptamers are chemically powerful. They are essentially configured to recover activity after exposure to factors such as heat and denaturing agents and can be stored as lyophilized powders at room temperature for extended periods (> 1 year). In contrast, antibodies must be stored refrigerated.

  In addition to the intrinsic stability of aptamers, the introduction of modified nucleotides (eg 2'-modified nucleotides) into aptamers can increase resistance to enzymatic, chemical, thermal and physical degradation. The introduction of modified nucleotides during the SELEX ™ process is often preferred over the introduction of modified nucleotides after aptamer identification, but has historically been difficult due to low transcription yields. Furthermore, it is beneficial to increase the chemical and structural diversity of the pool or library from which the aptamer is selected. The present invention provides improved materials and methods that meet this and other needs.

Tucker et al. Chromatography B.M. (1999) 732: 203-212.

  Currently relates to T7 RNA polymerase, which can be purified, isolated, and / or recombinant. As used herein, the term “isolated” includes a polymerase of the invention when recombinantly expressed in a cell or tissue. As used herein, the term “isolated” includes a nucleic acid sequence of the invention introduced into a cell or tissue. In one embodiment, a T7 RNA polymerase comprising modified amino acids at positions 639 and 784, where the modified amino acid at position 639 is not phenylalanine when the modified amino acid at position 784 is alanine is provided. In another embodiment, the T7 RNA polymerase described above is further provided that further comprises a modified amino acid at position 378. In another embodiment, the T7 RNA polymerase described above is further provided, further comprising a modified amino acid at position 266. In certain embodiments, the modified amino acid at position 639 is leucine and the modified amino acid at position 784 is alanine. In a further embodiment, the modified amino acid at position 266 is leucine. In a further embodiment, the modified amino acid at position 378 is arginine. As used herein, the term “modified amino acid” is modified relative to the corresponding wild-type amino acid at the specified position.

  In a preferred embodiment, the modified amino acid is a nucleotide triphosphate wherein the guanidine triphosphate is 2'-OMe guanidine triphosphate, preferably all nucleotide triphosphates are 2'-OMe nucleotide triphosphates. Increase the transcription yield of nucleic acids containing 2′-OMe modification by polymerase in a transcription reaction containing In certain embodiments, the increase in transcription yield is relative to T7 RNA polymerase lacking a modified amino acid when transcription is performed for both the modified amino acid T7 RNA polymerase and the T7 RNA polymerase lacking the modified amino acid under the same transcription conditions. It is. In another embodiment, the modified amino acid reduces discrimination against 2'-OMe nucleotide triphosphates, particularly 2'-OMe guanidine triphosphate. In certain embodiments, the reduced discrimination for 2′-OMe nucleotide triphosphates may be greater for T7 RNA polymerase lacking modified amino acids, particularly wild type T7 RNA polymerase, when both polymerases are used under the same transcription conditions. It is against. In certain embodiments of this aspect, the T7 RNA polymerase lacking the modified amino acid is a wild type T7 RNA polymerase, except that the amino acid at position 639 is changed to phenylalanine and the amino acid at position 784 is changed to alanine, or Wild-type amino acid sequence except that phenylalanine is replaced with tyrosine at position 639, alanine is replaced with histidine at position 784, and arginine residue is replaced with a lysine residue at position 378 (Y639F / H784A / K378R) Is a mutant polymerase.

  In certain embodiments, an isolated polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6. In certain embodiments, a kit is provided that includes a container comprising a T7 RNA polymerase of the invention.

  In some embodiments, a method of transcribing a single stranded nucleic acid is provided comprising incubating the mutant T7 RNA polymerase of the invention with a template nucleic acid under reaction conditions sufficient to effect transcription. In some embodiments, the template nucleic acid, also referred to as an oligonucleotide transcription template, is at least partially double stranded. For example, the template nucleic acid is at least 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, over the entire length of the template. 95%, 96%, 97%, 98% or 99% duplex. In some embodiments, the template nucleic acid is completely double stranded over the entire length of the template.

  In another embodiment, an isolated nucleic acid encoding a polypeptide of the invention is provided. In certain embodiments, a nucleic acid encoding a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6 is provided. In certain embodiments, a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37 is provided. In some embodiments, a vector comprising the isolated nucleic acid sequence of the present invention is provided. In certain embodiments, an expression vector comprising a nucleic acid of the invention operably linked to a promoter is provided. In another embodiment of the invention, a cell comprising the expression vector of the invention is provided. In certain embodiments, cells are provided in which a mutant T7 RNA polymerase of the invention is expressed. In some embodiments, a kit is provided that includes a container comprising a nucleic acid encoding a T7 RNA polymerase of the invention. In some embodiments, provided is a method of producing a mutant T7 polymerase of the invention, eg, a polymerase comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6. The In some embodiments, the production method includes expressing an expression vector encoding a mutant T7 polymerase of the invention in a cell. In some embodiments, the production method further comprises purifying the expressed mutant T7 polymerase of the invention.

  In another embodiment, a) incubating a template nucleic acid in a reaction mixture under conditions comprising a mutant RNA polymerase, a nucleic acid transcription template, and a nucleoside triphosphate, wherein the nucleoside triphosphate is 2′-OMe. And b) transcribing the transcription reaction mixture to yield a single stranded nucleic acid, wherein the first nucleotide of the transcript (ie, the 5 ′ terminal nucleotide) may not be 2 ′ modified. Except that all nucleotides of the single stranded nucleic acid are 2′-OMe modified, and a method of transcribing a complete 2′-OMe nucleic acid is provided. In some embodiments, the first nucleoside of the transcript can be 2'-OH guanidine. In some embodiments of the method, the mutant RNA polymerase is a mutant T7 RNA polymerase comprising modified amino acids at positions 639 and 784, particularly a T7 RNA polymerase comprising modified amino acids at positions 639 and 784, wherein the modified amino acid at position 784. When T is alanine, the modified amino acid at position 639 is not a phenylalanine, particularly a T7 RNA polymerase further comprising a modified amino acid at position 378 and / or a modified amino acid at position 266. In certain embodiments, the modified amino acid at position 639 of the polymerase used in the methods of the invention is leucine and the modified amino acid at position 784 is alanine. In a further embodiment, the modified amino acid at position 266 of the polymerase used in the methods of the invention is leucine. In a further embodiment, the modified amino acid at position 378 of the polymerase used in the method of the invention is arginine. In certain embodiments, an isolated polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6 is provided. In some embodiments, the template nucleic acid, also referred to as an oligonucleotide transcription template, is at least partially double stranded. For example, the template nucleic acid is at least 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, over the entire length of the template. 95%, 96%, 97%, 98%, or 99% duplex. In some embodiments, the template nucleic acid is completely double stranded over the entire length of the template.

  In some embodiments of the method of the present invention, the transcription reaction further comprises magnesium ions. In another embodiment, the transcription reaction further comprises manganese ions. In some embodiments, the transcription reaction further includes both magnesium and manganese ions. In another embodiment, the magnesium ions are present in the transcription reaction at a concentration that is 3.0 to 3.5 times higher than the manganese ions. In another embodiment where each nucleotide triphosphate is present in the transcription reaction at a concentration of 1.0 mM, the concentration of magnesium ions is 6.5 mM and the concentration of manganese ions is 2.0 mM. In another embodiment where each nucleotide triphosphate is present in the transcription reaction at a concentration of 1.5 mM, the magnesium ion concentration is 8 mM and the manganese ion concentration is 2.5 mM. In another embodiment where each nucleotide triphosphate is present in the transcription reaction at a concentration of 2.0 mM, the magnesium ion concentration is 9.5 mM and the manganese ion concentration is 3.0 mM.

  In another embodiment, the transcription reaction includes non-2′-OMe guanosine non-triphosphate residues, particularly guanosine monophosphate, guanosine diphosphate, 2′fluoroguanosine monophosphate, 2′fluoroguanosine diphosphate. Non-2'- selected from the group consisting of phosphoric acid, 2'-aminoguanosine monophosphate, 2'-aminoguanosine diphosphate, 2'-deoxyguanosine monophosphate, and 2'-deoxyguanosine diphosphate Further included are OMe guanosine non-triphosphate residues. In another embodiment, the transcription template includes a T7 RNA polymerase promoter. In another embodiment, the transcription reaction further comprises polyethylene glycol. In another embodiment, the transcription reaction includes inorganic pyrophosphatase.

  In another aspect of the invention, a method for identifying aptamers is provided. In one embodiment, a) providing a transcription reaction mixture comprising a mutant polymerase of the invention and one or more nucleic acid transcription templates; and) producing a candidate mixture of single stranded nucleic acids. A step wherein all but one of the nucleotides of the single stranded nucleic acid are 2 ′ modified, c) contacting the candidate mixture with the target molecule, and d) the candidate mixture Separating the nucleic acid with increased affinity for the target molecule relative to the affinity of the target from the candidate mixture; and e) amplifying the nucleic acid with increased affinity to obtain a mixture enriched in the aptamer. The aptamer to the target molecule is all 2'-modified nucleotides, except that the first nucleotide of the aptamer can be 2'-unmodified Including including the step of, but is identified, methods of identifying aptamers is provided. In some embodiments, the amplification step f) includes (i) selectively dissociating nucleic acids with increased affinity from the target; and ii) increasing affinity dissociated from the nucleic acid-target complex. Reverse transcription of the nucleic acid, iii) amplifying the reverse transcribed nucleic acid with increased affinity; (ii) containing the amplified reverse transcribed nucleic acid with increased affinity as a transcription template The steps include preparing a transcription reactant mixture and transferring the transcription mixture.

  In some embodiments of the aptamer identification method of the invention, the mutant RNA polymerase is a mutant T7 RNA polymerase comprising modified amino acids at positions 639 and 784, particularly T7 RNA polymerase comprising modified amino acids at positions 639 and 784; When the modified amino acid at position 784 is alanine, the modified amino acid at position 639 is not a phenylalanine, particularly a T7 RNA polymerase further comprising a modified amino acid at position 378 and / or a modified amino acid at position 266. In certain embodiments, the modified amino acid at position 639 of the polymerase used in the methods of the invention is leucine and the modified amino acid at position 784 is alanine. In a further embodiment, the modified amino acid at position 266 of the polymerase used in the methods of the invention is leucine. In a further embodiment, the modified amino acid at position 378 of the polymerase used in the method of the invention is arginine. In certain embodiments, an isolated polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6 is used in the aptamer identification method of the invention. In some embodiments, the template nucleic acid, also referred to as an oligonucleotide transcription template, is at least partially double stranded. For example, the template nucleic acid is at least 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, over the entire length of the template. 95%, 96%, 97%, 98%, or 99% duplex. In some embodiments, the template nucleic acid is fully double stranded over the entire length of the template.

  In some embodiments, all nucleotide triphosphates in the transcription reaction are 2'-OMe modified. In one embodiment, the one or more nucleic acid transcription templates include a T7 RNA polymerase promoter and a leader sequence immediately 3 'to the T7 RNA polymerase promoter. In some embodiments of this aspect, the method includes iteratively repeating steps a) -e).

  In some embodiments, the transcription reactant further comprises magnesium ions. In another embodiment, the transcription reaction further comprises manganese ions. In another embodiment, magnesium ions are present in the transcription reaction at a concentration that is 3.0 to 3.5 times higher than manganese ions. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction at a concentration of 1.0 mM, the concentration of magnesium ions is 6.5 mM and the concentration of manganese ions is 2.0 mM. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction at a concentration of 1.5 mM, the magnesium ion concentration is 8 mM and the manganese ion concentration is 2.5 mM. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction at a concentration of 2.0 mM, the magnesium ion concentration is 9.5 mM and the manganese ion concentration is 3.0 mM.

  In another embodiment of this aspect, the transcription reaction for use in the aptamer identification method of the invention includes non-2′-OMe guanosine non-triphosphate residues, particularly guanosine monophosphate, guanosine diphosphate. 2 'fluoroguanosine monophosphate, 2' fluoroguanosine diphosphate, 2'-aminoguanosine monophosphate, 2'-aminoguanosine diphosphate, 2'-deoxyguanosine monophosphate, and 2'-deoxyguanosine Further included are non-2′-OMe guanosine non-triphosphate residues selected from the group consisting of diphosphates. In another embodiment, the transcription template includes a T7 RNA polymerase promoter. In another embodiment, the transcription reaction further comprises polyethylene glycol. In another embodiment, the transcription reaction includes inorganic pyrophosphatase. In some embodiments, the transcription reaction further includes both magnesium and manganese ions.

  In another embodiment, a) incubating the template nucleic acid in a reaction mixture comprising a mutant polymerase, a nucleic acid transcription template, and a nucleoside triphosphate, wherein the guanidine triphosphate is 2′-OMe; B) transcribing the transcription reaction mixture under conditions that give rise to single-stranded nucleic acid, except for the first nucleotide of the transcript, all guanidine nucleotides of the resulting single-stranded nucleic acid are 2′-OMe modified. A method of transcribing a nucleic acid comprising the steps of:

  In some embodiments of the method, the mutant RNA polymerase is a mutant T7 RNA polymerase comprising modified amino acids at positions 639 and 784, particularly a T7 RNA polymerase comprising modified amino acids at positions 639 and 784, wherein the modified amino acid at position 784. When T is alanine, the modified amino acid at position 639 is not a phenylalanine, particularly a T7 RNA polymerase further comprising a modified amino acid at position 378 and / or a modified amino acid at position 266. In certain embodiments, the modified amino acid at position 639 of the polymerase used in the methods of the invention is leucine and the modified amino acid at position 784 is alanine. In a further embodiment, the modified amino acid at position 266 is leucine. In a further embodiment, the modified amino acid at position 378 of the polymerase used in the method of the invention is arginine. In a particular embodiment, the transcription method of the invention uses an isolated polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the template nucleic acid, also referred to as an oligonucleotide transcription template, is at least partially double stranded. For example, the template nucleic acid is at least 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, over the entire length of the template. 95%, 96%, 97%, 98%, or 99% duplex. In some embodiments, the template nucleic acid is fully double stranded over the entire length of the template.

  In some embodiments of the transcription method, where all guanidine triphosphates in the reaction mixture are 2'-OMe, the cytidine triphosphate in the reaction mixture is 2 'deoxy. In some embodiments of the method, wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, the thymidine triphosphate in the reaction mixture is 2 'deoxy. In some embodiments of the method where all guanidine triphosphates in the reaction mixture are 2'-OMe, the uridine triphosphate in the reaction mixture is 2'-OH. In some embodiments of the method where all guanidine triphosphates in the reaction mixture are 2'-OMe, the uridine triphosphate in the reaction mixture is 2'-deoxy. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, cytidine triphosphate is 2'-deoxy and thymidine and adenosine triphosphate are 2'-deoxy. OMe. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, cytidine triphosphate is 2'-deoxy and uridine and adenosine triphosphate are 2'-deoxy. OMe. In some embodiments of the method, wherein all guanidine triphosphates in the reaction mixture are 2′-OMe, thymidine triphosphate 2′-deoxy, and uridine and adenosine triphosphate are 2′-OMe. It is. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, uridine triphosphate is 2'-deoxy and uridine and adenosine triphosphate are 2'-deoxy. OMe. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, uridine triphosphate is 2'-OH and uridine and adenosine triphosphate are 2'-OMe. OMe.

  In some embodiments of the transfer method of the present invention, the transcription reaction mixture further comprises magnesium ions. In another embodiment, the transcription reaction further comprises manganese ions. In another embodiment, the transcription reaction further includes both magnesium and manganese ions. In another embodiment, magnesium ions are present in the transcription reaction at a concentration that is 3.0 to 3.5 times higher than manganese ions. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction mixture at a concentration of 1.0 mM, the concentration of magnesium ions is 6.5 mM and the concentration of manganese ions is 2.0 mM. In another embodiment where each nucleotide triphosphate is present in the transcription reaction mixture at a concentration of 1.5 mM, the concentration of magnesium ions is 8 mM and the concentration of manganese ions is 2.5 mM. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction mixture at a concentration of 2.0 mM, the concentration of magnesium ions is 9.5 mM and the concentration of manganese ions is 3.0 mM.

  In another embodiment, the transcription reaction mixture comprises non-2′-OMe guanosine non-triphosphate residues, in particular guanosine monophosphate, guanosine diphosphate, 2 ′ fluoroguanosine monophosphate, 2 ′ fluoroguanosine diphosphate. Non-2'- selected from the group consisting of phosphoric acid, 2'-aminoguanosine monophosphate, 2'-aminoguanosine diphosphate, 2'-deoxyguanosine monophosphate, and 2'-deoxyguanosine diphosphate OMe guanosine non-triphosphate residues are included. In another embodiment, the transcription template includes a T7 RNA polymerase promoter. In another embodiment, the transcription reaction further comprises polyethylene glycol. In another embodiment, the transcription reaction includes inorganic pyrophosphatase. In some embodiments, the transcription reaction further includes both magnesium and manganese ions.

  In another embodiment, a) preparing a transcription reaction mixture comprising a mutant polymerase, one or more nucleic acid transcription templates, and a nucleoside triphosphate, wherein the guanidine triphosphate is 2′-OMe. And b) transcribing the transcription reaction mixture under conditions that yield a candidate mixture of single stranded nucleic acids, wherein all but one of the guanosine nucleotides of the single stranded nucleic acid are selectively 2′-modified. C) contacting the candidate mixture with the target molecule; d) separating nucleic acids having increased affinity for the target molecule relative to the affinity of the candidate mixture from the candidate mixture; e) aptamers Is a step of amplifying nucleic acid with increased affinity to obtain a concentrated mixture, all 2 'except for the first nucleotide of the aptamer. Comprising the step of aptamer to the target molecule containing a modified guanidine nucleotide is identified, methods of identifying aptamers is provided. In one embodiment, the amplification step comprises (i) selectively dissociating the nucleic acid with increased affinity from the target; and ii) the nucleic acid with increased affinity released from the nucleic acid-target complex, Reverse transcription; iii) amplifying the reverse transcribed nucleic acid with increased affinity; (ii) a transcription reaction mixture comprising the amplified reverse transcribed nucleic acid with increased affinity as a transcription template. Preparing and transferring the transfer mixture.

  In some embodiments of the aptamer identification method of the invention, the mutant RNA polymerase is a mutant T7 RNA polymerase comprising modified amino acids at positions 639 and 784, particularly T7 RNA polymerase comprising modified amino acids at positions 639 and 784; When the modified amino acid at position 784 is alanine, the modified amino acid at position 639 is not a phenylalanine, particularly a T7 RNA polymerase further comprising a modified amino acid at position 378 and / or a modified amino acid at position 266. In certain embodiments, the modified amino acid at position 639 of the polymerase used in the methods of the invention is leucine and the modified amino acid at position 784 is alanine. In a further embodiment, the modified amino acid at position 266 is leucine. In a further embodiment, the modified amino acid at position 378 is arginine. In a particular embodiment, the aptamer identification method of the invention uses a polymerase comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the template nucleic acid, also referred to as an oligonucleotide transcription template, is at least partially double stranded. For example, the template nucleic acid is at least 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, over the entire length of the template. 95%, 96%, 97%, 98%, or 99% duplex. In some embodiments, the template nucleic acid is fully double stranded over the entire length of the template.

  In some embodiments of the aptamer identification method where all guanidine triphosphates in the reaction mixture are 2'-OMe, the cytidine triphosphate in the reaction mixture is 2 'deoxy. In some embodiments of the method, wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, the thymidine triphosphate in the reaction mixture is 2 'deoxy. In some embodiments of the method where all guanidine triphosphates in the reaction mixture are 2'-OMe, the uridine triphosphate in the reaction mixture is 2'-OH. In some embodiments of the method where all guanidine triphosphates in the reaction mixture are 2'-OMe, the uridine triphosphate in the reaction mixture is 2'-deoxy. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, cytidine triphosphate is 2'-deoxy and thymidine and adenosine triphosphate are 2'-deoxy. OMe. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, cytidine triphosphate is 2'-deoxy and uridine and adenosine triphosphate are 2'-deoxy. OMe. In some embodiments of the method, wherein all guanidine triphosphates in the reaction mixture are 2′-OMe, thymidine triphosphate 2′-deoxy, and uridine and adenosine triphosphate are 2′-OMe. It is. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, uridine triphosphate is 2'-deoxy and uridine and adenosine triphosphate are 2'-deoxy. OMe. In some embodiments of the method wherein all guanidine triphosphates in the reaction mixture are 2'-OMe, uridine triphosphate is 2'-OH and uridine and adenosine triphosphate are 2'-OMe. OMe.

  In some embodiments of the aptamer identification method of the invention, the transcription reaction mixture further comprises magnesium ions. In another embodiment, the transcription reaction further comprises manganese ions. In another embodiment, the transcription reaction further includes both magnesium and manganese ions. In another embodiment, the magnesium ions are present in the transcription reaction at a concentration that is 3.0 to 3.5 times higher than the manganese ions. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction mixture at a concentration of 1.0 mM, the concentration of magnesium ions is 6.5 mM and the concentration of manganese ions is 2.0 mM. In another embodiment where each nucleotide triphosphate is present in the transcription reaction mixture at a concentration of 1.5 mM, the concentration of magnesium ions is 8 mM and the concentration of manganese ions is 2.5 mM. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction mixture at a concentration of 2.0 mM, the concentration of magnesium ions is 9.5 mM and the concentration of manganese ions is 3.0 mM.

  In another embodiment, the transcription reaction mixture contains non-2′-OMe guanosine non-triphosphate residues, in particular guanosine monophosphate, guanosine diphosphate, 2′fluoroguanosine monophosphate, 2′fluoroguanosine. Non-2'- selected from the group consisting of diphosphate, 2'-aminoguanosine monophosphate, 2'-aminoguanosine diphosphate, T-deoxyguanosine monophosphate, and 2'-deoxyguanosine diphosphate Further included are OMe guanosine non-triphosphate residues. In another embodiment, the transcription template includes a T7 RNA polymerase promoter. In another embodiment, the transcription reaction further comprises polyethylene glycol. In another embodiment, the transcription reaction includes inorganic pyrophosphatase. In some embodiments, the transcription reaction further includes both magnesium and manganese ions.

  In another embodiment, a) preparing a transcription reaction mixture comprising a mutant T7 RNA polymerase, at least one nucleic acid transcription template, and a nucleoside triphosphate, wherein the nucleotide triphosphate comprises guanidine triphosphate. All guanidine triphosphates are 2′-OMe modified; and b) transcribe the transcription reaction mixture under conditions that yield single stranded nucleic acids, all of the single stranded nucleic acids obtained A method of transcribing a nucleic acid is provided, comprising the steps of: wherein one of the guanidine nucleotides is 2′-OMe modified, with the exception of one. In another embodiment, a) providing a transcription reaction mixture comprising a mutant T7 RNA polymerase, at least one nucleic acid transcription template, and a nucleotide triphosphate, wherein the nucleotide triphosphate comprises guanidine triphosphate. All guanidine triphosphates are 2′-OMe modified; and b) transcribing the transcription reaction mixture under conditions resulting in a candidate mixture of single stranded nucleic acids, A step wherein all but one of the guanidine nucleotides are selectively 2 ′ modified, c) contacting the candidate mixture with the target molecule, and d) affinity for the target molecule relative to the affinity of the candidate mixture. Separating the increased nucleic acid from the candidate mixture; and e) increasing the affinity to obtain a mixture enriched in the aptamer. The step of amplifying the aptamer, wherein an aptamer to a target molecule comprising all 2'-OMe-modified guanidine nucleotides, with the exception of one of the aptamer guanidine nucleotides, is identified. A method of identifying is provided. In some embodiments, the amplification step includes (i) selectively dissociating the nucleic acid with increased affinity from the target; and ii) increasing affinity with the nucleic acid-target complex being dissociated. Reverse transcription of the nucleic acid; iii) amplifying the reverse transcribed nucleic acid with increased affinity; and (ii) a transcription reaction comprising the amplified reverse transcribed nucleic acid with increased affinity as a transcription template. The method further includes preparing a product mixture and transferring the transfer mixture. In some embodiments, the method further includes the step of iteratively repeating steps a) -e).

  In some embodiments of this aspect of the methods of the invention, the mutant T7 RNA polymerase comprises a modified amino acid at positions 639 and 784 relative to wild type T7 polymerase, and the modified amino acid at position 784 is alanine. The modified amino acid at position 639 is not phenylalanine, and in particular mutant T7 RNA polymerase contains leucine at position 639 and alanine at position 784. In some embodiments, the mutant T7 RNA polymerase further comprises a modified amino acid at position 378 and / or 266 relative to the wild type. In a further embodiment, the modified amino acid at position 266 is leucine. In a further embodiment, the modified amino acid at position 378 is arginine. In certain embodiments, an isolated polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6 is used in the methods of the invention. In some embodiments, the template nucleic acid, also referred to as an oligonucleotide transcription template, is at least partially double stranded. For example, the template nucleic acid is at least 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, over the entire length of the template. 95%, 96%, 97%, 98%, or 99% duplex. In some embodiments, the template nucleic acid is fully double stranded over the entire length of the template.

  In some embodiments, the transcription reaction mixture comprises at least two additional different types of nucleotide triphosphates selected from the group consisting of cytidine triphosphate, uridine triphosphate, thymidine triphosphate and adenosine triphosphate. Further comprising phosphate, in particular at least two additional different types of nucleotide triphosphates are 2′-OMe modified, in particular all of the different types of nucleotide triphosphates contained in the transcription reaction mixture are 2 ′ -OMe modified.

  In some embodiments of this aspect of the method of the present invention, at least one nucleotide triphosphate comprises a nucleobase modified with a side chain. As used herein, a side chain includes an organic side chain containing at least two atoms, wherein at least one atom is carbon and at least one other atom is not monovalent. In one embodiment, the nucleobase is uridine 5- and 6-position, thymidine 5- and 6-position, cytidine 5- and 6-position and exocyclic amine, adenosine 2-, 7- and 8- Modified at a position selected from the group consisting of the position and exocyclic amine, the 7- and 8-positions of guanosine and the exocyclic amine, in particular a nucleobase, for example pyrimidine, is modified at the 5 position, in particular the modified nucleobase is uracil Or cytosine. In some embodiments, the side chain is a hydrophobic and / or aromatic side chain, particularly a benzyl or indolyl side chain.

In some embodiments, the nucleobase is modified directly at the side chain, but in some embodiments, the nucleobase is modified by a linker that is covalently attached to the side chain, and the linker is -O-, Selected from the group consisting of —NH—, —CO— (carboxy) and —CH ₂ —, —CO—O— and —CO—NH— and —CH ₂ —CO—NH— (CH ₂ ) _n —NH—. , N is 1-6, and in particular the linker is selected from the group consisting of —CO— (carboxy) and —CH ₂ — and —CO—NH—.

  In some embodiments, the nucleotide comprising the modified nucleobase is 2'-OMe. In certain embodiments, all different types of nucleotide triphosphates included in the transcription reaction mixture are 2'-OMe modified, including types of nucleotides that include modified nucleobases. In certain embodiments, the nucleotide comprising the modified nucleobase is 2'-O-methyl-5-indolylmethylene U or 2'-O-methyl-5-benzyl U. In other embodiments, the nucleotide comprising the modified nucleobase is 2'-deoxy or 2'-OH.

  In some embodiments, the transcription reactant further comprises magnesium ions. In another embodiment, the transcription reaction further comprises manganese ions. In some embodiments, the transcription reaction further includes both magnesium and manganese ions. In another embodiment, the magnesium ions are present in the transcription reaction at a concentration that is 3.0 to 3.5 times higher than the manganese ions. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction at a concentration of 1.0 mM, the concentration of magnesium ions is 6.5 mM and the concentration of manganese ions is 2.0 mM. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction at a concentration of 1.5 mM, the magnesium ion concentration is 8 mM and the manganese ion concentration is 2.5 mM. In another embodiment, where each nucleotide triphosphate is present in the transcription reaction at a concentration of 2.0 mM, the magnesium ion concentration is 9.5 mM and the manganese ion concentration is 3.0 mM.

  In some embodiments, the transcription reaction mixture contains non-2′-OMe guanosine non-triphosphate residues, in particular guanosine monophosphate, guanosine diphosphate, 2′fluoroguanosine monophosphate, 2′fluoro. Non-2 selected from the group consisting of guanosine diphosphate, 2′-aminoguanosine monophosphate, 2′-aminoguanosine diphosphate, 2′-deoxyguanosine monophosphate, and 2′-deoxyguanosine diphosphate '-OMe guanosine non-triphosphate residues are included. In another embodiment, the transcription template includes a T7 RNA polymerase promoter. In some embodiments, the nucleic acid transcription template included in the transcription reaction mixture includes a T7 RNA polymerase promoter and a leader sequence immediately 3 'to the T7 RNA polymerase promoter. In another embodiment, the transcription reaction further comprises polyethylene glycol. In another embodiment, the transcription reaction further comprises inorganic pyrophosphatase. In some embodiments, the transcription reaction further includes both magnesium and manganese ions.

  In some embodiments of this aspect of the method of the invention, the step of transcription results in a transcription yield of the single stranded nucleic acid, wherein the same transcription reaction contains the wild type T7 RNA polymerase. Except for inclusion, it is higher than the yield of the same transcription reaction product. Transcription yield is quantified by electrophoresis and visualization and can be expressed in transcript copy number and / or concentration, eg, μM or μg / ml. In this aspect of the method of the invention, the wild type T7 polymerase comprises the amino acid sequence according to SEQ ID NO: 33.

1 is a schematic of an in vitro aptamer selection (SELEX ™) process from a pool of random sequence oligonucleotides. FIG. FIG. 2 shows various PEGylation strategies representing standard mono-PEGylation, multiple PEGylation, and dimerization by PEGylation. It is a nucleic acid (SEQ ID NO: 32). It is the amino acid sequence of wild type T7 RNA polymerase (SEQ ID NO: 33). It is the nucleic acid sequence of the mutant T7 RNA polymerase Y639L / H784A (SEQ ID NO: 34). It is the nucleic acid sequence of T7 mutant polymerase Y639L / H784A / K378R (SEQ ID NO: 35). It is the nucleic acid sequence of the mutant T7 polymerase P266L / Y639L / H784A (SEQ ID NO: 36). It is the nucleic acid sequence of the mutant T7 polymerase P266L / 639L / H784A / K378R (SEQ ID NO: 37). Relative transcripts quantified from PAGE-gel analysis UV-shadowing of ARC2118 and ARC2119 using titration of YG639L / H784A / K37SR mutant T7 RNA polymerase and rGTP (2'-OH GTP) in the transcription mixture Yield is shown. * The given yield is for ARC2118 transcribed with 20 uM rGTP, the highest yield quantified by UV-shadow. Y639L / H784A / K37SR mutant T7 RNA polymerase in the transcription mixture and various concentrations of 2'-OMe NTPs (2'-OMe ATP, 2'-OMe UTP, 2'-OMe CTP, and 2'-OMe GTP), FIG. 6 shows relative transcript yields as determined from UV-shadowing of PAGE-gel analysis of ARC2119 with MgCl ₂ and MnCl ₂ and without rGTP (2′-OH GTP). The yields given are for transcription conditions of 1 mM 2′-OMe NTP, 6.5 mM MgCl ₂ , and 2 mM MnCl ₂ each. Complete 2′-OMe transcription (100% 2′-OMe ATP, 2 ′) by Y639L / H784A / K378R mutant T7 RNA polymerase compared to fidelity of total RNA or 2′-OMe transcription using Y639F / K378R mutant T7 RNA polymerase -OMe UTP, 2'-OMe CTP, and 2'-OMe GTP) nucleotide insertion, deletion and substitution analysis. In the table, (1) indicates “Direct in Vitro Selection of a 2′-O-Methyl Atomer to VEGF” Burmeister et al., (2005) Chemistry and Biology, 12:25. Data from 33 are shown, (2) shows that transcription was performed with LAR T7 mutant polymerase. Complete 2'-OMe transcript before and after one round of complete 2'-OMe transcription with Y639L / H784A / K378R mutant T7 RNA polymerase followed by DNase treatment, reverse transcription, springed ligation, and PCR amplification 2 is a table showing the analysis of% nucleotide composition of (100% 2′-OMe ATP, 2′-OMe UTP, 2′-OMe CTP, and 2′-OMe GTP). 3'-Schematic of a minimized MNA anti-IgE aptamer shown in the 5 'to 3' direction with a cap at the end (dark ball). FIG. 4 is a schematic of a minimized MNA anti-IgE aptamer, a minimized MNA anti-IgE aptamer with two deoxy substitutions, and a minimized MNA anti-IgE aptamer with one deoxy substitution and a phosphorothioate substitution, each in the 5′-3 ′ direction. Each having a cap at the 3'-end (black ball). FIG. 2 shows the level of transcription that introduces 2'-OMe UTP, 2'-OMe ATP, 2'-OMe GTP, 2'-OMe CTP or 2'-OH NTPs using modified and wild type T7 polymerase.

  The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods or materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. Other features, objects and advantages of the invention will be apparent from the description. In the specification, the singular includes the plural unless the context clearly indicates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will prevail.

SELEX ™ Method A preferred method for generating aptamers is shown generally in FIG. 1 and is referred to as “in vitro selection,” “Systemic Evolution of Ligands by Expensive Enrichment” (“SELEX ™”). )). The SELEX ™ process is a method for in vitro evolution of nucleic acid molecules with high specificity binding to a target molecule, for example, the currently abandoned US patent application filed on June 11, 1990. 07/536428, US Pat. No. 5,475,096 entitled “Nucleic Acid Ligands” and US Pat. No. 5,270,163 entitled “Nucleic Acid Ligands” (see also WO 91/19813) Has been. SELEX ™ is used to obtain an aptamer, also referred to herein as a “nucleic acid ligand”, having any desired level of target binding affinity by performing an iterative cycle of selection and amplification. it can.

  The SELEX ™ process acts as a ligand for virtually any chemical compound, whether monomeric or polymeric, with sufficient capacity for nucleic acids to form a variety of 2D and 3D structures (ie, specific binding) Based on the unique insight of having sufficient chemical convertibility in their monomers available to form pairs). Molecules of any size or composition can be targeted.

  The SELEX ™ process is based on the ability to bind a target. Thus, aptamers obtained from the SELEX ™ procedure have the property of binding to the target. However, mere target binding does not provide information on the functional effects that can be exerted on the target by aptamer binding, if any.

  Changing the properties of the target molecule requires the aptamer to bind at a certain position on the target in order to effect a change in the properties of the target. Theoretically, the SELEX ™ method can lead to the identification of multiple aptamers where each aptamer binds to a different site on the target. In practice, aptamer-target binding interactions often occur at one or a relatively small number of preferred binding sites on the target that provide a stable and available structural interface for the interaction. Furthermore, when the SELEX ™ method is performed on a physiological target molecule, the technician typically cannot control the position of the aptamer relative to the target. Thus, the location of the aptamer binding site on the target may or may not be one of multiple potential binding sites that may lead to the desired effect or not affect the target molecule. There is also.

  Even if an aptamer is found to have an effect due to its target binding ability, there is no way to predict the existence of the effect or to predict what the effect will be. In carrying out a SELEX ™ experiment, the technician is only sure that the aptamer has the property of binding the target to the extent that an aptamer to the target can be obtained. Although SELEX (TM) can be performed in the hope that some of the aptamers identified will also have an effect beyond binding to the target, it is uncertain.

  The SELEX ™ process relies on a large library or pool of single stranded oligonucleotides containing randomized sequences as starting points. Oligonucleotides can be modified or unmodified DNA, RNA or DNA / RNA hybrids. In some examples, the pool comprises 100% degenerate or partially degenerate oligonucleotides. In other examples, the pool includes degenerate or partially degenerate oligonucleotides that include at least one fixed and / or conserved sequence introduced into the randomized sequence. In other examples, the pool includes at least one fixed and / or conserved sequence at the 5 ′ and / or 3 ′ end that may include sequences shared by all molecules of the oligonucleotide pool. Includes partially degenerate oligonucleotides. The fixed sequence includes a CpG motif described later, a hybridization site of a PCR primer, a promoter sequence of RNA polymerase (eg, T3, T4, T7, and SP6), a restriction site, or a homopolymer sequence such as a poly A region or a poly T region, Introduced for preselected purposes such as catalytic nuclei, sites of selective binding to affinity columns, leader sequences that promote transcription, and other sequences that facilitate cloning and / or sequencing of the oligonucleotide of interest Sequence common to the pooled oligonucleotides.

  The pool oligonucleotides preferably include a fixed sequence as well as a degenerate sequence portion necessary for efficient amplification. Typically, the starting pool oligonucleotide comprises fixed 5 'and 3' terminal sequences flanking an internal region of 30-40 random nucleotides. Degenerate nucleotides can be made from randomly cleaved cellular nucleic acids by a number of methods including chemical synthesis and size selection. It is also possible to introduce or increase sequence variations in the test nucleic acid by mutagenesis before or during the selection / amplification iteration.

The degenerate sequence portion of the oligonucleotide can be of any length, can include ribonucleotides and / or deoxyribonucleotides, and can include modified or unnatural nucleotides or nucleotide analogs. For example, U.S. Patent Nos. 5,958,691; 5,660,985; 5,958,691; 5,698,687; 5,817,635; 5,672,695. No. and International Publication No. WO 92/07065. Degenerate oligonucleotides can be synthesized from phosphodiester-linked nucleotides using oligonucleotide solid phase synthesis techniques well known in the art. For example, Froehler et al., Nucl. Acid Res. 14: 5399-5467 (1986) and Froehler et al., Tet. Lett. 27: 5575-5578 (1986). Random oligonucleotides can also be synthesized using a liquid phase method such as a triester synthesis method. See, for example, Sood et al., Nucl. Acid Res. 4: 2557 (1977) and Hirose et al., Tet. Lett. 28: 2449 (1978). A typical synthesis performed on an automated DNA synthesizer yields 10 ¹⁶ to 10 ¹⁷ individual molecules, which is sufficient for most SELEX ™ experiments. A sufficiently large region of degenerate sequence in the sequence design increases the likelihood that each synthetic molecule represents a unique sequence.

  The starting library of oligonucleotides can be generated by automated chemical synthesis on a DNA synthesizer. To synthesize degenerate sequences, a mixture of all four nucleotides is added at each nucleotide addition step during the synthesis process to allow for the stochastic introduction of nucleotides. As described above, in one embodiment, a random oligonucleotide includes a fully degenerate sequence, while in other embodiments, a degenerate oligonucleotide can include a sequence of non-random or partially random sequences. By adding four nucleotides at different molar ratios at each addition step, a partially random sequence can be generated.

  When an RNA library is used as the starting library, typically, the DNA library is synthesized, selectively PCR amplified, and then transcribed in vitro using T7 RNA polymerase or modified T7 RNA polymerase, It is generated by purifying the generated library. The RNA or DNA library is then mixed with the target under conditions favorable for binding, and stepwise iterations of binding, separation and amplification are performed using the same general selection scheme, with virtually any binding capacity and selectivity. Desired criteria are achieved. More particularly, the SELEX ™ method starts with a mixture comprising an initiating pool of nucleic acids, (a) contacting the mixture with a target under conditions favorable for binding; (b) specifically binding to a target molecule. Separating unbound nucleic acid from the treated nucleic acid; (c) selectively dissociating the nucleic acid-target complex; and (d) nucleic acid-target complex to obtain a ligand-enriched nucleic acid mixture. Amplifying nucleic acid dissociated from the body; (e) binding, separation, dissociation, and amplification steps in the required number of cycles to obtain a high specificity, high affinity nucleic acid ligand for the target molecule. Including repeating steps. In the case where an RNA aptamer is selected, the SELEX ™ method comprises (i) reverse transcribing the nucleic acid dissociated from the nucleic acid-target complex prior to amplification in step (d); (ii) The method further includes the step of transcribing the amplified nucleic acid from step (d) before resuming the process.

  Within a nucleic acid mixture containing a large number of potential sequences and structures, there are various binding affinities for a given target. Those having a high affinity for the target (low dissociation constant) are most likely to bind to the target. After separation, dissociation and amplification, a second nucleic acid mixture is generated and candidates with higher binding affinity are enriched. Until the resulting nucleic acid mixture is mainly composed of only one or several sequences, the optimal ligand is gradually selected through additional rounds of selection. These can then be cloned, sequenced and individually tested for the ability to affect 1) target binding affinity; and / or 2) target function as ligands or aptamers.

The selection and amplification cycle is repeated until the desired goal is achieved. In the most general case, selection / amplification is continued until no significant improvement in binding strength is achieved by repeated cycles. The method is typically used for sampling about 10 ¹⁴ different nucleic acid species, but can also be used for sampling as many as about 10 ¹⁸ different nucleic acid species. In general, nucleic acid aptamer molecules are selected in a 5-20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stage and does not occur throughout the replication process.

  In one embodiment of the SELEX ™ method, the selection process is very efficient in isolating the nucleic acid ligand that binds most strongly to the selected target, so that the selection and amplification cycle is performed once. It ’s enough. Such an efficient selection is, for example, a chromatographic type in which the ability of the nucleic acid to bind to the target bound to the column works in such a way that the column is well tolerated for the separation and isolation of the highest affinity nucleic acid ligands. Can occur in the process.

  In many cases, it is not always desirable to perform iterative steps of the SELEX ™ process until a single nucleic acid ligand is identified. A target-specific nucleic acid ligand solution can comprise a family of nucleic acid structures or motifs having a large number of conserved sequences and a number of sequences that can be substituted or added without significantly affecting the affinity of the nucleic acid ligand for the target. By terminating the SELEX ™ process prior to completion, it is possible to determine the sequence of multiple members of the nucleic acid ligand solution family.

  It is known that there are primary, secondary and tertiary structures of various nucleic acids. The structures or motifs most commonly shown to be involved in non-Watson-Crick interactions are called hairpin loops, symmetric and asymmetric bulges, pseudoknots, and myriad combinations thereof. Almost all of the known cases of such motifs suggest that they can be formed in nucleic acid sequences of only 30 nucleotides or less. For this reason, a SELEX ™ procedure using consecutive randomized segments is initiated with a nucleic acid sequence comprising a randomized segment of about 20-50, in some embodiments between about 30-40 nucleotides. Often it is preferred. In one example, the 5'-fixed: random: 3'-fixed sequence comprises a random sequence of about 30 to about 40 nucleotides.

  The basic SELEX (TM) method has been modified to achieve many specific objectives. For example, US Pat. No. 5,707,796 describes the use of the SELEX ™ process combined with gel electrophoresis to select nucleic acid molecules having specific structural characteristics such as bent DNA. US Pat. No. 5,763,177 describes a SELEX ™ -based method for selecting nucleic acid ligands that contain a photoreactive group that can bind to and / or photocrosslink and / or inactivate the target molecule. Describes the method. US Pat. Nos. 5,567,588 and 5,861,254 describe SELEX ™ that achieves very efficient separation of oligonucleotides with high and low affinity for the target molecule. The base method is described. US Pat. No. 5,496,938 describes a method for obtaining improved nucleic acid ligands after the SELEX ™ process has been performed. US Pat. No. 5,705,337 describes a method for covalently binding a ligand to a target.

  The SELEX ™ method can also be used to obtain nucleic acid ligands that bind to one or more sites on the target molecule and to obtain nucleic acid ligands that contain non-nucleic acid species that bind to specific sites on the target . The SELEX ™ method includes macromolecules and small molecules, such as nucleic acid binding proteins and proteins that are not known to bind to nucleic acids as part of their biological function, and cofactors and other small molecules Means are provided for isolating and identifying nucleic acid ligands that bind to any possible target. For example, US Pat. No. 5,580,737 discloses nucleic acid sequences identified by the SELEX ™ method that can bind with high affinity to caffeine and its closely related analog, theophylline.

  The Counter-SELEX ™ process is a method for improving the specificity of a nucleic acid ligand for a target molecule by eliminating nucleic acid ligand sequences that have cross-reactivity to one or more non-target molecules. The Counter-SELEX ™ process includes (a) preparing a candidate mixture of nucleic acids; and (b) contacting the candidate mixture with a target, with increased affinity for the target compared to the candidate mixture. Selecting a nucleic acid having increased affinity from the target; and (c) separating nucleic acid having increased affinity from the remainder of the candidate mixture; and (d) selecting nucleic acid having increased affinity from the target. (E) the increased affinity nucleic acid with one or more non-target molecules such that a nucleic acid ligand with specific affinity for the non-target nucleic acid molecule (s) is removed. (F) only the target molecule to obtain a mixture of nucleic acid sequences enriched for nucleic acid sequences having a relatively high affinity and specificity for binding to the target molecule; Amplifying a nucleic acid having a specific affinity for include step. As described above for the SELEX ™ method, the selection and amplification cycles are repeated as necessary until the desired goal is achieved.

One potential problem faced in the use of nucleic acids as therapeutic agents and vaccines is that the oligonucleotides in their phosphodiester form can be activated before intracellular enzymes such as endonucleases and exonucleases and extracellular before the desired effect appears. It can be rapidly degraded in body fluids by enzymes. Thus, the SELEX ™ method involves the identification of high affinity nucleic acid ligands containing modified nucleotides that give the ligand improved properties such as improved in vivo stability or improved delivery properties. Examples of such modifications include chemical substitutions at the sugar and / or phosphate and / or base positions. SELEX ™ containing modified nucleotides-US Pat. No. 6,036,095 describes an oligonucleotide wherein the identified nucleic acid comprises a nucleotide derivative chemically modified, for example, at the 2 ′ position of ribose, the 5 position of pyrimidine, and the 8 position of purine No. 5,660,985, US Pat. No. 5,756,703 describing oligonucleotides containing various 2′-modified pyrimidines, and 2′-amino (2′-NH ₂ ), 2′-fluoro (2 US Pat. No. 5,580,737 describing highly specific nucleic acid ligands comprising one or more nucleotides modified with a '-F) and / or 2'-O-methyl (2'-OMe) substituents In the issue.

Nucleic acid ligand modifications contemplated in the present invention include nucleic acid ligand bases or other chemical groups that introduce additional charge, polarity, hydrophobicity, hydrogen bonding, electrostatic interactions, and fluidity into the nucleic acid ligand as a whole. Are provided, but are not limited to these. Modifications to generate a population of oligonucleotides that are resistant to nucleases can also include one or more substituted internucleotide linkages, modified sugars, modified bases, or combinations thereof. Such modifications include 2′-position sugar modification, 5-position pyrimidine modification, 8-position purine modification, modification with exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodouracil; Backbone modifications, phosphorothioate or alkyl phosphate modifications, methylation, and unusual base pair combinations such as, but not limited to, the isobases isocytidine and isoguanosine. Modifications can also include 3 ′ and 5 ′ modifications such as capping. Modifications can also include 3 ′ and 5 ′ modifications such as capping, eg, addition of a 3′-3′-dT cap to increase exonuclease resistance (eg, each herein by reference in its entirety). (See US Pat. Nos. 5,674,685; 5,668,264; 6,207,816; and 6,229,002, which are incorporated herein by reference.)
In one embodiment, the P (O) O group is P (O) S (“thioate”), P (S) S (“dithioate”), P (O) NR ₂ (“amidate”), P (O ) R, P (O) OR ′, CO or CH ₂ (“formacetal”), or oligonucleotides substituted with 3′-amine (—NH—CH ₂ —CH ₂ —) are provided, each R or R ′ is independently H or substituted or unsubstituted alkyl. A linking group can be attached to an adjacent nucleotide via a —O—, —N—, or —S— linkage. Not all bonds in an oligonucleotide need be identical.

  In further embodiments, the oligonucleotide comprises a modified sugar group, eg, one or more hydroxyl groups are substituted with a halogen, an aliphatic group, or functionalized as an ether or amine. In one embodiment, the 2 'position of the furanose residue is substituted with either an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group. Methods for synthesizing 2'-modified sugars are described in, for example, Sproat et al., Nucl. Acid Res. 19: 733-738 (1991); Cotton et al., Nucl. Acid Res. 19: 2629-2635 (1991); and Hobbs et al., Biochemistry 12: 5138-5145 (1973). Other modifications are known to those skilled in the art. Such modifications can be pre-SELEX ™ process modification or post-SELEX ™ process modification (modification of previously identified unmodified ligands) or can be made by incorporation into the SELEX ™ process.

  What is made by SELEX (TM) pre-modification or incorporation into the SELEX (TM) process allows for a nucleic acid ligand that has both specificity for its SELEX (TM) target and improved stability, e.g., in vivo stability. Arise. A SELEX ™ post-process modification to a nucleic acid ligand (eg, a previously identified ligand cleavage, deletion, substitution or additional nucleotide modification introduced with a nucleotide by a SELEX ™ pre-process modification) It can lead to improved stability, eg in vivo stability, without adversely affecting the binding capacity of the ligand.Optionally, aptamers in which modified nucleotides have been introduced by SELEX ™ pre-modification can be used as SELEX ™ It can be further modified by post-process modification (ie SELEX ™ post-modification process after SELEX).

  The SELEX ™ method can be used to select selected oligonucleotides from other selected oligonucleotide and non-oligonucleotide functions as described in US Pat. Nos. 5,637,459 and 5,683,867. Including the step of combining with the unit. The SELEX ™ method uses selected nucleic acid ligands for diagnostics as described, for example, in US Pat. Nos. 6,011,020, 6,051,698, and WO 98/18480. Or further comprising combining with a lipophilic or non-immunogenic high molecular weight compound in a therapeutic complex. These patents and patent applications teach a wide range of shapes and combinations of other properties, along with efficient amplification and replication properties of oligonucleotides, and desired properties of other molecules.

  The identification of nucleic acid ligands for small flexible peptides by the SELEX ™ method has also been investigated. Small peptides have a flexible structure and are usually present in solution in the equilibrium state of multiple conformers, so that binding affinity can be limited by loss of conformational entropy when binding flexible peptides. At first it was thought. However, the feasibility of identifying nucleic acid ligands for small peptides in solution was shown in US Pat. No. 5,648,214. In this patent, a high affinity RNA nucleic acid ligand for substance P of the 11 amino acid peptide was identified.

  As part of the SELEX ™ process, the sequence selected to bind to the target is then selectively minimized to determine the minimum sequence with the desired binding affinity. The selected sequence and / or the minimized sequence can be used for random or directed mutagenesis of the sequence, for example to increase binding affinity or to determine which positions in the sequence are essential for binding activity. Is selectively modified.

Modified Nucleotide SELEX ™ Method For aptamers to be suitable for use as therapeutic agents and / or certain types of diagnostic agents, it is preferred that the synthesis be inexpensive, safe and stable in vivo. Wild-type RNA and DNA aptamers are not stable in vivo because they are usually susceptible to degradation by nucleases. Introduction of a modifying group at the 2′-position can greatly increase resistance to nuclease degradation.

  In addition, the introduction of chemical modifications to the oligonucleotide library from which the aptamer is selected increases the structural and chemical diversity of the oligonucleotide library, resulting in high affinity and highly specific aptamers with particularly desirable properties The number potentially increases. For example, in many applications it is desirable to obtain complete 2′-OMe aptamers, but such aptamers can be combined into a single aptamer that modulates, eg, inhibits, the function of the target to a sufficient degree in a given application. It may not always be possible to obtain from a pool of compositions, eg, oligonucleotides that are all 2'-OMe except the first nucleotide. In this case, it is desirable to select aptamers from other pool compositions such as a pool containing oligonucleotides in which all but one nucleotide is 2'-OMe, such as a library obtained from a dCmD transcription composition. Let ’s go. It is also desirable, and in another embodiment, the present invention provides a method for selecting aptamers from a pool composition comprising an oligonucleotide having a 2'-OMe modification and a side chain modification at the nucleobase position.

  2'-fluoro and 2'-amino groups have been successfully introduced into the oligonucleotide pool from which aptamers have been selected. However, these modifications greatly increase the cost of synthesis of the resulting aptamer, and the modified nucleotide can be reused in the host DNA by degradation of the modified oligonucleotide and subsequent use of the nucleotide as a substrate for DNA synthesis. In some cases, this can lead to safety issues.

  Aptamers comprising 2'-O-methyl ("2'-OMe") nucleotides provided herein overcome many of these disadvantages. Oligonucleotides containing 2'-OMe nucleotides are nuclease resistant and inexpensive to synthesize. Although 2'-OMe nucleotides are ubiquitous in biological systems, natural polymerases do not accept 2'-OMe NTPs as a substrate under physiological conditions and are therefore concerned with the reuse of 2'-OMe nucleotides into host DNA. There are no safety issues. The SELEX ™ method used to generate 2′-modified aptamers is described, for example, in US Provisional Application No. 60 / 430,761, filed on Dec. 3, 2002, on Jul. 15, 2003. U.S. Provisional Application No. 60 / 487,474, U.S. Provisional Application No. 60 / 517,039, filed Nov. 4, 2003, U.S. Patent Application No. 10 / filed on Dec. 3, 2003 No. 729,581, U.S. Patent Application No. 10 / 873,856 filed June 21, 2004, entitled "Method for in vitro Selection of 2'-O-methyl Substituted Nucleic Acids", June 2005. Materials and Methods for the Generalatio, filed on 30th n of Fully 2'-Modified Nucleic Acid Transscripts, US Provisional Application No. 60 / 696,292, and Materials and Methods for the Fully of Full 2 ' No. 11 / 480,188, entitled Transscripts, each of which is incorporated herein by reference in its entirety.

  The present invention is an aptamer that binds to an aptamer target and modulates its function to make the oligonucleotide more stable against enzymatic and chemical degradation, and thermal and physical degradation than unmodified oligonucleotides. Aptamers are included, including modified nucleotides (eg, nucleotides having modifications at the 2′-position). The present invention is an aptamer that binds to an aptamer target and regulates its function, modified with a 2 ′ position, eg, a 2′-OMe modification, and a nucleobase position, eg, a side chain modification at position 5 of a pyrimidine Aptamers that contain nucleotides are also included. There are several examples of aptamers containing 2′-OMe in the literature (eg, Ruckman et al., J. Biol. Chem, 1998 273, 20556-20567-695), which contain 2′-C and U residues. It was generated by in vitro selection of a library of modified transcripts that were fluoro (2'-F) substituted and the A and G residues were 2'-OH. Once the functional sequence is identified, the tolerance of each A and G residue to 2'-OMe substitution is tested and all A and G residues that are tolerant to 2'-OMe substitution as 2'-OMe residues are tested. Aptamers with groups were resynthesized. Most A and G residues of aptamers generated in this two-step manner are tolerant of substitution by 2'-OMe residues, but on average do not allow about 20%. Thus, aptamers produced using this method tend to contain 2-4 2'-OH residues, resulting in a loss of stability and synthesis costs. The methods of the invention are used in oligonucleotide pools where aptamers are selected and enriched by the SELEX ™ method (and / or any variation and improvement thereof, including those described herein). Need to stabilize selected aptamer oligonucleotides by re-synthesizing aptamer oligonucleotides with 2'-OMe modified nucleotides by introducing modified nucleotides into the transcription reaction that produce stabilized oligonucleotides Is lost.

In one embodiment, the present invention provides an aptamer comprising a combination of 2'-OH, 2'-F, 2'-deoxy, and 2'-OMe modifications of ATP, GTP, CTP, TTP, and UTP nucleotides. To do. In another embodiment, the present invention provides ATP, GTP, CTP, TTP, and UTP nucleotides 2′-OH, 2′-F, 2′-deoxy, 2′-OMe, 2′-NH ₂ , and An aptamer comprising a combination of T-methoxyethyl modifications is provided. In preferred embodiments, the present invention provides aptamers comprising ATP, GTP, CTP, TTP, and / or UTP nucleotides that are all or substantially all 2′-OMe modified. In another preferred embodiment, the present invention provides aptamers in which all nucleotide triphosphate groups, ATP, GTP, CTP, TTP and / or UTP are 2′-OMe modified except for one. For example, aptamers that include 2′-OMe ATP, GTP, TTP, but CTP is deoxy. The present invention further provides aptamers comprising nucleobase modifications by side chains in combination with any preceding 2 'modification combination.

  In another preferred embodiment, the present invention provides three 2′-OMe modified nucleotide triphosphate groups and one deoxy or 2′-OH nucleotide, except that the starting nucleotide need not be 2 ′ modified. An aptamer containing a triphosphate group is provided. In certain embodiments, the aptamer comprises deoxycytidine nucleotide triphosphates, and the remaining nucleotide triphosphates are 2'-OMe modified except that the starting nucleotide need not be 2'modified. In another embodiment, the aptamer comprises deoxythymidine nucleotide triphosphates, and the remaining nucleotide triphosphates are 2'-OMe modified except that the starting nucleotide need not be 2'modified. In another embodiment, the aptamer comprises a 2′-OH uridine nucleotide triphosphate, and the remaining nucleotide triphosphate is 2′-OMe modified, except that the starting nucleotide need not be 2 ′ modified. ing. In another embodiment, the aptamer comprises deoxyguanosine nucleotide triphosphates and the remaining nucleotides are modified with 2'-OMe except that the starting nucleotide need not be 2'modified. In another embodiment, the aptamer comprises deoxyadenosine nucleotide triphosphates and the remaining nucleotides are 2'-OMe modified, except that the starting nucleotide need not be 2'modified.

Modified Polymerase The 2′-modified aptamer of the present invention is produced using a modified polymerase having a higher introduction rate of a modified nucleotide having a large substituent at the 2′-position of furanose than a wild-type polymerase, for example, wild-type T7 polymerase, such as a mutant T7 polymerase. It is done. For example, a mutant T7 polymerase in which the tyrosine residue at position 639 is changed to phenylalanine (Y639F) readily utilizes 2′deoxy, 2′amino-, and 2′fluoro-nucleotide triphosphates (NTPs) as substrates. However, it is widely used to synthesize modified RNAs for various uses. However, it has been reported that this mutant T7 polymerase cannot readily utilize (ie introduce) NTPs with large 2′-substituents such as 2′-OMe or 2′-azido (2′-N ₃ ) substituents. ing. In addition to the Y639F mutation, a mutant T7 polymerase in which histidine at position 784 has been changed to an alanine residue has been described (Y639F / H784A) in order to introduce large 2 ′ substituents, introducing modified pyrimidine NTPs. Because of this, it is used in limited situations. Padilla, R .; And Sousa, R .; , Nucleic Acids Res. 2002, 30 (24): 138. A mutant T7 RNA polymerase (Y639F / H784A / K378R) in which the tyrosine residue at position 639 is changed to phenylalanine, the histidine residue at position 784 is changed to alanine, and the lysine residue at position 378 is changed to arginine is modified. Used in limited circumstances to introduce purine and pyrimidine NTPs, such as 2'-OMe NTPs, but includes 2'-OH GTP spikes for transcription. See Burmeister et al. (2005) Chemistry and Biology, 12: 25-33. Inclusion of 2'-OH GTP spikes for transcription can result in aptamers that can depend entirely on the presence of 2'-OH GTP but not 2'-OMe.

  A mutant T7 polymerase in which histidine at position 784 has been changed to an alanine residue (H784A) has also been described. Padilla et al., Nucleic Acids Research, 2002, 30: 138. In both the Y639F / H784A mutation and the H784A mutant T7 polymerase, changes to smaller amino acid residues such as alanine allow the introduction of larger nucleotide substrates, such as 2'-OMe substituted nucleotides. Chellery, K.M. And Ellington, A .; D. (2004) Nature Biotech, 9: 1155-60. Additional T7 RNA polymerases with mutations in the active site of T7 RNA polymerase, such as a mutant T7 RNA polymerase in which the tyrosine residue at position 639 was changed to leucine (Y639L), which more easily introduce large 2 ′ modified substrates, are described. ing. However, the increase in substrate specificity conferred by such mutations often sacrifices activity and reduces transcription yield. Padilla R and Sousa, R .; (1999) Nucleic Acids Res. 27 (6): 1561. The T7 RNA polymerase mutation P266L has been described to promote promoter clearance (Guillerez et al. (2005) Proc. Nat. Acad. Sci. USA, 102 (17) 5958). The polymerase changes from an initial conformation bound to the promoter to an unbound extension conformation. None of the above mutant polymerases has been reported to yield complete 2'-OMe transcripts.

  The present invention provides materials and methods for increasing the transcription yield of oligonucleotides. In one embodiment, the present invention is for using modified T7 RNA polymerase (modification of T7 RNA polymerase to wild-type T7 RNA polymerase, SEQ ID NO: 33) to enzymatically introduce modified nucleotides into oligonucleotides. Provide methods and conditions. In a preferred embodiment, the mutant T7 RNA polymerase used in the transcription method of the present invention does not require the presence of 2'-OH GTP. In a preferred embodiment, the modified polymerase is a mutant T7 RNA polymerase in which the tyrosine residue at position 639 is changed to a leucine residue and the histidine residue at position 784 is changed to an alanine residue (Y639L / H784A). In another preferred embodiment, the modified polymerase comprises a tyrosine residue at position 639 is changed to a leucine residue, a histidine residue at position 784 is changed to an alanine residue, and a lysine residue at position 378 is an arginine residue. (Y639L / H784A / K378R), a mutant T7 RNA polymerase. In another embodiment, the modified polymerase used in the methods of the present invention has a tyrosine residue at position 639 changed to a leucine residue (Y639L), and in yet another embodiment, the mutant T7 RNA polymerase has position 639. The tyrosine residue was changed to a leucine residue, and the lysine residue at position 378 was changed to an arginine residue (Y639L / K378R). Without being bound by any theory, the K378R mutation is not near the active site of the polymerase and is therefore considered to be a silent mutation. In another embodiment, the modified polymerase used in the method of the present invention is such that the proline residue at position 266 is changed to leucine, the tyrosine residue at position 639 is changed to leucine, and the histidine residue at position 784 is alanine. (P266L / Y639L / H784A) mutated T7 RNA polymerase, and in yet another embodiment, the mutated T7 RNA polymerase has a proline residue at position 266 changed to leucine and a tyrosine residue at position 639 Is changed to a leucine residue, a histidine residue at position 784 is changed to an alanine residue, and a lysine residue at position 378 is changed to an arginine residue (P266L / Y639L / H784A / K378R).

  The amino acid sequence of the mutant T7 RNA polymerase is shown below.

２’修飾（例えば２’―ＯＭｅまたは２’―ＯＭｅおよび核酸塩基側鎖修飾）ＲＮＡ転写物のプールを生成するために、変異ポリメラーゼが２’―修飾ＮＴＰｓを許容する条件下における、Ｙ６３９Ｆ、Ｙ６３９Ｆ／Ｋ３７８Ｒ、Ｙ６３９Ｆ／Ｈ７８４Ａ、Ｙ６３９Ｆ／Ｈ７８４Ａ／Ｋ３７８Ｒ、Ｙ６３９Ｌ／Ｈ７８４Ａ、Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ、Ｙ６３９Ｌ、Ｙ６３９Ｌ／Ｋ３７８Ｒ、Ｐ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４ＡまたはＰ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼが使用されうる。好ましいポリメラーゼは、Ｙ６３９Ｌ／Ｈ７８４Ａ変異Ｔ７ＲＮＡポリメラーゼである。別の好ましいポリメラーゼは、Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼである。本発明における別の好ましいポリメラーゼは、Ｐ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４ＡまたはＰ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼである。他のＴ７ＲＮＡポリメラーゼ、特に大きな２’―置換基に対する高い寛容性をもつものも、本発明の方法で使用できる。本明細書に開示される条件を用いたテンプレート指向性重合において使用される場合には、Ｙ６３９Ｌ／Ｈ７８４Ａ、Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ、Ｐ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４ＡまたはＰ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼが、Ｙ６３９Ｆ、Ｙ６３９Ｆ／Ｋ３７８Ｒ、Ｙ６３９Ｆ／Ｈ７８４Ａ、Ｙ６３９Ｆ／Ｈ７８４Ａ／Ｋ３７８Ｒ、Ｙ６３９Ｌ、またはＹ６３９Ｌ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼを用いて達成されるより高い転写収率で、２’―ＯＭｅ ＧＴＰを含み、核酸塩基の位置で側鎖修飾もされた２’―ＯＭｅ ＮＴＰＳも含む、全て２’―ＯＭｅのＮＴＰｓの導入のために使用されうる。Ｙ６３９Ｌ／Ｈ７８４Ａ、Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ、Ｐ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４ＡまたはＰ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼは、２’―修飾、例えば２’―ＯＭｅを含むオリゴヌクレオチドの高収率を達成するために、２’―ＯＨ ＧＴＰを伴って使用されうるがその必要はない。

Y639F, Y639F, under conditions where mutant polymerases tolerate 2′-modified NTPs to generate a pool of 2′-modified (eg, 2′-OMe or 2′-OMe and nucleobase side chain modifications) RNA transcripts / K378R, Y639F / H784A, Y639F / H784A / K378R, Y639L / H784A, Y639L / H784A / K378R, Y639L, Y639L / K378R, P266L / Y639L / H784A or P266L / Y639L / Y739 / 783 A preferred polymerase is Y639L / H784A mutant T7 RNA polymerase. Another preferred polymerase is Y639L / H784A / K378R mutant T7 RNA polymerase. Another preferred polymerase in the present invention is P266L / Y639L / H784A or P266L / Y639L / H784A / K378R mutant T7 RNA polymerase. Other T7 RNA polymerases, particularly those with high tolerance to large 2′-substituents, can also be used in the methods of the invention. When used in template-directed polymerization using the conditions disclosed herein, Y639L / H784A, Y639L / H784A / K378R, P266L / Y639L / H784A or P266L / Y639L / H784A / K378R mutant T7 RNA polymerase is Including 2'-OMe GTP with higher transcription yields achieved using Y639F, Y639F / Y378F / K378R, Y639F / H784A, Y639F / H784A / K378R, Y639L, or Y639L / K378R mutant T7 RNA polymerase, All 2′-OMe NTPs can also be used for introduction, including 2′-OMe NTPS with side chain modifications in position. Y639L / H784A, Y639L / H784A / K378R, P266L / Y639L / H784A or P266L / Y639L / H784A / K378R mutant T7 RNA polymerase to achieve high yields of oligonucleotides including 2'-modifications, eg 2'-OMe Can be used with 2'-OH GTP, but this is not necessary.

  In a preferred embodiment, the Y639L / H784A or Y639L / H784A / K378R mutant T7 RNA polymerase of the present invention is used with a MNA transcription mixture to promote higher full 2'-OMe transcription yields. In some embodiments, at least one oligonucleotide of the MNA transcription mixture also includes a side chain change at the nucleobase position. In another preferred embodiment, the Y639L / H784A or Y639L / H784A / K378R mutant T7 RNA polymerase of the present invention has a higher transcription in which all guanosine triphosphates other than the first nucleotide of the transcript are 2′-OMe. Used with dCmD, dTmV, rTmV, rUmV or dUmV transcription mixture to facilitate yield. In some embodiments, the Y639L / H784A or Y639L / H784A / K378R mutant T7 RNA polymerase of the invention is used with a dCmD, dTmV, rTmV, rUmV or dUmV transcription mixture and all but the first nucleotide of the transcript. In order to promote higher transcription yields where the guanosine triphosphate is 2'-OMe, at least one nucleotide type in the mixture also includes a side chain modification at its nucleobase position, and one nucleotide type is a nucleic acid Also includes base side chain modifications. In some embodiments, Y639L / H784A or Y639L / H784A / K378R mutant T7 RNA polymerase can be used with rRmY, dRmY, rGmH, fGmH, dGmH, dAmB, rRdY, dRdY or rN transcription mixtures. In some embodiments, any one of Y639L / H784A, Y639L / H784A / K378R, P266L / Y639L / H784A or P266L / Y639L / H784A / K378R mutant T7 RNA polymerase is dCmD, dTmV, rUmV, dHV, It can be used in rGmH, alternating mixtures, fGmH, rAmB or dAmB transcription reaction mixtures. In certain embodiments, any one of Y639L / H784A, Y639L / H784A / K378R, P266L / Y639L / H784A or P266L / Y639L / H784A / K378R mutant T7 RNA polymerase is transcribed in a dCmD, dTmV, rUmV or dUmV reaction. Can be used.

  As used herein, 2'-OMe A, G, C and U triphosphates (2'-OMe ATP, 2'-OMe UTP, 2'-OMe CTP, and 2'-OMe GTP) A transcription mixture that contains only an MNA mixture and aptamers selected therefrom are called MNA aptamers and contain only 2'-O-methyl nucleotides. A transcription mixture containing 2'-OMe C and U and 2'-OH A and G is referred to as an "rRmY" mixture, and aptamers selected therefrom are referred to as "rRmY". A transcription mixture containing deoxy A and G, and 2'-OMe U and C is referred to as a "dRmY" mixture, and aptamers selected therefrom are referred to as "dRmY" aptamers. A transcription mixture containing 2'-OMe A, C and U, and 2'-OH G is referred to as an "rGmH" mixture, and aptamers selected therefrom are referred to as "rGmH" aptamers. Transcription mixtures containing 2′-OMe A, C, U and G, and 2′-OMe A, U and C, and 2′-FG alternately are called “alternate mixtures” and aptamers selected therefrom Are called “alternate mixture” aptamers. A transcription mixture containing 2'-OMe A, U, and C, and 2'-FG is referred to as an "fGmH" mixture, and aptamers selected therefrom are referred to as "fGmH" aptamers. A transcription mixture containing 2'-OMe A, U, and C, and deoxy G is referred to as a "dGmH" mixture, and aptamers selected therefrom are referred to as "dGmH" aptamers. A transcription mixture containing deoxy A and 2'-OMe C, G and U is referred to as a "dAmB" mixture, and aptamers selected therefrom are referred to as "dAmB" aptamers. A transcription mixture containing 2'-OH A and 2'-OMe C, G and U is referred to as a "rAmB" mixture, and aptamers selected therefrom are referred to as "rAmB" aptamers. A transcription mixture containing 2'-OH adenosine triphosphate and guanosine triphosphate and deoxycytidine triphosphate and thymidine triphosphate is referred to as an rRdY mixture, and aptamers selected therefrom are referred to as "rRdY" aptamers. A transcription mixture containing 2'-OMe A, U or T, and G, and deoxy C is referred to as a "dCmD" mixture, and aptamers selected therefrom are referred to as "dCmD" aptamers. A transcription mixture containing 2'-OMe A, G, and C, and deoxy T is referred to as a "dTmV" mixture, and aptamers selected therefrom are referred to as "dTmV" aptamers. A transcription mixture comprising 2'-OMe A, C, and G, and 2'-OH U is referred to as a "rUmV" mixture, and aptamers selected therefrom are referred to as "rUmV" aptamers. A transcription mixture containing 2'-OMe A, C, and G, and 2'-deoxy U is referred to as a "dUmV" mixture, and aptamers selected therefrom are referred to as "dUmV" aptamers. A transcription mixture containing all 2′-OH nucleotides is called an “rN” mixture, aptamers selected therefrom are called “rN”, “rRrY” or RNA aptamers, and a transcription mixture containing all deoxynucleotides is “ Aptamers, referred to as “dN” mixtures, are selected as “dN” or “dRdY” or DNA aptamers.

  2'-modified oligonucleotides can be synthesized from fully modified nucleotides or from a subset of modified nucleotides. All nucleotides can be modified and all can contain the same modification. All nucleotides are modified but may contain different modifications. For example, all nucleotides containing the same base can have one type of modification, and nucleotides containing other bases can have different types of modifications. All purine nucleotides have one type of modification (or no modification) and all pyrimidine nucleotides can have another (or unmodified) different type of modification. In this manner, any combination of modifications can be used to produce a transcript or transcription, including, for example, ribonucleotide (2′-OH), deoxyribonucleotide (2′-deoxy), 2′-F, and 2′-OMe nucleotides. A pool of products is generated. Furthermore, modified oligonucleotides can include nucleotides with two or more modifications at the same time, such as internucleotide linkages (eg, phosphorothioates) and sugars (eg, 2'-OMe) and bases (eg, inosine). In another embodiment, a nucleotide having two or more modifications simultaneously comprises, for example, a 2'-OMe modification and a side chain modification at the nucleobase position.

Transcription Conditions A number of factors have been determined to be important for the transcription conditions of the 2′-modified SELEX ™ method. Factors described for transcription conditions with 2 ′ modifications, in particular transcription mixtures comprising 2′-OMe modified nucleotides, are the transcription methods of the present invention with transcription mixtures comprising 2 ′ modified and side chain nucleobase modified nucleotide triphosphates. Also used in conjunction with. The side chain modified nucleotide triphosphates used in the present invention have been described. For example, Vaught JD, Deway T, Eaton BE., Each of which is incorporated herein by reference in its entirety. J Am Chem Soc. 2004; 126: 11231-11237; Nieuwlandt D, West M, Cheng X, Kirshenheuter G, Eaton BE. Chembiochem. 2003, 4, 651-654; Ito T, Ueno Y, Komatsu Y, Matsuda A. et al. Nucleic Acids Res. 2003; 31, 2514-2523; Dewey, TM, Mundt, AA, Crouch, GJ, Zyzniewski, MC, Eaton, BE. J Am Chem Soc 1995, 117, 8474-8475; Jager S, Rasched G, Kornreich-Leshem H, Engeser M, Thum O, Famulok M .; J Am Chem Soc. 2005, 127, 15071-15082; Tarasow ™, Tarasow SL, Eaton BE. Nature. 1997, 389, 54-57; Liu D, Gugliotti LA, Wu T, Dolska M, Tkachenko AG, Shipton MK, Eaton BE, Feldheim DL. Langmuir. 2006, 22, 5862-5866; Gugliotti LA, Feldheim DL, Eaton BE. J Am Chem Soc. 2005, 127, 17814-17818; Gugliotti LA, Feldheim DL, Eaton BE. Science. 2004, 304, 850-852; Kuwahara M, Hanawa K, Ohsawa K, Kitata R, Ozaki H, Sawai H .; Bioorg Med Chem. 2006, 14, 2518-2526; Saitoh H, Nakamura A, Kuwahara M, Ozaki H, Sawai H. et al. Nucleic Acids Res Suppl. 2002, 2, 215-216; Kuwahara M, Ohbayashi T, Hanawa K, Shoji A, Ozaki AN, Ozaki H, Sawai H. et al. Nucleic Acids Res Suppl. 2002, 2, 83-84. Mehedi Masud M, Ozaki-Nakamura A, Kuwahara M, Ozaki H, Sawai H Chembiochem. 2003, 4, 584-548; Masud MM, Ozaki-Nakamura A, Satou F, Ohbayashi T, Ozaki H, Sawai H. et al. Nucleic Acids Res Suppl. 2001; (l): 21-2; Obayashi T, Masud MM, Ozaki AN, Ozaki H, Kuwahara M, Sawai H .; Bioorg Med Chem Lett. 2002, 12, 1167-70; Sawai H, Ozaki-Nakamura A, Mine M, Ozaki H. . Bioconjug Chem. 2002, 2, 309-316; Ohbayashi T, Kuwahara M, Hasegawa M, Kasamatsu T, Tamura T, Sawai H Org Biomol Chem. 2005, 3, 2463-2468; Shoji A, Hasegawa T, Kuwahara M, Ozaki H, Sawai H. et al. Bioorg Med Chem Lett. 2006 Oct 28; [Epub ahead of print] Kuwahara M, Nagashima J, Hasegawa M, Tamura T, Kitagata R, Hanawa K, Hoshima Shimas, KasatamaS. Nucleic Acids Res. 2006, 34, 5383-5394; Nucleic Acids Sym Ser. 997, 37, 259-260; Schoetzau T, Langner J, Moyroud E, Roehl I, Vonoff S, Klussmann S. Bioconjug Chem. 2003, 5, 919-926; Mehedi Masud M, Ozaki-Nakamura A, Kuwahara M, Ozaki H, Sawai H .; Chembiochem. 2003, 4, 584-588; and Ebara Y, Kaihatsu K, Ueji S .; Nucleic Acids Sym Ser. 1999, 42, 175-176.

  For example, an increase in yield of modified transcript can be observed under some conditions where a particular leader sequence / mutant polymerase combination is used. A leader sequence is a sequence that can be introduced at the 3 'end of a fixed sequence at the 5' end of a DNA transcription template. The leader sequence is typically 6-15 nucleotides in length and is made up of a predetermined nucleotide composition, for example, all purines or a specific mixture of purine and pyrimidine nucleotides.

  Examples of templates that can be used with the mutant polymerases and transcription conditions of the present invention, particularly in combination with Y639L / H784A, Y639L / H784A / K378R, P266L / Y639L / H784A or P266L / Y639L / H784A / K378R, include ARC2118 (SEQ ID NO: 7 ), ARC2119 (SEQ ID NO: 31), and as shown in the Examples below. The template leader sequence used with these mutant polymerases is selective.

  Furthermore, the presence of 2'-OH GTP has historically been an important factor in obtaining transcripts incorporating modified nucleotides. Transcription can be divided into two phases: the first phase is the beginning, during which NTP is added to the 3′-end of GTP (or another substituted guanosine) to give a dinucleotide, which is about 10 The second phase is extension, during which transcription proceeds beyond the addition of the first about 10-12 nucleotides. A small amount of 2'-OH GTP and an excess of 2'OMe GTP added to a transcription mixture containing a Y639F / K378R mutation or a Y639F / H784A / K378R mutant T7 RNA polymerase initiate transcription with 2'-OH GTP by the polymerase. Was previously found to be sufficient to enable (and resulted in a higher transcription yield of transcripts containing 2'-OMe than without 2'-OH GTP) , The introduction of 2'-OMe GTP is mainly allowed due to the reduced sorting between 2'-OMe and 2'-OH GTP and the excess of 2'OMe GTP relative to 2'-OH GTP Is done.

  The present invention provides a mutant T7 RNA polymerase that does not require 2′-OH GTP in the transcription mixture for high yield 2′-OMe transcription, resulting in a transcript in which GTP is 2′-OMe except for the starting nucleotide, For example, Y639L / H784A, Y639L / H784A / K378R, P266L / Y639L / H7S4A or P266L / Y639L / H784A / K378R are provided. In one embodiment, the high yield is on average at least one transcript per input transcription template.

Another factor in the introduction of 2′-OMe substituted nucleotides into the transcript is the use of divalent magnesium and manganese (Mn ²⁺ ) in the transcription mixture. Different combinations of magnesium chloride and manganese chloride concentrations have been found to affect the yield of 2-'O-methylated transcripts, the best concentration of magnesium chloride and manganese chloride being complex with divalent metal ions Depends on the concentration of NTPs in the transcription reaction mixture that forms the body. To obtain the highest yield of all 2-'O-methylated transcripts (ie total 2'-OMe ATP, 2'-OMe UTP, 2'-OMe CTP, and 2'-OMe GTP nucleotides) When each NTP is present at a concentration of 0.5 mM, a magnesium chloride concentration of about 5 mM and a manganese chloride concentration of about 1.5 mM are preferred. When the concentration of each NTP is 1.0 mM, a magnesium chloride concentration of about 6.5 mM and a manganese chloride concentration of 2.0 mM are preferred. When each NTP is present at a concentration of 1.5 mM, a concentration of about 8 mM magnesium chloride and 3.0 mM manganese chloride is preferred. When the concentration of each NTP is 2.0 mM, magnesium chloride at a concentration of about 9.5 mM and manganese chloride at a concentration of 3.0 mM are preferred. In any case, a maximum doubling from these concentrations still gives significant amounts of modified transcripts.

  It is also important for transcription mixtures that do not contain 2'-OH GMP to include 2'-OH GMP, guanosine, or other 2'-OH guanosine substituted at positions other than the 2'-OH sugar position. . This effect arises from the specificity of the polymerase for the starting nucleotide. As a result, the 5'-terminal nucleotide of any transcript produced in this manner is likely 2'-OH guanosine (2'-OH G). A preferred concentration of GMP (or guanosine) is 0.5 mM, more preferably 1 mM. It has also been found that including PEG, preferably PEG-8000, in the transcription reaction is useful to maximize the introduction of modified nucleotides.

Maximum of 2'-OMe ATP (100%), 2'-OMe UTP (100%), 2'-OMe CTP (100%) and 2'-OMe GTP (100%) ("MNA") on transcripts For limiting introduction, the following conditions may be used: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w / v), Triton X-100 0.01% (w / v), MgCl ₂ 8 mM, MnCl ₂ 3.0 mM, 2′-OMe NTP (each) 1.5 mM, 2′-OH GMP 1 mM, pH 7.5, Y639L / H784A / K378R mutant T7 RNA polymerase 200 nM, inorganic pyrophosphatase 0.025 unit / ml, and DNA template. In some embodiments, the DNA template may be present at a concentration of preferably about 5-500 nM.

2′-OMe ATP (100%), 2′-OMe UTP (100%) or 2′-OMe TTP (100%), 2′-OMe GTP (100%) and 2′-deoxy CTP ( 100%) (“dCmD”) for maximal introduction, the following conditions may be used: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w / v), Triton X-100 01% (w / v), MgCl ₂ 8 mM, MnCl ₂ 3.0 mM, 2′-OMe NTP (ATP, UTP or TTP, GTP) 1.5 mM, 2-deoxy CTP 1.5 mM, 2′-OH GMP 1 mM , PH 7.5, Y639L / H784A / K378R mutant T7 RNA polymerase 200 nM, inorganic pyrophosphatase 0.0 5 units / ml, and DNA templates. In some embodiments, the DNA template may be present at a concentration of preferably about 5-500 nM.

Maximum of 2'-OMe ATP (100%), 2'-OMe CTP (100%), 2'-OMe GTP (100%) and 2'-deoxy TTP (100%) ("dTmV") on transcripts For limiting introduction, the following conditions may be used: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w / v), Triton X-100 0.01% (w / v), MgCl ₂ 8 mM, MnCl ₂ 2.5 mM, 2′-OMe NTP (ATP, CTP, GTP) 1.5 mM, 2-deoxy TTP 1.5 mM, 2′-OH GMP 1 mM, pH 7.5, Y639L / H784A / K378R mutant T7 RNA Polymerase 200 nM, inorganic pyrophosphatase 5 units / ml, and DNA template. In some embodiments, the DNA template may be present at a concentration of preferably about 5-500 nM.

Maximum of 2'-OMe ATP (100%), 2'-OMe CTP (100%), 2'-OMe GTP (100%) and 2'-deoxy UTP (100%) ("dUmV") to the transcript For limiting introduction, the following conditions may be used: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w / v), Triton X-100 0.01% (w / v), MgCl ₂ 8 mM, MnCl ₂ 3.0 mM, 2′-OMe NTP (ATP, CTP, GTP) 1.5 mM, 2-deoxy UTP 1.5 mM, 2′-OH GMP 1 mM, pH 7.5, Y639L / H784A / K378R mutant T7 RNA Polymerase 200 nM, inorganic pyrophosphatase 0.025 units / ml, and DNA template. In some embodiments, the DNA template may be present at a concentration of preferably about 5-500 nM.

Maximum of 2'-OMe ATP (100%), 2'-OMe CTP (100%), 2'-OMe GTP (100%) and 2'-OH UTP (100%) ("rUmV") on transcripts For limiting introduction, the following conditions may be used: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w / v), Triton X-100 0.01% (w / v), MgCl ₂ 8 mM, MnCl ₂ 3.0 mM, 2′-OMe NTP (ATP, CTP, GTP) 1.5 mM, 2′-OH UTP 1.5 mM, 2′-OH GMP 1 mM, pH 7.5, Y639L / H784A / K378R mutation T7 RNA template 200 nM, inorganic pyrophosphatase 0.025 units / ml, and DNA template. In some embodiments, the DNA template may be present at a concentration of preferably about 5-500 nM.

  Optionally, the DNA template used in the transcription conditions described above increases the transcription yield relative to a template that does not include such a leader sequence when both templates are transcribed under the same conditions. All contain the purine leader sequence. In another embodiment, the leader sequence is a mixture of purines and pyrimidines that, when both are transcribed under the same conditions, increases transcription yield relative to a template that does not contain such leader sequence. . As used herein, a unit of inorganic pyrophosphatase is defined as the amount of enzyme that liberates 1.0 mole of inorganic orthophosphate per minute at pH 7.2 and 25 ° C. The reaction can be performed for about 1 to 24 hours.

  In each case, the transcript can then be used as input to the SELEX ™ process to identify aptamers and / or determine conserved sequences with binding specificity for a given target. The resulting sequence is already stabilized and this step is removed from the SELEX ™ post-modification process, resulting in a more stabilized aptamer.

  As will be described later, useful yields of transcripts incorporating 2'-substituted nucleotides can be obtained under conditions other than those described above. For example, changes to the above transfer conditions include:

  The concentration of HEPES buffer can range from 0 to 1M. The present invention also contemplates the use of other buffers having a pKa between 5 and 10, including for example tris-hydroxymethyl-aminomethane.

  The concentration of DTT can range from 0 to 400 mM. The method of the present invention also provides for the use of other reducing agents including, for example, mercaptoethanol.

  The concentration of spermidine and / or spermine can range from 0 to 20 mM.

  The concentration of PEG-8000 can range from 0 to 50% (w / v). The methods of the present invention also provide for the use of other hydrophilic polymers including, for example, other molecular weights of PEG or other polyalkylene glycols.

  The concentration of Triton X-100 can range from 0 to 0.1% (w / v). The methods of the present invention also provide for the use of other non-ionic surfactants including other surfactants including, for example, other Triton-X surfactants.

The concentration of MgCl ₂ can range from 0.5 mM to 50 mM. The concentration of MnCl ₂ can range from 0.15 mM to 15 mM. Both MgCl ₂ and MnCl ₂ must be within the stated ranges, and in a preferred embodiment, MgCl ₂ : MnCl ₂ is present in a ratio of about 10 to about 3, preferably the ratio is about 3-5: 1, more preferably the ratio is about 3-4: 1.

  The concentration of 2'-OMe NTP (each NTP) can range from 5 μM to 5 mM.

  The concentration of deoxyNTPs (each NTP) can range from 5 μM to 5 mM.

  The concentration of 2'-OH NTP (each NTP) can range from 5 μM to 5 mM.

  The concentration of 2'-OH GTP (when 2'-OMe GTP is in the range of 5 μM to 5 mM in the reaction mixture) can range from 0 μM to 300 μM. In a preferred embodiment, transcription occurs in the absence of 2'-OH GTP (0 μM).

  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. If 2'-OH GTP is not included in the reaction, 2'-OH GMP is required and can range from 5 μM to 5 mM.

  The concentration of the DNA template can range from 5 nM to 5 μM.

  The concentration of the mutant polymerase can range from 2 nM to 20 μM.

  Inorganic pyrophosphatase can range from 0 to 100 units / ml.

  The pH can range from pH 6 to pH 9. The methods of the invention can be practiced within the pH range of the activity of most polymerases that have introduced modified nucleotides.

  The transcription reaction can be performed for about 1 hour to several weeks, preferably about 1 to about 100 hours.

  Furthermore, the methods of the invention provide for selective use of chelating agents in transcription reaction conditions, including, for example, EDTA, EGTA, and DTT.

Aptamer Medicinal Chemistry
Once an aptamer that binds to the desired target has been identified, several techniques can be selectively performed to further increase the binding and / or functional properties of the identified aptamer sequence. Aptamers that bind to the desired target identified by the SELEX ™ process (eg, 2′-modified SELEX ™), eg, MNA, dCmD, dTmV, rUmV, dUmV aptamer, are desired binding and / or functional properties. Can be selectively cleaved to obtain a minimal aptamer sequence (also referred to herein as a “minimized construct”). One way to achieve this is to use folding programs and sequence analysis (eg, alignment of clone sequences resulting from selection to look for conserved motifs and / or covariations) to know the design of the minimized construct by. Biochemical probing experiments can also be performed to determine the 5 'and 3' boundaries of aptamer sequences in order to know the design of the minimized construct. The minimized constructs can then be chemically synthesized and tested for binding and functional properties compared to the resulting non-minimized sequences. Variants of aptamer sequences containing a series of 5 ′, 3 ′ and / or internal deletions are also directly chemically synthesized and tested for binding and / or functional properties compared to the resulting non-minimized aptamer sequences. Can be done.

  In addition, using dope reselection, single active aptamer sequences such as MNA, dCmD, dTmV, rUmV or dUmV aptamers (ie, identified by the SELEX ™ process, including the (2'-modified SELEX ™ process) Aptamers that bind to the desired target) or sequence requirements within a single minimized aptamer sequence, using a synthetic degenerate pool designed based on a single sequence of interest, Dope reselection takes place, the level of degeneracy typically varies from 70% to 85% from the single sequence of interest, i.e. a single sequence of interest, generally sequences with neutral mutations are identified by the dope reselection process However, in some cases, sequence changes can result in improved affinity, and clones identified using dope reselection Using a synthetic sequence information, to identify the minimal binding motif, it may help medicinal chemistry research.

  Aptamer and / or minimized aptamer sequences identified using the SELEX ™ process (including 2'-modified SELEX process and dope reselection), such as MNA, dCmD, dTmV, rUmV or dUmV aptamers Or use aptamer medicinal chemistry to make directed mutations to increase binding affinity and / or functional properties, or to determine which positions in the sequence are essential for binding activity and / or functional properties. And may be selectively modified by SELEX ™ post-selection.

  Aptamer medicinal chemistry is an aptamer improvement technique in which a series of mutant aptamers are chemically synthesized. These series of variants typically differ from the parent aptamer by the introduction of a single substituent and differ from each other by the position of this substituent. These variants are then compared against each other and the parent. The improvement in properties can be large enough that inclusion of a single substituent may be sufficient to achieve a particular therapeutic criterion.

  Alternatively, information collected from a single mutant set can be used to design additional mutant sets in which two or more substituents are introduced simultaneously. In one design strategy, all single-substituent variants are ranked and the top 4 is selected, and all four possible double (6), triple (4 ) And quadruple (1) combinations are synthesized and tested. In the second design strategy, the best single substituent variant is considered the new parent, and all possible double substituent variants including this highest rank single substituent variant are synthesized and tested. The Other strategies may be used, and these strategies can be repeated so that the number of substituents increases stepwise while continuing to identify further improved variants.

  Aptamer medicinal chemistry can be used in particular as a method for investigating local but not global introduction of substituents. As aptamers are found in libraries generated by transcription, any substituents introduced during the SELEX ™ process must be introduced globally. For example, if it is desired to introduce a phosphorothioate bond between nucleotides, it can only be introduced (entirely substituted) for all A (or all G, C, T, U, etc.). Aptamers that require phosphorothioate for some A (or some G, C, T, U, etc.) (locally substituted) but are not tolerated by others cannot be easily discovered by this process .

  The types of substituents that can be utilized by aptamer medicinal chemistry processes are limited only by the ability to generate them as solid phase synthesis reagents and introduce them into oligomer synthesis schemes. Of course, the process is not limited to nucleotides. Aptamer medicinal chemistry schemes include steric size, hydrophobicity, hydrophilicity, lipophilicity, oleophobicity, positive charge, negative charge, neutral charge, zwitterion, polarizability, nuclease resistance, structural rigidity, structure Substituents that introduce structural flexibility, protein binding properties, mass, etc. may be included. Aptamer medicinal chemistry schemes can include base-modification, sugar-modification or phosphodiester bond-modification.

When considering the types of substituents that can be beneficial in the context of therapeutic aptamers, it is desirable to introduce substitutions that fall into one or more of the following categories:
(1) Substituents already present in the body, such as 2'-deoxy, 2'-ribo, 2'-O-methylpurine or pyrimidine or 5-methylcytosine.

  (2) Substituents that are part of an already approved therapeutic, such as phosphorothioate-linked oligonucleotides.

  (3) Substituents that hydrolyze or decompose into one of the above two categories, such as methylphosphonate-linked oligonucleotides.

  The aptamers of the present invention include aptamers developed by the aptamer medicinal chemistry described herein.

The target binding affinity of the aptamer of the present invention can be assessed by a series of binding reactions between the aptamer and the target (eg, protein), and the trace ³² P-labeled aptamer is incubated with the target dilution system in buffered medium, followed by vacuum filtration Analyzed by nitrocellulose filtration using a manifold. This method, referred to herein as the dot blot binding assay, uses a three-layer filtration medium consisting of nitrocellulose (from top to bottom), nylon filter, and gel blot paper. RNA bound to the target is captured by the nitrocellulose filter, and RNA not bound to the target is captured by the nylon filter. Gel blot paper is included as a support medium for other filters. After filtration, the filter layer is separated, dried and exposed to a fluorescent screen and quantified using a phosphorescent imaging system. The aptamer binding curve can be generated using the quantitative result, and the dissociation constant (K _D ) can be calculated therefrom. In a preferred embodiment, the buffer medium used to perform the binding reaction is 1X Dulbecco's PBS (Ca ⁺⁺ and Mg ⁺⁺ ) and 0.1 mg / mL BSA.

  In general, the ability of an aptamer to modulate the functional activity of a target, ie, the functional activity of an aptamer, can be assessed using in vitro and in vivo models depending on the biological function of the target. In some embodiments, the aptamer of the present invention can inhibit the known biological function of the target, and in other embodiments, the aptamer of the present invention can stimulate the known biological function of the target. . In vitro and in vivo models designed to measure the known function of aptamer targets can be used to assess the functional activity of the aptamers of the invention.

  The aptamers of the present invention can be routinely employed for diagnostic purposes by any number of techniques used by those skilled in the art. Diagnostic applications can include both in vivo and in vitro diagnostic applications. The diagnostic agent need only allow the user to identify the presence of a given target at a particular location or concentration. Simply the ability to form a binding pair with the target may be sufficient to elicit a positive signal for diagnostic purposes. One skilled in the art can adapt any aptamer by methods known in the art to introduce a labeled tag to track the presence of such a ligand. Such tags can be used in a number of diagnostic methods.

Aptamers with immunostimulatory motifs Recognition of bacterial DNA by the vertebrate immune system is based on the recognition of unmethylated CG dinucleotides in a specific sequence context ("CpG motif"). One receptor that recognizes such motifs is Toll-like receptor 9 (“TLR9”), a member of the family of Toll-like receptors (-10 members) involved in innate immune responses by recognizing different microbial components. ]). TLR9 binds unmethylated oligodeoxynucleotide ("ODN") CpG sequences in a sequence specific manner. Recognition of the CpG motif triggers a defense mechanism, leading to innate and ultimately acquired immune responses. For example, activation of TLR9 in mice induces antigen-presenting cell activation, upregulation of MHC class I and II molecules, and expression of key costimulatory molecules and cytokines including IL-12 and IL-23. This activation enhances B and T cell responses directly and indirectly, including strong up-regulation of the TH1 cytokine IFN-gamma. Responses to CpG sequences collectively lead to protection from infection, improved immune response to the vaccine, effective response to asthma, and improved antibody dependence-cell mediated cytotoxicity. Thus, CpG ODNs provide protection from infections and can function as immune adjuvants or cancer therapeutics (monotherapy or in combination with mAbs or other therapies) to reduce asthma and allergic reactions.

  Aptamers of the invention, such as MNA, dCmD, dTmV, rUmV or dUmV aptamers, can include one or more CpG or other immunostimulatory sequences. Such aptamers can be identified or generated by a variety of strategies, for example using the SELEX ™ process described herein. In general, strategies can be divided into two groups. In Group 1, the strategy aims to identify or generate aptamers that contain both a binding site for the target as well as a CpG motif or other immunostimulatory sequence, where the target (hereinafter “non-CpG target”) is CpG Recognizing motifs or other immunostimulatory sequences are targets other than those known to stimulate immune responses when binding to CpG motifs. The first strategy in this group uses oligonucleotide pools in which a CpG motif is introduced into each member of the pool as a fixed region or part thereof (eg, in some embodiments, a randomized region of pool members). Includes a fixed region into which a CpG motif has been introduced), performing SELEX ™ to obtain an aptamer to a specific non-CpG target, and identifying an aptamer containing a CpG motif. A second strategy in this group is to perform a SELEX ™ to obtain an aptamer to a specific non-CpG target, preferably the target, and after selection add a CpG motif to the 5 ′ and / or 3 ′ end or add a CpG motif to the aptamer A step of incorporating into the region, preferably a non-essential region. The third strategy in this group is to perform SELEX ™ to obtain aptamers to specific non-CpG targets, during which the CpG motif in the randomized region of each member of the pool is enriched. As such, the step includes biasing the molar ratio of the various nucleotides in one or more nucleotide addition steps and identifying an aptamer comprising a CpG motif. The fourth strategy in this group includes performing SELEX ™ to obtain aptamers against specific non-CpG targets and identifying aptamers that contain CpG motifs. The fifth strategy of this group includes performing SELEX ™ to obtain aptamers to specific non-CpG targets and identifying aptamers that stimulate an immune response upon binding but do not contain a CpG motif.

  In Group 2, the strategy is to identify or generate aptamers containing CpG motifs and / or other sequences that are bound by a receptor of the CpG motif (eg, TLR9 or other toll-like receptor) and stimulate an immune response upon binding. It aims to. The first strategy in this group is to use an oligonucleotide pool in which a CpG motif is introduced into each member of the pool as a fixed region or part thereof (eg, in some embodiments, a randomized region of pool members). Includes a fixed region into which a CpG motif has been introduced), aptamers for targets known to perform SELEX ™ to bind to CpG motifs or other immunostimulatory sequences and to stimulate immune responses upon binding And identifying an aptamer containing a CpG motif. The second strategy in this group is to perform SELEX ™ to obtain aptamers to targets that are known to bind to CpG motifs or other immunostimulatory sequences and stimulate an immune response upon binding, Adding a CpG motif to the 5 'and / or 3' end or incorporating the CpG motif into a region of the aptamer, preferably a non-essential region. The third strategy in this group is to perform SELEX ™ to obtain aptamers to targets that bind to CpG motifs or other immunostimulatory sequences and are known to stimulate immune responses upon binding. During the synthesis of the pool, biasing the molar ratio of the various nucleotides in one or more nucleotide addition steps such that the randomized region of each member of the pool is enriched in the CpG motif; Identifying an aptamer containing the motif. A fourth strategy in this group is to perform SELEX ™ to obtain aptamers to targets that are known to bind to CpG motifs or other immunostimulatory sequences and stimulate an immune response upon binding. , Identifying an aptamer comprising a CpG motif. The fifth strategy in this group is to perform SELEX ™ to obtain aptamers to targets known to bind to CpG motifs or other immunostimulatory sequences, and to stimulate immune responses upon binding Identifying an aptamer that does not contain a CpG motif.

  Various classes of CpG motifs have been identified, each resulting from recognition in a cascade of different events, release of cytokines and other molecules, activation of certain cell types. See, for example, CpG Motifs in Bacterial DNA and Their Immuno Effects, Annu. Rev. Immunol. 2002, 20: 709-760. Additional immunostimulatory motifs are disclosed in the following US patents, each incorporated herein by reference: US Pat. Nos. 6,207,646; 6,239,116; 6,429 199; 6,214,806; 6,653,292; 6,426,434; 6,514,948 and 6,498,148. These CpGs or other immunostimulatory motifs can be introduced into aptamers. The choice of aptamer depends on the disease or disorder being treated. Preferred immunostimulatory motifs are as follows (indicated from 5 ′ to 3 ′ from left to right): “r” represents purine, “y” represents pyrimidine, “X” represents any nucleotide. Represent:

，ｒＣＧｙ；ｒｒＣＧｙｙ，ＸＣＧＸ，ＸＸＣＧＸＸ，およびＸ_１がＧまたはＡであり、Ｘ_２がＣでなく、Ｙ_１がＧでなく、Ｙ_２が好ましくはＴであるＸ_１Ｘ_２ＣＧＹ_１Ｙ_２。

, RCGy; rrCGy, XCGX, XXCGXX, and X ₁ X ₂ CGY ₁ Y ₂ , wherein X ₁ is G or A, X ₂ is not C, Y ₁ is not G, and Y ₂ is preferably T.

  When a CpG motif is introduced into an aptamer that binds to a specific target other than a target known to bind to and stimulate an immune response upon binding (“non-CpG target”), CpG is It is preferably located in a non-essential region of the aptamer. Non-essential regions of aptamers can be identified by site-directed mutagenesis, deletion analysis and / or substitution analysis. However, any position that does not significantly interfere with the ability of the aptamer to bind to a non-CpG target can be used. CpG motifs can be attached to aptamers, such as added to either or both of the 5 'and 3' ends in addition to being embedded in the aptamer sequence. Any attachment location or means can be used, as long as the ability of the aptamer to bind to the non-CpG target is not significantly hindered.

  As used herein, “stimulation of an immune response” refers to (1) a specific response (eg, induction of a Th1 response) or the production of certain molecules, or (2) a specific response (eg, Th2 Inhibition or suppression of the reaction) or inhibition or suppression of certain molecules.

Modulation of pharmacokinetics and biodistribution of aptamer therapeutics It is important that the pharmacokinetic properties of all oligonucleotide-based therapeutics, including aptamers, are adjusted to suit the desired pharmaceutical application. Although aptamers directed against extracellular targets do not have the problems associated with intracellular delivery (as in the case of antisense and RNAi-based therapeutics), such aptamers are still distributed in the target organs and tissues and desired It should be possible to remain in the body for a period consistent with the drug regimen.

  Accordingly, the present invention provides materials and methods for influencing the pharmacokinetics of aptamer compositions, and in particular the ability to modulate aptamer pharmacokinetics. The tunability of the aptamer pharmacokinetics (ie, the possibility of modulation) is achieved by attaching a modifying moiety (eg PEG polymer) to the aptamer and / or introducing a modified nucleotide (eg 2′-fluoro or 2′-O-methyl). This is achieved by changing the chemical composition of The ability to adjust aptamer pharmacokinetics is used in improving existing therapeutic applications or in developing new therapeutic applications. In some therapeutic applications, it is desirable to reduce the residence time of aptamers in the circulation, for example in the case of anti-tumor or emergency treatment where rapid drug clearance or turn-off may be required. Alternatively, in other therapeutic applications, such as maintenance therapy where systemic circulation of the therapeutic agent is required, it may be desirable to increase the residence time of the aptamer in the circulation.

  Furthermore, the adjustability of aptamer pharmacokinetics is used to alter the biodistribution of aptamer therapeutics 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 specific type of tissue or a specific organ (or series of organs). In these applications, aptamer therapeutics preferentially accumulate in a particular tissue or organ (s). In other therapeutic applications, target tissues that exhibit cellular markers or symptoms associated with a given disease, cell injury, or other abnormal condition so that aptamer therapeutics preferentially accumulate in the affected tissue May be desired. For example, US Provisional Application No. 60/550790, entitled “Controlled Modulation of the Pharmacokinetics and Biodistribution of Polymer Therapeutics”, filed Mar. 5, 2004, and filed March 7, 2005 PEGylation of aptamer therapeutics (eg PEGylation with a 20 kDa PEG polymer) as described in US patent application Ser. No. 10 / -------------------------- PEGylated aptamer therapeutics preferentially accumulate in inflamed tissues In so that, as used in the targeting of inflamed tissue.

Various parameters are monitored to determine the pharmacokinetic and biodistribution profile of aptamer therapeutics (eg, chemically altered aptamers such as aptamer conjugates or modified nucleotides). Such parameters include, for example, half-life (t _1/2 ), plasma clearance (Cl), volume of distribution (Vss), area under the concentration-time curve (AUC), maximum value of serum or plasma concentration (C _max ) and the average residence time (MRT) of the aptamer composition. As used herein, the term “AUC” refers to the area under a plot of plasma concentration of an aptamer therapeutic against time after aptamer administration. The AUC value is used to determine the bioavailability of a given aptamer therapeutic (ie, the proportion of administered aptamer therapeutic in the circulation after aptamer administration) and / or the total clearance (Cl) (ie, The rate of removal) is estimated. The volume of distribution relates the plasma concentration of the aptamer therapeutic to the amount of aptamer present in the body. The larger Vss, the more aptamers are found outside the plasma (ie more extravasation).

  The present invention relates to aptamers such as MNA, dCmD, dTmV, rUmV or dUmV aptamers in vivo by linking them to regulatory moieties such as small molecules, peptides or polymer end groups, or by introducing modified nucleotides into aptamers. Provides materials and methods for modulating the pharmacokinetics and biodistribution of stabilized aptamer compositions, such as MNA, dCmD, dTmV, rUmV or dUmV aptamers, in a controlled manner. As described herein, modification of the modifying moiety and / or alteration of the nucleotide (s) chemical composition alters fundamental aspects of aptamer residence time in circulation and distribution to tissues.

  In addition to nuclease clearance, oligonucleotide therapeutics are removed by renal filtration. As such, nuclease resistant oligonucleotides administered intravenously typically have an in vivo half-life of <10 minutes unless filtration can be blocked. This can be achieved by promoting rapid distribution from the bloodstream to the tissue or by increasing the apparent molecular weight of the oligonucleotide above the effective cut-off size of the glomerulus. Conjugation of small therapeutic agents to PEG polymers, described below (PEGylation), can dramatically extend the residence time of aptamers in the circulation, thereby reducing dosing frequency and increasing efficacy against vascular targets.

Aptamers are high molecular weight polymers such as PEG; peptides such as Tat (13-amino acid fragment of HIV Tat protein (Vives et al. (1997), J. Biol. Chem. 272 (25): 16010-7)), Ant (Drosophila). antennapedia homeodomain tick proteins third helix derived 16 amino acid sequence of (Pietersz, etc. (2001), Vaccine 19 (11-12 ): 1397-405)) and is composed of Arg ₇ (polyarginine (Arg ₇₎ Short positively charged cell penetrating peptides (Rothbard et al. (2000), Nat. Med. 6 (11): 1253-7; Rothbard, J et al. (2002), J. Med. Chem. 45 (17): 3612- 8)); and subdivision , For example, lipophilic compounds such as cholesterol, etc., can be coupled to a variety of modifying moieties. Among the various conjugates described herein, the in vivo properties of aptamers are most altered by complex formation with PEG groups. For example, complexing a mixed 2′F and 2′-OMe modified aptamer therapeutic with a 20 kDa PEG polymer prevents renal filtration and promotes aptamer distribution to healthy and inflamed tissues. Furthermore, the 20 kDa PEG polymer-aptamer conjugate is almost as effective as the 40 kDa PEG polymer in preventing renal filtration of the aptamer. One effect of PEGylation is on aptamer clearance, but the extended systemic exposure caused by the presence of the 20 kDa moiety also promotes the distribution of aptamers to tissues, especially to tissues with high perfusion and sites of inflammation. The aptamer-20 kDa PEG polymer conjugate directs the aptamer distribution to the inflamed site so that the PEGylated aptamer preferentially accumulates in the inflamed tissue. In some cases, the 20 kDa PEGylated aptamer conjugate can access the interior of a cell, such as a kidney cell.

  Modified nucleotides can also be used to modulate the plasma clearance of aptamers. For example, unbound aptamers that introduce 2′-F and 2′-OMe stabilization chemistry typical of current generation aptamers due to their high nuclease stability in vivo and in vitro are unmodified aptamers and In comparison, it shows a rapid decrease from plasma (ie rapid plasma clearance) and a rapid distribution to tissues, mainly the kidney.

PEG-derivatized nucleic acids As mentioned 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. Have. Preferred changes in activity may include increased resistance to nuclease degradation, decreased renal filtration, decreased exposure to the immune system, and altered biodistribution of therapeutic agents.

The aptamer compositions of the present invention can be derivatized with a polyalkylene glycol (“PAG”) moiety. Examples of PAG-derivatized nucleic acids can be found in US patent application Ser. No. 10 / 718,833, filed Nov. 21, 2003, which is incorporated herein by reference in its entirety. Typical polymers used in the present invention include polyethylene glycol (“PEG”), also known as polyethylene oxide (“PEO”), and polypropylene glycol (including polyisopropylene glycol). In addition, random or block copolymers of different alkylene oxides (eg, ethylene oxide and propylene oxide) can be used in many applications. In its most common form, polyalkylene glycols such as PEG are linear polymers terminated with hydroxyl groups at each end: HO—CH ₂ CH ₂ O— (CH ₂ CH ₂ O) _n —CH ₂ CH ₂ -OH. The polymer, alpha -, omega - dihydroxylated polyethylene glycol, can also be represented as HO-PEG-OH, -PEG- symbol is understood to represent the following structural _unit: -CH ₂ CH ₂ O- (CH _{2 CH 2 O) n -CH 2} CH 2 -, n is typically in the range of from about 4 to about 10,000.

  As shown, PEG molecules are bifunctional and are sometimes referred to as “PEG diols”. The terminal part of the PEG molecule is a relatively non-reactive hydroxy moiety, the —OH group, that can be converted to an activated or functional moiety for attachment of PEG to other compounds at the reactive site of the compound. Such activated PEG diols are referred to herein as diactivated PEGs. For example, the terminal portion of a PEG diol can be activated by replacing the relatively non-reactive hydroxy moiety, —OH, with a succinimidyl active ester moiety from N-hydroxysuccinimide for selective reaction with the amino moiety. Functionalized as a nate ester.

In many applications, it is desirable to cap the PEG molecule at one end with an essentially non-reactive moiety so that the PEG molecule is monofunctional (or mono-activated). In general, in the case of protein therapeutics with multiple reactive sites for activated PEGs, bifunctional activated PEGs lead to extensive cross-linking and produce aggregates with low functionality. To generate mono-activated PEGs, one hydroxy moiety on the terminus of the PEG diol molecule is typically replaced with a non-reactive methoxy terminal moiety, —OCH ₃ . The other uncapped end of the PEG molecule is typically converted to a reactive end portion that can be activated for attachment at a reactive site on the surface or molecule such as a protein.

  PAGs are polymers that typically have solubility, non-toxic, and non-immunogenic properties in water and many organic solvents. One use of PAGs is to covalently attach polymers to insoluble molecules to make the resulting PAG-molecule “conjugate” soluble. For example, the water-insoluble drug paclitaxel has been shown to become water soluble when linked to PEG. Greenwald et al. Org. Chem. 60: 331-336 (1995). PAG conjugates are often used not only to increase solubility and stability, but also to increase the blood circulation half-life of the molecule.

  The polyalkylated compounds of the present invention are typically 5-80 kDa in size, but any size can be used and the choice depends on the aptamer and application. Other PAG compounds of the present invention are 10-80 kDa in size. Still other PAG compounds of the present invention are 10-60 kDa in size. For example, the PAG polymer can be at least 10, 20, 30, 40, 50, 60, or 80 kDa in size. Such polymers can be linear or branched polymers. In some embodiments, the polymer is PEG.

  In contrast to biologically expressed protein therapeutics, nucleic acid therapeutics are typically chemically synthesized from activated monomeric nucleotides. PEG-nucleic acid conjugates can be prepared by introducing PEG using the same iterative monomer synthesis. For example, PEGs activated by conversion to the phosphoramidite form can be introduced into solid phase oligonucleotide synthesis. Alternatively, oligonucleotide synthesis can be completed by site-specific introduction of reactive PEG attachment sites. Most commonly this has been achieved by the addition of a free primary amine at the 5'-end (introduced with the modifier phosphoramidite in the last coupling step of the solid phase synthesis). Using this approach, a reactive PEG (eg, one that has been activated to react to form a bond with an amine) is combined with a purified oligonucleotide and a conjugation reaction is performed in solution.

  The ability of a PEG linkage to alter the biodistribution of a therapeutic agent is related to a number of factors, including the apparent size of the conjugate (eg, measured in terms of hydrodynamic radius). Larger conjugates (> 10 kDa) are known to more effectively block filtration through the kidney and consequently increase the blood half-life of small macromolecules (eg, peptides, antisense oligonucleotides). Yes. The ability of PEG conjugates to block filtration has been shown to increase with the size of PEG up to about 50 kDa (the beneficial effect of further increase is because the half-life is defined by macrophage-mediated metabolism rather than renal removal) Is minimal).

  Production of high molecular weight PEGs (> 10 kDa) can be difficult, inefficient and expensive. As a route to the synthesis of high molecular weight PEG-nucleic acid conjugates, past research has focused on the generation of higher molecular weight activated PEGs. One method of generating such molecules involves the formation of branched activated PEGs, in which two or more PEGs are attached to a central core carrying activated groups. The ends of these high molecular weight PEG molecules, i.e., the relatively non-reactive hydroxyl (-OH) moiety, are activated or attached for attachment of one or more PEGs at reactive sites on the compound to other compounds It can be converted into a functional part. Branched activated PEGs have two or more termini, and where two or more termini are activated, such activated high molecular weight PEG molecules are multi-active herein. Called PEGylated PEGs. In some cases, not all ends of the branched PEG molecule are activated. In the case where any two ends of a branched PEG molecule are activated, such PEG molecules are referred to as di-activated PEGs. In some cases where only one end of a branched PEG molecule is activated, such a PEG molecule is referred to as single activation. As an example of this approach, an activated PEG prepared by the attachment of two monomethoxy PEGs to a lysine core that is subsequently activated for the reaction has been described (Harris et al., Nature, vol. 2: 214- 221, 2003).

The present invention provides another cost effective route for the synthesis of high molecular weight PEG-nucleic acid (preferably aptamer) conjugates comprising multiple PEGylated nucleic acids. The invention also includes PEG-linked multimeric oligonucleotides, such as dimerized aptamers. The present invention relates to a nucleic acid-PEG-nucleic acid (-PEG-nucleic acid) wherein the PEG stabilizing moiety is a linker that separates different parts of the aptamer, for example, the linear arrangement of the high molecular weight aptamer composition is, for example, n is 1 or more. It also relates to high molecular weight compositions in which PEG is attached in a single aptamer sequence, such as _n .

  High molecular weight compositions of the present invention include those having a molecular weight of at least 10 kDa. The composition typically has a molecular weight in the size of 10-80 kDa. The high molecular weight composition of the present invention is at least 10, 20, 30, 40, 50, 60, or 80 kDa in size.

  A stabilizing moiety is a molecule or portion of a molecule that improves the pharmacokinetic and pharmacodynamic properties of the high molecular weight aptamer composition of the present invention. In some cases, the stabilizing moiety approximates two or more aptamers or aptamer domains or provides a reduction in the overall rotational freedom of the high molecular weight aptamer composition of the invention. It is. The stabilizing moiety can be a polyalkylene glycol, a polyethylene glycol such as 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. The stabilizing moiety can be an integral part of the aptamer composition, ie, a moiety that is covalently attached to the aptamer.

  The compositions of the present invention include high molecular weight aptamer compositions in which two or more nucleic acid moieties are covalently linked to at least one polyalkylene glycol moiety. The polyalkylene glycol moiety serves as a stabilizing moiety. In a composition in which the polyalkylene glycol is covalently attached at either end of the aptamer so that the polyalkylene glycol binds the nucleic acid moiety in one molecule, the polyalkylene glycol moiety is said to be a linking moiety. In such compositions, the primary structure of the covalent molecule includes a linear arrangement of nucleic acid-PAG-nucleic acid. One example is a composition having the primary structure of nucleic acid-PEG-nucleic acid. Another example is a linear arrangement: nucleic acid-PEG-nucleic acid-PEG-nucleic acid.

To produce a nucleic acid-PEG-nucleic acid conjugate, the nucleic acid is first synthesized to have a single reaction site (eg, single activation). In a preferred embodiment, this reactive site is an amino group that is introduced at the 5'-terminus by the addition of the modifier phosphoramidite in the final step of the solid phase synthesis of the oligonucleotide. After 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 comprises 200 mM NaHCO ₃ -buffer at pH 8.3. The synthesis of the conjugate is initiated by the slow stepwise addition of highly purified bifunctional PEG. In a preferred embodiment, PEG diol is activated (di-activated) at both ends by derivatization with succinimidylpropionic acid. After the reaction, the PEG-nucleic acid conjugate is purified by gel electrophoresis or liquid chromatography to separate complete-, partial-, and non-linked species. Multiple linked PAG molecules or small PAG chains (eg, as random or block copolymers) can be linked to achieve various lengths (or molecular weights). Non-PAG linkers can be used between PAG chains of various lengths.

  2'-O-methyl, 2'-fluoro and other modified nucleotide modifications stabilize the aptamer against nucleases and increase its in vivo half-life. The 3'-3'-dT cap also increases exonuclease resistance. See, for example, US Pat. Nos. 5,674,685; 5,668,264; 6,207,816; and 6,229,002, each incorporated herein by reference in its entirety. reference.

PAG-derivatization of reactive nucleic acids High molecular weight PAG-nucleic acid-PAG conjugates can be prepared by reacting a nucleic acid containing two or more reactive sites with a monofunctional activated PEG. In one embodiment, the nucleic acid is direactive or diactivated and has two reaction sites: a 5′-amino group and a 3′-amino group introduced into the oligonucleotide by conventional phosphoramidite synthesis, such as shown in FIG. 3′-5′-di-PEGylation as described above. In an alternative embodiment, a reactive site can be introduced at an internal position, for example using the 5-position of pyrimidine, the 8-position of purine or the 2′-position of ribose as the site for attachment of the primary amine. In such embodiments, the nucleic acid can have several activation or reactive sites and is said to be multiple activated. After synthesis and purification, the modified oligonucleotide is combined with a mono-activated PEG under conditions that promote selective reaction with the oligonucleotide reaction site while minimizing spontaneous hydrolysis. In a preferred embodiment, monomethoxy-PEG is activated with succinimidylpropionic acid and the conjugation reaction is performed at pH 8.3. To induce synthesis of the disubstituted PEG, a stoichiometric excess of PEG relative to the oligonucleotide is provided. After the reaction, the PEG-nucleic acid conjugate is purified by gel electrophoresis or liquid chromatography to separate complete, partially, and unbound species.

  The linking domain can also have one or more polyalkylene glycol moieties attached thereto. Such PAGs can be of various lengths and can be used in appropriate combinations to achieve the desired molecular weight of the composition.

  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 stereochemical and / or ionic interactions with the target prevents complex formation between the aptamer and the target. Longer linkers necessary to connect the distances between nucleic acids can reduce binding stability by reducing the effective concentration of ligand. Therefore, it is often necessary to optimize the linker composition and length in order to maximize the affinity of the aptamer for the target.

  All publications and patent documents cited herein are hereby incorporated by reference as if each such publication and document were specifically indicated to be individually incorporated herein by reference. Incorporated into. Citations of publications and patent literature are not intended as an admission that any is pertinent prior art, and do not constitute any admission as to content or date. Although the invention has been described above, it will be appreciated by persons skilled in the art that the invention may be practiced in various embodiments, and the foregoing description and the following examples are for illustrative purposes only and are It does not limit the range.

Example 1 Polymerase Expression and Purification Mutant T7 RNA polymerase used in the method of the present invention can be prepared as follows. T7 RNA polymerase (shown in FIGS. 3A and 3B, respectively, Bull, JJ et al., J. Mol. Evol., 57 (3), 241-248 (2003)) LA mutant (Y639L / H784A), LAR mutant (Y639L / H784A / K378R), LLA mutant (P266L / Y639L / H784A) or LLAR mutant P266L / Y639L / H784A / K378R) may be generated. T7 RNA polymerase is included in an expression vector (examples of T7 RNA polymerase expression vectors are described in US Pat. No. 5,869,320, incorporated herein by reference in its entirety), or expression vector after mutagenesis. Can be inserted. Mutant T7 RNA polymerase can be engineered to selectively include His-tags for ease during protein purification.

  Complementary oligonucleotide sequence containing mutation of leucine at position 639

が、合成されうる。Ｐ２６６Ｌ変異のための相補的オリゴヌクレオチド配列

Can be synthesized. Complementary oligonucleotide sequence for the P266L mutation

が、合成されうる。Ｈ７８４Ａ変異のための相補的オリゴヌクレオチド配列

Can be synthesized. Complementary oligonucleotide sequence for the H784A mutation

が、合成されうる。Ｋ３７８Ｒ変異のための相補的オリゴヌクレオチド配列

Can be synthesized. Complementary oligonucleotide sequence for the K378R mutation

が、合成されうる。部位特異的変異誘発が、ＱｕｉｋＣｈａｎｇｅ（登録商標）Ｓｉｔｅ―Ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ、カリフォルニア州ラホーヤ）を用いて、製造者の指示に従って実行されて、上述の変異の組み合わせをもつ変異ポリメラーゼをコードする核酸配列（図４）を生じうる。本発明の変異ポリメラーゼをコードする得られた核酸配列が、発現および精製のための標準的な技術を用いて、所望の発現ベクターに挿入されうる。

Can be synthesized. Site-directed mutagenesis is performed using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) According to the manufacturer's instructions to encode a nucleic acid encoding a mutant polymerase with a combination of the above mutations An array (FIG. 4) can be generated. The resulting nucleic acid sequence encoding the mutant polymerase of the invention can be inserted into the desired expression vector using standard techniques for expression and purification.

Expression and Purification Expression vectors containing mutant T7 polymerase nucleic acid sequences are transformed into BL21 (DE3) competent cells (Stratagene, Calif.) And incubated on ice for 20 minutes. A heat shock is performed by placing the tube at 42 ° C. for 2 minutes. After placing the tube on ice for 1 minute, 1 ml of L broth (“LB”) is added and incubated on a 37 ° C. shaker for 45 minutes. 100 ul of culture is plated on LB + Amp agar plates and incubated overnight at 37 ° C.

  Single colonies from plates cultured overnight are inoculated overnight at 37 ° C. in 100 ml LB-Amp + (150 ug / ml). On the second day, two 4 liter flasks containing 2 liters of preheated LB + Amp were planted with 50 ml of overnight culture and grown at 37 ° C. until OD600 reached between 0.6 and 0.8 Be made. 200ul of 1M IPTG is added to each 2L cell culture at a final concentration of 100uM and allowed to grow for an additional 3 hours at 37 ° C. Spinning at 5000 rpm for 10 minutes pellets the cells. The cells are resuspended in 200 ml of lysis buffer (lysis buffer: 50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM imidazole, beta mercaptoethanol (“BME”) 5 mM), 6 Divided into 50 ml conical tubes. For each tube, the cells are sonicated at power level 8, 3 × 30 ″, bacterial debris is sedimented at 11,000 rpm for 60 minutes, and the supernatant is filtered through a 0.22 uM filter. Imidazole is added to the filter at a final concentration of 10 mM.

  The filtrate is loaded onto a 5 ml Ni-NTA column (GE Healthcare Bio-Sciences, NJ) with a sample pump. The column is washed with 10 column volumes (CV) of buffer A containing 20 mM imidazole (buffer A: 50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% glycerol, 10 mM imidazole, BME 10 mM). The column is then washed with 10 CV buffer with a linear gradient of 40 mM to 70 mM imidazole in buffer A. The protein is eluted with 6 CV of buffer B (buffer B: 50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% glycerol, 250 mM imidazole, BME 10 mM). After collating the collected fractions with 5 μl sample on 4-12% SDS-PAGE, all the desired fractions were combined and placed in 1 L dialysis buffer (dialysis buffer: 50 mM Tris-Cl, pH 7.9, Dialyze overnight (100 mm NaCl, 50% glycerol, 0.1 mM EDTA, 0.1% Triton X-100, BME 20 mM) (dialysis tubing: Spectrum Spectra / Por Molecular Porous Membrane (VWR) MWCO: 12-14000). The dialysis buffer is changed after 12 hours and the dialysis is carried out for a further 4 hours. The concentration of T7 RNA polymerase was determined by Bradford, M .; M.M. (1976) Anal. Biochem. 72, 248, measured using the Bradford assay.

Example 2 Transcription Introducing 100% 2′-O-Methyl Nucleotide Example 2A: 2′-O-Methyl Transcription without 2′-OH GTP Using 2′-OH GTP titration, 2 ′ Experiments were performed to test the sensitivity of the Y639L / H784A / K378R mutant polymerase to the concentration of —OH GTP.

  Transcription of Y639L / H784A / K378R mutant T7 RNA polymerase against 2'-OH GTP concentration using two libraries, ARC2118 and ARC2119, which showed high transcription yield when used with Y639L / H784A / K378R mutant T7 RNA polymerase The sensitivity of was tested. The sequence of ARC2118 is given in Example 2C below, and the sequence of ARC2119 is given directly in the 5 'to 3' direction.

１Ｘ転写バッファー（ＨＥＰＥＳ２００ｍＭ、ＤＴＴ４０ｍＭ、スペルミジン２ｍＭ、ＴｒｉｔｏｎＸ―１００ ０．０１％ｗ／ｖ）、〜２００ｎＭテンプレート、２’―ＯＭｅ ＡＴＰ、ＣＴＰ、ＵＴＰ、およびＧＴＰ各１ｍＭ、ＭｇＣｌ_２６．５ｍＭ、ＭｎＣｌ_２２．０ｍＭ、ＰＥＧ―８０００ｗ／ｖ １０％、ＧＭＰ１ｍＭ、無機ピロホスファターゼ５単位／ｍＬ、Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼ２００ｎＭと、２’―ＯＨ ＧＴＰ（０〜１６０ｕＭ）の滴定を用いて、一晩３７℃で転写が行われた。

1X transcription buffer (HEPES 200 mM, DTT 40 mM, spermidine 2 mM, Triton X-100 0.01% w / v), -200 nM template, 2'-OMe ATP, CTP, UTP, and GTP 1 mM each, MgCl ₂ 6.5 mM, MnCl ₂ 2.0 mM, PEG-8000 w / v 10%, GMP 1 mM, inorganic pyrophosphatase 5 units / mL, Y639L / H784A / K378R mutant T7 RNA polymerase 200 nM and 2′-OH GTP (0-160 uM) titration The transfer was performed at 37 ° C in the evening.

  Transcripts under each condition were assayed by PAGE-gel analysis using 200 uL of reaction mixture and from Image-UV UV-shadowing for each condition using ImageQuant 5.2 software (Molecular Dynamics). Transcripts were quantified. FIG. 5 summarizes the quantitative results of the PAGE-gel analysis, and shows the increase rate of the transcription yield according to each condition with respect to the background. As seen in FIG. 5, the yield in the absence of ARC2118 and ARC2119, and 2′-OH GTP transcribed by Y639L / H784A / K378R under all conditions without 2′-OH GTP, This was equivalent to the transcription yield when 2′-OH GTP was included in the reaction mixture. These results show that, in contrast to Y639F / H784A / K378R mutant T7 RNA polymerase, which requires 2′-OH GTP for transcription (data not shown), Y639L / H784A / K378R mutant T7 RNA polymerase shows transcription yield Indicates that the presence of 2′-OH GTP is not required for increase.

  Experiments were then performed to determine the optimal transcription conditions used with the Y639L / H784A / K378R mutant T7 RNA polymerase. ARC2119 was used with Y639L / H784A / K378R mutant T7 RNA polymerase to test the effect of changing 2'-OMe NTP, magnesium and manganese concentrations on transcription yield.

1X transcription buffer (HEPES 200 mM, DTT 40 mM, spermidine 2 mM, Triton X-100 0.01%), ~ 200 nM template, 2'-OMe ATP, CTP, UTP, and GTP (0.5 mM, 1 mM, 1.5 mM, and 2 mM, respectively) ), MgCl ₂ (5 mM, 6.5 mM, 8 mM, and 9.5 mM), MnCl ₂ (1.5 mM, 2 mM, 2.5 mM, 3 mM), PEG-8000 w / v 10%, GMP 1 mM, inorganic pyrophosphatase 5 units / ML, Y639L / H784A / K378R mutant T7 RNA polymerase 200 nM was used for transcription at 37 ° C. overnight.

Transcripts under each condition were assayed by PAGE-gel analysis using a 200 uL reaction mixture, and transcripts under each condition were UV-shadowed for PAGE-gel analysis using ImageQuant version 5.2 software (Molecular Dynamics). Quantified from Ing. FIG. 6 summarizes the quantification results of the PAGE-gel analysis, and shows the increase rate of transcription yield according to each condition with respect to the background. Based on the cost of 2′-OMe NTPs and the results of this experiment, each 1.5 mM 2′-OMe NTP (and 8 mM MgCl ₂ , 2.5 mM MnCl ₂ ) was converted to ARC2119 and the Y639L / H784A / K378R mutation of the present invention. Adopted as a preferred condition for T7 RNA polymerase.

Example 2B: Fidelity and bias of MNA transcription using Y639L / H784A / K378R mutant T7 RNA polymerase:
Additional experiments were performed to assess the fidelity and bias of MNA transcription using Y639L / H784A / K378R mutant T7 RNA polymerase and no 2′-OH GTP. To test fidelity, a single clone sequence was amplified by PCR, used to program MNA transcription without 2'-OH GTP using Y639L / H784A / K378R polymerase, and purified by PAGE The remaining DNA template was digested with RQl DNase (and the absence of DNA template was assayed by PCR) and the transcript was reverse transcribed (Thermoscript, Invitrogen, Carlsbad, Calif.) And amplified by PCR. After this PCR product was sequenced, the number of deletions, insertions and substitutions was calculated. Of the 1300 bases sequenced in this experiment, no deletions and insertions were observed, and three substitutions were observed (see FIG. 7). These numbers suggest that there is a 93% chance that the sequence information encoded within the 30-nucleotide degenerate region will be faithfully transmitted to the next round of SELEX ™. Higher than the number measured for wild-type RNA.

To test for nucleotide bias, library ARC2118 was tested under the following conditions: HEPES 200 mM, DTT 40 mM, spermidine 2 mM, Triton X-100 0.01% w / v, ˜200 nM template, 2′-OMe ATP, CTP, UTP, And GTP 1 mM each (without 2′-OH GTP), MgCl ₂ (6.5 mM), MnCl ₂ (2 mM), PEG-8000 w / v 10%, GMP 1 mM, inorganic pyrophosphatase 5 units / mL, Y639L / H784A / K378R Transcribed with mutant T7 RNA polymerase 200 nM overnight at 37 ° C., purified by PAGE, the remaining DNA template digested with RQ1 DNase (the absence of DNA template is then assayed by PCR) and transferred. Object is reverse transcribed and amplified using PCR before cloning and sequencing. Forty-eight clones from the amplified library and 48 clones from the starting library were sequenced. To see if a bias occurred, the value of nucleotide frequency in the degenerate region was examined. As shown by FIG. 8, the percentage of nucleotide composition after transcription is very similar to the percentage of nucleotide composition of the starting library in which the percentage of each nucleotide (A, T, C and G) is approximately equal, Y639L / H784A / K378R mutant T7 RNA polymerase was used for transcription, indicating no nucleotide bias.

Example 2C: MNA transcription using P266L / Y639L / H784A / K378R mutant T7 RNA polymerase The following DNA templates and primers were used to program the polymerase chain reaction to generate a double-stranded transcription template. N indicates degenerate positions with approximately equal probability for each of the ATGCs and all sequences are listed in the 3 ′ to 5 ′ direction: PCR template (ARC2118)

その後、得られた二本鎖転写テンプレートを用いて、以下のように各サンプルの２００ｕＬ転写混合物がプログラムされた：ＨＥＰＥＳ（２００ｍＭ）、ＤＴＴ（４０ｍＭ）、スペルミジン（２ｍＭ）、ＴｒｉｔｏｎＸ―１００（０．０１％ｗ／ｖ）、ＭｇＣｌ_２（８ｍＭ）、ＭｎＣｌ_２（２．５ｍＭ）、ＰＥＧ―８０００（１０％ｗ／ｖ）、各１．５ｍＭの２’―ＯＭｅ ＮＴＰ、ＧＭＰ １ｍＭ、１００―２００ｎＭ転写テンプレート、無機ピロホスファターゼ（１単位）、ｐＨ７．５、Ｔ７変異ポリメラーゼＰ２６６Ｌ／Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒが下記のように希釈された。転写混合物は、３７℃で一晩インキュベートされた（１６ｈ）。

The resulting double-stranded transcription template was then used to program a 200 uL transcription mixture for each sample as follows: HEPES (200 mM), DTT (40 mM), spermidine (2 mM), Triton X-100 (0. 01% w / v), MgCl ₂ (8 mM), MnCl ₂ (2.5 mM), PEG-8000 (10% w / v), 1.5 mM each 2′-OMe NTP, GMP 1 mM, 100-200 nM transcription Template, inorganic pyrophosphatase (1 unit), pH 7.5, T7 mutant polymerase P266L / Y639L / H784A / K378R was diluted as follows. The transcription mixture was incubated overnight at 37 ° C. (16 h).

  After incubation, the mixture was precipitated with isopropanol and the resulting pellet was dissolved and quantified using modified PAGE (12.5% acrylamide) for 60 minutes at 25W. Samples were visualized and quantified at 260 nm with UV shadows. The transcription yield is given in Table 1 below.

（実施例３）２’―Ｏ―メチルＡＴＰ、ＧＴＰ、およびＵＴＰヌクレオチド、および２’―デオキシＣＴＰヌクレオチドを導入する転写
ｉｎ ｖｉｔｒｏ転写を用いて２’―Ｏ―メチルヌクレオチドおよび２’―デオキシヌクレオチドの両者を導入するＹ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異ポリメラーゼの能力をテストするために実験が行われた。

Example 3 Transcription Introducing 2′-O-Methyl ATP, GTP, and UTP Nucleotides, and 2′-Deoxy CTP Nucleotides Using 2 vitro-transcription of 2′-O-methyl and 2′-deoxy nucleotides Experiments were performed to test the ability of the Y639L / H784A / K378R mutant polymerase to introduce both.

Using DNA templates ARC4206 and ARC4212, and their respective primers (sequences given in the 5 ′ to 3 ′ direction below), one 2′-deoxy CTP nucleotide and 2′-O-methyl ATP, GTP, And the ability of Y639L / H784A / K378R mutant T7 RNA polymerase to introduce the other three nucleotides, UTP, was tested. 200mM HEPES, 40mM DTT, 2.0mM _{spermidine, 0.01% w / v TritonX-} 100,10% PEG-8000,8mM MgCl 2, 2.5mM MnCl 2, 1.5mM dCTP, 1.5mM mUTP, 1. Overnight at 37 ° C. with 5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01 unit / μL inorganic pyrophosphatase and ˜9 μg / mL mutant T7 polymerase (Y639L / H784A / K378R) and 0.3 μM template DNA Transcription was performed to generate ARC4206 and ARC4212 pools consisting of 2′-OMe ATP, GTP, and UTP nucleotides and 2′-deoxy CTP nucleotides (hereinafter “dCmD”). At the same time, 50 mM HEPES, 10 mM DTT, 0.5 mM spermidine, 0.0025% Triton X-100, 10% PEG-8000, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1.5 mM mCTP, 1.5 mM mUTP, 1.5 mM Transcription overnight at 37 ° C. with mGTP, 1.5 mM mATP, 1 mM GMP, 0.01 unit / μL inorganic pyrophosphatase and ˜9 μg / mL mutant T7 polymerase (Y639L / H784A / K378R) and 0.3 μM template DNA Was performed to generate ARC4206 and ARC4212 pools consisting of 2'-OMe purine and pyrimidine nucleotides (hereinafter "MNA"). Transcripts under each condition set were assayed by PAGE-gel analysis using a 400 uL reaction mixture, and transcripts for each condition were visualized from UV-shadowing of the PAGE-gel. Table 2 shows quantification of transcription yield based on pools isolated after elution from PAGE-gel overnight in 300 mM NaOAc, 20 mM EDTA at 37 ° C., ethanol precipitation followed by quantification by UV spectroscopy. Summarize the results.

Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼを用いたＭＮＡおよびｄＣｍＤ転写の忠実度を評価するために、追加的な実験が行われた。忠実度をテストするために、上で生成されたＡＲＣ４２０６およびＡＲＣ４２１２ＭＮＡおよびｄＣｍＤプールが、逆転写され（Ｔｈｅｒｍｏｓｃｒｉｐｔ、Ｉｎｖｉｔｒｏｇｅｎ、カリフォルニア州カールズバッド）、ＰＣＲにより増幅され、Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒポリメラーゼを用いた追加的なＭＮＡおよびｄＣｍＤ転写をプログラムするために使用された。プールはＰＡＧＥにより精製され、転写物がＰＡＧＥ―ゲルのＵＶ―シャドウイングから視覚化された。表３は、３００ｍＭ ＮａＯＡｃ、２０ｍＭ ＥＤＴＡ中での６５℃での４．５時間のＰＡＧＥ―ゲルからの溶出、エタノール沈殿、その後のＵＶ分光法による定量後に単離されたプールに基づく、転写収率の定量結果をまとめる。

Additional experiments were performed to assess the fidelity of MNA and dCmD transcription using Y639L / H784A / K378R mutant T7 RNA polymerase. To test fidelity, the ARC4206 and ARC4212MNA and dCmD pools generated above were reverse transcribed (Thermoscript, Invitrogen, Carlsbad, Calif.), Amplified by PCR, and added with Y639L / H784A / K378R polymerase Were used to program MNA and dCmD transcription. The pool was purified by PAGE, and transcripts were visualized from PAGE-gel UV-shadowing. Table 3 shows transcription yields based on pools isolated after elution from PAGE-gel for 4.5 hours at 65 ° C. in 300 mM NaOAc, 20 mM EDTA, ethanol precipitation followed by quantification by UV spectroscopy. Summarize the quantitative results.

テンプレートは、その各々のＤＮＡプライマにより増幅された：

The template was amplified by its respective DNA primer:

（実施例４）Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼを用いたアプタマー選択
２’―ＯＭｅプリンおよびピリミジンヌクレオチド（以下に「ＭＮＡ」）からなるプールを用いて、ヒトＡｎｇ２（以下に「ｈ―Ａｎｇ２」）に対するアプタマーを同定するために、選択が実行された。選択戦略は、ｈ―Ａｎｇ２に特異的な高親和性アプタマーをもたらした。

Example 4 Aptamer Selection Using Y639L / H784A / K378R Mutant T7 RNA Polymerase Using a pool consisting of 2′-OMe purine and pyrimidine nucleotides (hereinafter “MNA”), human Ang2 (hereinafter “h-Ang2”) Selection was performed to identify aptamers for The selection strategy resulted in a high affinity aptamer specific for h-Ang2.

  Human Ang2 is available from R & D Systems, Inc. (Minneapolis, Minnesota). T7 RNA polymerase (Y639L / H784A / K378R) was expressed and purified as described in Example 1 above. 2'-OMe purine and pyrimidine nucleotides were purchased from TriLink BioTechnologies (San Diego, CA).

Ang2 aptamer selection Pool preparation Sequence

のＤＮＡテンプレートが、ＡＢＩ ＥＸＰＥＤＩＴＥ（商標）（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、カリフォルニア州フォスターシティ）ＤＮＡ合成装置を用いて合成され、標準法により脱保護された。テンプレートは、プライマ

DNA templates were synthesized using an ABI EXPEDITE ™ (Applied Biosystems, Foster City, CA) DNA synthesizer and deprotected by standard methods. Template is a primer

により増幅されてから、Ｔ７ＲＮＡポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）によるｉｎ ｖｉｔｒｏ転写のためのテンプレートとして使用された。２００ｍＭ Ｈｅｐｅｓ、４０ｍＭ ＤＴＴ、２ｍＭスペルミジン、０．０１％ｗ／ｖ ＴｒｉｔｏｎＸ―１００、１０％ＰＥＧ―８０００、８ｍＭ ＭｇＣｌ_２、２．５ｍＭ、ＭｎＣｌ_２、１．５ｍＭ ｍＣＴＰ、１．５ｍＭ ｍＵＴＰ、１．５ｍＭ ｍＧＴＰ、１．５ｍＭ ｍＡＴＰ、１ｍＭ ＧＭＰ、０．０１単位／μＬ無機ピロホスファターゼ、および〜９μｇ／ｍＬ Ｔ７ポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）および０．５μＭテンプレートＤＮＡを用いて転写が行われて、ＡＲＣ２１１８ｍＲｍＹプールが生成された。

And then used as a template for in vitro transcription with T7 RNA polymerase (Y639L / H784A / K378R). 200mM Hepes, 40mM DTT, 2mM _{spermidine, 0.01% w / v TritonX-} 100,10% PEG-8000,8mM MgCl 2, 2.5mM, MnCl 2, 1.5mM mCTP, 1.5mM mUTP, 1.5mM Transcription was performed using mGTP, 1.5 mM mATP, 1 mM GMP, 0.01 unit / μL inorganic pyrophosphatase, and ˜9 μg / mL T7 polymerase (Y639L / H784A / K378R) and 0.5 μM template DNA, and ARC2118 mRmY A pool was created.

Selection Selection was initiated by incubating 100 pmol of protein and 330 pmol (2 × 10 ¹⁴ molecules) of MNA ARC2118 pool in 100 μL final volume of selection buffer (1X Dulbecco's PBS (DPBS)) for 1 hour at room temperature. . RNA-protein complexes and unbound RNA molecules were separated using a 0.45 micron nitrocellulose spin column (Schleicher and Schuell, Keene, NH). The column was pretreated with KOH (column filter soaked in 1 mL 0.5 M KOH, 15 min RT; spin; filter soaked in 1 mL dH 2 O 5 min RT; spin), washed twice with 1 mL 1 × PBS, then pooled : A solution containing Ang2 complex was added to the column and centrifuged at 1500 xg for 2 minutes. The filter was washed twice with 500 μL DPBS to remove non-specific binders. RNA was eluted by the addition of 2 × 100 μL elution buffer (7M urea, 100 mM sodium acetate, 3 mM EDTA, preheated to 95 ° C.) and then precipitated with ethanol. RNA was reverse transcribed with the ThermoScript RT-PCR ™ system (Invitrogen, Carlsbad, Calif.) Using primer SEQ ID NO: 27 according to the manufacturer's instructions. The cDNA was amplified by PCR with Taq polymerase (New England Biolabs, Beverly, Mass.) Using SEQ ID NO: 26 and SEQ ID NO: 27 according to the manufacturer's instructions. Templates were transferred as described above for pool preparation and purified on a denaturing polyacrylamide gel.

  Round 2 was performed in the same way as round 1. Rounds 3-12 were performed with h-Ang2 immobilized on a hydrophobic plate. Each selection round was initiated by immobilizing 20 pmol h-Ang2 on the surface of a Nunc Maxisorp hydrophobic plate for 1 hour at room temperature in 100 μL 1 × DPBS. The plate was washed 5 × with 120 μL DPBS and then incubated with blocking buffer (1 × DPBS, and 0.1 mg / mL BSA) for 1 hour. The supernatant was then removed and the wells were washed 5 times with 120 μL 1 × DPBS. Pooled RNA was incubated in empty wells for 1 hour at room temperature and then for 1 hour in wells previously blocked with 100 μL blocking buffer. From round 3 onwards, wells with immobilized targets were placed in 100 μL blocking buffer (1 × PBS, 0.1 mg / mL tRNA, 0.1 mg / mL ssDNA and 0.1 mg / mL BSA) prior to the positive selection step. Blocked for 1 hour at room temperature. In all cases, pooled RNA bound to immobilized h-Ang2 was added to the selection plate by the addition of a reverse transcription (“RT”) mixture (3 ′ primer, SEQ ID NO: 27, and Thermoscript RT, Invitrogen, Carlsbad, Calif.). After direct reverse transcription, it was incubated at 65 ° C. for 1 hour. The resulting cDNA was used as a template for PCR (Taq polymerase, New England Biolabs, Beverly, Mass.) And transcription as described for Round 1. Table 4 shows the conditions for each round.

ＭＮＡアプタマー結合分析
プールのタンパク質結合親和性をモニタするために、選択の全体にわたりドットブロット結合アッセイが行われた。トレース^３２Ｐ―エンド標識したプールＲＮＡが、ｈ―Ａｎｇ２と組み合わされ、ＤＰＢＳバッファー中で３０μＬの最終的体積で、３０分間室温でインキュベートされた。混合物がドットブロット装置に適用され（Ｍｉｎｉｆｏｌｄ―１Ｄｏｔ Ｂｌｏｔ、Ａｃｒｙｌｉｃ、Ｓｃｈｌｅｉｃｈｅｒ ａｎｄ Ｓｃｈｕｅｌｌ、ニューハンプシャー州キーン）、（上から下へ）ニトロセルロース、ナイロン、およびゲルブロット膜とアセンブルされた。タンパク質に結合されたＲＮＡはニトロセルロースフィルタに捕らえられ；非タンパク結合ＲＮＡはナイロンフィルタに捕らえられる。ラウンド９でｈ―Ａｎｇ２結合の濃縮の開始が見られた。ラウンド９、１０および１２のプールテンプレートが、ＴＯＰＯ ＴＡクローニングキット（Ｉｎｖｉｔｒｏｇｅｎ、カリフォルニア州カールズバッド）を使用して製造者の指示に従ってクローニングされ、化学合成のために２６のユニーククローンが選択され、解離定数（Ｋ_Ｄ）が決定された。簡単にいうと、合成ＲＮＡｓがγ―^３２Ｐ ＡＴＰで５’末端標識され、ドットブロットアッセイおよび１Ｘ ＤＰＢＳ（ｗ／Ｃａ^２＋およびＭｇ^２＋）（Ｇｉｂｃｏ、Ｃａｔａｌｏｇ＃１４０４０、Ｉｎｖｉｔｒｏｇｅｎ、カリフォルニア州カールズバッド）のバッファー条件を用いてＫ_Ｄ値が決定された。式：ＲＮＡ結合画分＝振幅^＊（（（ＡｐｔＣｏｎｃ＋［ｈ―Ａｎｇ２］＋Ｋ_Ｄ）−ＳＱＲＴ（（ＡｐｔＣｏｎｃ＋［ｈ―Ａｎｇ２］＋Ｋ_Ｄ）^２−４（ＡｐｔＣｏｎｃ^＊［ｈ―Ａｎｇ２］）））／（２^＊ＡｐｔＣｏｎｃ））＋バックグラウンドにデータをあてはめてＫ_Ｄｓが推定された。結果は、下表５にレポートされる。

MNA Aptamer Binding Analysis A dot blot binding assay was performed throughout the selection to monitor the protein binding affinity of the pool. Trace ³² P-endo labeled pool RNA was combined with h-Ang2 and incubated in DPBS buffer with a final volume of 30 μL for 30 minutes at room temperature. The mixture was applied to a dot blot apparatus (Minifold-1 Dot Blot, Acrylic, Schleicher and Schuell, Keene, NH) (from top to bottom) and assembled with nitrocellulose, nylon, and gel blot membranes. RNA bound to protein is captured on a nitrocellulose filter; non-protein bound RNA is captured on a nylon filter. In round 9, the beginning of enrichment of h-Ang2 binding was observed. Round 9, 10 and 12 pool templates were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, 26 unique clones were selected for chemical synthesis, and the dissociation constant ( K _D ) was determined. Briefly, synthetic RNAs are 5 ′ end labeled with γ- ³² P ATP, buffered with dot blot assay and 1 × DPBS (w / Ca ²⁺ and Mg ²⁺ ) (Gibco, Catalog # 14040, Invitrogen, Carlsbad, Calif.). The _KD value was determined using the conditions. Formula: RNA bound fraction = amplitude _{^{* (((AptConc + [h}} -Ang2] + K D) -SQRT ((AptConc + [h-Ang2] + K D) 2 -4 (AptConc * [h-Ang2]))) / ( 2 ^* ApConc)) + K _D s was estimated by fitting data to the background. The results are reported in Table 5 below.

  Of the 26 unique sequences, 8 shared similar motifs and had similar binding and inhibitory activities. These sequences are identified as Family I. Family II contains two sequences sharing a motif with similar binding and inhibitory activity.

Analysis of MNA aptamer function Elisa assay Several aptamers were tested in an ELISA assay prepared to measure the ability to prevent Ang2 binding to the Tie2 receptor. To capture the Tie2 receptor, 150 ng of Tie2-Fc (R & D systems 313-TI-100-CF, Minneapolis, MN) in 100 μL of PBS (pH 7.4) was added to a 96-well Maxisorb plate (NUNC # 446612, New York). And incubate at 4 ° C. overnight. During capture, 50 μL of various concentrations of synthetic RNA is 50 μL of 3.6 nM Ang2 (200 ng / mL) (R & D systems, 623-AN-025 / CF, Minneapolis, MN) (in PBS with 0.2% BSA) And a final Ang2 concentration of 1.8 nM (100 ng / mL) in PBS with 0.1% BSA and incubated for 1 hour at room temperature. After overnight incubation, the capture solution was removed and the plate was washed three times with 200 μL TBST (25 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.01% Tween 20). The plate was then blocked with 200 μL TBST containing 5% non-fat dry milk for 30 minutes at room temperature. After blocking, the plate was washed again three times with 200 μL TBST at room temperature and the synthetic RNA: Ang2 mixture was added to the plate and incubated for 1 hour at room temperature. Plates were then washed three times with 200 μL TBST and 100 μL of biotinylated goat anti-Ang2 antibody (1: 1000; R & D Systems BAF623, Minneapolis, MN) was added and incubated for 1 hour at room temperature. After three washes with 200 μL TBST, 100 μL HRP-linked streptavidin (1: 200; R & D systems # DY998, Minneapolis, MN) was added and incubated for 0.5 hours at room temperature. The plate was then washed again three times with 200 μL TBST, 100 μL TMP solution (Pierce, # 34028) was added and incubated for 5 minutes at room temperature in the dark. The reaction was stopped by adding 100 μL of a solution containing 2N H ₂ SO ₄ and the plate was read at 450 nm with a SpectroMax. The results are given in the final column of Table 5 below.

FACS assay Human umbilical vein endothelial cells (“HUVEC”) (ATCC) and K293 cells, a cell line that overexpresses human Tie2 receptor, is used to inhibit the binding of specific MNA Ang2 aptamer to Tie2 receptor on the cell membrane. IC ₅₀ was determined. Briefly, recombinant mammalian expression vector pCDNA3.1-Tie2 was transfected into 293 cells (ATCC, Manassas, VA) and stable clones were obtained after selection with G418 (Invitrogen, Carlsbad, CA). . Flow cytometry showed the expression of Tie2 protein in both HUVEC and K293 cells. The Ang2 titration assay further determined the amount of Ang2 (R & D Systems, Minneapolis, MN) in the cellular aptamer inhibition assay for HUVEC and K293 cells, 1 and 0.1 μg / mL, respectively.

In the flow cytometry binding assay, HUVEC and K293 cells (2 × 10 ⁵ cells / well) are pelleted into V-bottom 96 well plates and then resuspended in MNA aptamer / Ang2 solution and incubated for 2 hours. It was. The aptamer / Ang2 solution has different doses of aptamer (100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, Ang2 in FAC buffer (1% BSA in PBS, 0.2% sodium azide). 0.411 nM, 0.137 nM, and 0.0456 nM) were prepared by preincubation on ice for 30 minutes. After three washes with FACs buffer, cells were incubated with biotinylated anti-human Ang2 antibody (5 μg / mL; R & D Systems, Minneapolis, Minn.) For 30 minutes before streptavidin PE (1:10; BD Biosciences, California Incubation for another 30 minutes was performed. FACS analysis was completed using a FACScan (BD Biosciences, San Jose, CA). The results are reported in Table 5 below.

（実施例５）Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ変異Ｔ７ＲＮＡポリメラーゼを用いたアプタマー選択
２’―ＯＭｅプリンおよびピリミジンヌクレオチドからなるプール（以下に「ｍＲｍＹ」）を用いて、ヒトＩｇＥ（以下に「ｈ―ＩｇＥ」）に対するアプタマーを同定するために、選択が行われた。選択戦略は、ｈ―ＩｇＥに特異的な高親和性アプタマーをもたらした。

(Example 5) Aptamer selection using Y639L / H784A / K378R mutant T7 RNA polymerase Human IgE (hereinafter "h-IgE") was pooled with 2'-OMe purine and pyrimidine nucleotides (hereinafter "mRmY"). A selection was made to identify aptamers for The selection strategy resulted in a high affinity aptamer specific for h-IgE.

  Human IgE was purchased from Athens Research & Technology (Cat. # 16-16-090705 Athens, GA). T7 RNA polymerase (Y639L / H784A / K378R) was expressed and purified as described above in Example 1. 2'-OMe purine and pyrimidine nucleotides were purchased from TriLink BioTechnologies (San Diego, CA).

IgE aptamer selection Pool preparation Sequence

のＤＮＡテンプレートが、ＡＢＩ ＥＸＰＥＤＩＴＥ（商標）（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、カリフォルニア州フォスターシティ）ＤＮＡ合成装置用いて合成され、標準法により脱保護された。テンプレートは、プライマ

DNA templates were synthesized using an ABI EXPEDITE ™ (Applied Biosystems, Foster City, CA) DNA synthesizer and deprotected by standard methods. Template is a primer

により増幅され、Ｔ７ＲＮＡポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）によるｉｎ ｖｉｔｒｏ転写のテンプレートとして使用された。２００ｍＭ ＨＥＰＥＳ、４０ｍＭ ＤＴＴ、２．０ｍＭ スペルミジン、０．０１％ｗ／ｖ ＴｒｉｔｏｎＸ―１００、１０％ ＰＥＧ―８０００、８ｍＭ ＭｇＣｌ_２、２．５ｍＭ ＭｎＣｌ_２、１．５ｍＭ ｍＣＴＰ、１．５ｍＭ ｍＵＴＰ、１．５ｍＭ ｍＧＴＰ、１．５ｍＭ ｍＡＴＰ、１ｍＭ ＧＭＰ、０．０１単位／μＬ無機ピロホスファターゼ、および〜９μｇ／ｍＬ変異Ｔ７ポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）および０．３μＭテンプレートＤＮＡを用いて転写が行われ、ＡＲＣ２１１８ ＭＮＡプールが生成された。

And used as a template for in vitro transcription with T7 RNA polymerase (Y639L / H784A / K378R). 200 mM HEPES, 40 mM DTT, 2.0 mM spermidine, 0.01% w / v Triton X-100, 10% PEG-8000, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1.5 mM mCTP, 1.5 mM mUTP, 1. Transcription was performed using 5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01 unit / μL inorganic pyrophosphatase, and ˜9 μg / mL mutant T7 polymerase (Y639L / H784A / K378R) and 0.3 μM template DNA; An ARC2118 MNA pool was created.

Selection 330 pmol (2 × 10 ¹⁴ molecules) of MNA ARC2118 pool was added to 100 μL final volume of selection buffer (1 × Dulbecco's) with 24 pmol of protein bound to a BSA-blocked hydrophobic plate (Maxisorp plate, Nunc, Rochester, NY). Selection was initiated by incubating in PBS (DPBS) for 1 hour at room temperature. Wells were washed 4 times with 120 μL DPBS to remove non-specific binders. The RNA was eluted and reverse transcribed using Primer SEQ ID NO: 27 with the ThermoScript RT-PCR ™ system (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Using SEQ ID NO: 26 and SEQ ID NO: 27, cDNA was amplified by PCR using Taq polymerase (New England Biolabs, Beverly, Mass.) According to the manufacturer's instructions. The template was transferred as described above in the pool preparation and purified on a denaturing polyacrylamide gel.

  All rounds were performed with h-IgE immobilized on a hydrophobic plate. Each round of selection was initiated by immobilizing 24 pmol of h-IgE on the surface of a Nunc Maxisorp hydrophobic plate in 100 μL of 1X DPBS for 1 hour at room temperature. The plate was washed four times with 120 μL DPBS and then incubated for 1 hour in blocking buffer (1 × DPBS, and 0.1 mg / mL BSA). The supernatant was then removed and the wells were washed 4 times with 120 μL 1 × DPBS. Starting from round 2, the pool RNA was incubated for 1 hour at room temperature in empty wells and then for 1 hour in wells previously blocked with 100 μL blocking buffer. From round 2 onwards, non-specific competitors were added to the positive selection step (0.1 mg / mL tRNA, and 0.1 mg / mL ssDNA). In all cases, the pooled RNA bound to the immobilized h-IgE was 65 ° C. after addition of the reverse transcription (“RT”) mixture (3 ′ primer, SEQ ID NO: 27, and Thermoscript RT, Invitrogen, Carlsbad, Calif.). Was reverse transcribed directly on the selection plate by incubating for 1 hour. The resulting cDNA was used as a template for PCR (Taq polymerase, New England Biolabs, Beverly, Mass.) And transcription as described per round 1. Table 6 shows the conditions for each round.

ＭＮＡアプタマー結合分析
プールのタンパク質結合親和性をモニタするために、選択の全体にわたりドットブロット結合アッセイが行われた。トレース^３２Ｐ―エンド標識したプールＲＮＡが、ｈ―ＩｇＥと組み合わされ、ＤＰＢＳバッファー中で３０μＬの最終体積で、３０分間室温でインキュベートされた。混合物がドットブロット装置に適用され（Ｍｉｎｉｆｏｌｄ―１ Ｄｏｔ Ｂｌｏｔ、Ａｃｒｙｌｉｃ、Ｓｃｈｌｅｉｃｈｅｒ ａｎｄ Ｓｃｈｕｅｌｌ、ニューハンプシャー州キーン）、（上から下へ）ニトロセルロース、ナイロン、およびゲルブロット膜とアセンブルされた。タンパク質に結合されたＲＮＡは、ニトロセルロースフィルタに捕らえられ；非タンパク結合ＲＮＡは、ナイロンフィルタに捕らえられる。ラウンド８でｈ―ＩｇＥ結合の濃縮の開始が見られた。ラウンド５、８および１２のプールテンプレートが、ＴＯＰＯ ＴＡクローニングキット（Ｉｎｖｉｔｒｏｇｅｎ、カリフォルニア州カールズバッド）を使用して製造者の指示に従ってクローニングされた。配列決定データから、ラウンド８のプールが全配列の５９％を含む単一の主要クローンに収束したことが明らかになった。この主要クローンと三つの可能性のあるミニマーが化学合成のために選択され、解離定数（Ｋ_Ｄ）が決定された。簡単にいうと、合成ＲＮＡｓがγ―^３２Ｐ ＡＴＰで５’末端標識され、ドットブロットアッセイおよび１Ｘ ＤＰＢＳ（ｗ／Ｃａ２＋およびＭｇ２＋）（Ｇｉｂｃｏ、Ｃａｔａｌｏｇ＃１４０４０、Ｉｎｖｉｔｒｏｇｅｎ、カリフォルニア州カールズバッド）のバッファー条件を用いてＫＤ値が決定された。式：ＲＮＡ結合画分＝振幅^＊（（（ＡｐｔＣｏｎｃ＋［ｈ―ＩｇＥ］＋ＫＤ）−ＳＱＲＴ（（ＡｐｔＣｏｎｃ＋［ｈ―ＩｇＥ］＋ＫＤ）２−４（ＡｐｔＣｏｎｃ^＊［ｈ―ＩｇＥ］）））／（２^＊ＡｐｔＣｏｎｃ））＋バックグラウンドにデータをあてはめてＫＤｓが推定された。主要クローンは、約８００ｐＭのＫ_Ｄを有した。最善の結合のミニマーの、サルＩｇＥ（ｍ―ＩｇＥ）に対する結合もテストされたが、サルＩｇＥタンパク質に対する交差反応結合は示されなかった。この交差反応性の欠如は、ＥＬＩＳＡによっても確認された。３’末端に逆方向ｄＴを伴うミニマーが、医薬品化学プロセスの親分子として用いられた。

MNA Aptamer Binding Analysis A dot blot binding assay was performed throughout the selection to monitor the protein binding affinity of the pool. Trace ³² P-endo labeled pool RNA was combined with h-IgE and incubated in DPBS buffer at a final volume of 30 μL for 30 minutes at room temperature. The mixture was applied to a dot blot apparatus (Minifold-1 Dot Blot, Acrylic, Schleicher and Schuell, Keene, NH) (from top to bottom) and assembled with nitrocellulose, nylon, and gel blot membranes. RNA bound to protein is captured on a nitrocellulose filter; non-protein bound RNA is captured on a nylon filter. In round 8, the start of enrichment of h-IgE binding was seen. Round 5, 8, and 12 pool templates were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Sequencing data revealed that the round 8 pool converged to a single major clone containing 59% of the total sequence. This major clone and three potential minimers were selected for chemical synthesis and the dissociation constant (K _D ) was determined. Briefly, synthetic RNAs were 5 ′ end labeled with γ- ³² P ATP, and the buffer conditions of dot blot assay and 1 × DPBS (w / Ca2 + and Mg2 +) (Gibco, Catalog # 14040, Invitrogen, Carlsbad, Calif.) Were used. Used to determine the KD value. Formula: RNA binding fraction = amplitude ^* (((AptConc + [h-IgE] + KD) -SQRT ((ApTConc + [h-IgE] + KD) 2-4 (ApTConc ^* [h-IgE])))) / (2 ^* Opts)) + data was applied to the background to estimate KDs. Primary clones had a _{K D} of about 800 pM. The best binding minimers were also tested for binding to monkey IgE (m-IgE) but showed no cross-reactive binding to monkey IgE protein. This lack of cross-reactivity was also confirmed by ELISA. Minimers with reverse dT at the 3 ′ end were used as parent molecules for medicinal chemistry processes.

Medicinal Chemistry One chemical composition of IgE-specific MNA minimers (Figure 9) was modified to improve affinity and capacity while maintaining plasma stability of the compound. The process involves the design, synthesis and evaluation of a series of derivatives of minimized IgE aptamers, each series of derivatives having a single modification at each given nucleotide to determine which residues will tolerate substitution. Inclusive. The first modification set was the substitution of deoxynucleotides for each unique 2′-OMe nucleotide. In separate modification rounds, a series of derivatives were synthesized, each derivative containing a single phosphorothioate modification at a different internucleotide linkage position. Using the data generated in the first stages of these modifications, a structure-activity relationship (SAR) of minimized aptamers was established. In a later modification step, aptamers were synthesized and tested with complex substitution sets designed based on the initial SAR data. From the composite substitution panel, a 39 nucleotide long aptamer was identified in which a 2'-OMe to 2'-deoxy substitution was introduced into the composition. In addition, the resulting minimized modified aptamer, 39 nucleotides long, with one substitution from 2′-OMe to 2′-deoxy and four substitutions from phosphate to phosphorothioate introduced into the composition Identified. As shown in FIG. 10, this deoxy / phosphorothioate modified aptamer has binding affinity compared to both the minimized unmodified parent aptamer and the minimized parent aptamer with two 2′-OMe to deoxy substitutions. Shows an increase.

Serum stability Minimized unmodified parent aptamers and deoxy / phosphorothioate modified aptamers were assayed to determine stability in human, rat and monkey sera. Each aptamer was added to 1 ml of pooled serum to a final concentration of 5 μM in 90% serum. Aptamers were incubated at 37 ° C. with shaking and time points were taken at 0, 0.5, 1, 4, 24, 48, 72, and 98 hours. At each time point, 90 μl stock from the incubated sample was added to 10 μl 0.5 M EDTA and frozen at −20 ° C. for stability analysis after using a BIACORE 2000 system.

  All biosensor binding measurements were performed at 25 ° C. using a BIACORE 2000 (BIACORE Inc., Piscataway, NJ) equipped with a research grade CM5 biosensor chip. Purified recombinant human IgE (Athens Research & Technology, Athens, GA) was immobilized on the biosensor surface using amino-coupling chemistry. To achieve this, the surfaces of the two flow cells were first 1: 1 with 0.1 M NHS (N hydroxysuccinimide) and 0.4 M EDC (3- (N, N dimethylamine) propyl-N-ethylcarbodiimide). The mixture was activated for 7 minutes at a flow rate of 5 μl / min. After surface activation, 50 μg / ml IgE was injected into one flow cell at 10 μl / min for 20 minutes to allow establishment of covalent bonds to the activated surface. Next, 1M ethanolamine hydrochloride pH 8.5 was injected at 5 μl / min for 7 minutes to inactivate the residual ester. The flow cell used as a blank was injected with 1 M ethanolamine hydrochloride pH 8.5 for 7 minutes to inactivate the residual ester without protein injection.

  Before all time points were analyzed, a series of aptamer standards were passed through the prepared chip to generate a standard curve. To establish a standard curve, aptamers were serially diluted in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Surfactant 20) supplemented with 4% human serum and 50 mM EDTA. (From 200 nM to 12.5 nM). All diluted samples were injected into Biacore 2000 and bound at 20 μl / min for 5 minutes and placed for 3 minutes. To regenerate the chip, 1N NaCl was injected at 30 μl / min for 60 seconds. The final RU peak response of the binding stage was plotted against aptamer concentration and a standard curve was generated using the Four-Parameter logistic function. To measure active aptamer concentrations in human, rat, and monkey sera, timepoint samples were 22.5 times in HBS-P so that the final serum concentration was 4% just prior to injection into Biacore 2000. Diluted. The functional aptamer concentration for each serum incubation period was calculated by converting from RU reaction units to concentrations using the standard curve generated above. As an additional quality control measure, the two aptamer standards were tested independently at the end of the experiment to confirm that the deviation from the standard of concentration measured by BIACORE was less than 20%. The minimized unmodified parent aptamer and deoxy / phosphorothioate modified aptamer were both determined to be over 90% active in 98 hours in human, rat and monkey serum.

Example 6 Aptamer Selection Using Y639L / H784A / K378R Mutant T7 RNA Polymerase Using a pool of 2′-OMe ATP, GTP, and UTP nucleotides, and 2′-deoxy CTP nucleotides (hereinafter “dCmD”) Selections were made to identify aptamers to human von Willebrand factor (hereinafter “h-vWF”). The selection strategy resulted in a high affinity aptamer specific for h-vWF.

  Human vWF is available from EMD Biosciences Inc. (Cat. # 681300, La Jolla, CA). Human vWF Al domain was expressed and purified. A mutant polymerase (Y639L / H784A / K378R) was expressed and purified as described in Example 1 above. 2'-OMe ATP, GTP, and UTP nucleotides, and 2'-deoxy CTP nucleotides were purchased from TriLink BioTechnologies (San Diego, CA).

Selection of vWF aptamer Pool preparation Sequence

を伴うテンプレートが、ＡＢＩ ＥＸＰＥＤＩＴＥ（商標）（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、カリフォルニア州フォスターシティ）ＤＮＡ合成装置を使用して合成され、標準法により脱保護された。テンプレートは、プライマ

Were synthesized using an ABI EXPEDITE ™ (Applied Biosystems, Foster City, CA) DNA synthesizer and deprotected by standard methods. Template is a primer

により増幅されてから、Ｔ７ＲＮＡポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）によるｉｎ ｖｉｔｒｏ転写のためのテンプレートとして使用された。２００ｍＭ ＨＥＰＥＳ、４０ｍＭ ＤＴＴ、２ｍＭスペルミジン、０．０１％ｗ／ｖ ＴｒｉｔｏｎＸ―１００、１０％ＰＥＧ―８０００、８ｍＭ ＭｇＣｌ_２、２．５ｍＭ ＭｎＣｌ_２、１．５ｍＭ ｄＣＴＰ、１．５ｍＭ ｍＵＴＰ、１．５ｍＭ ｍＧＴＰ、１．５ｍＭ ｍＡＴＰ、１ｍＭ ＧＭＰ、０．０１単位／μＬ無機ピロホスファターゼ、および〜９μｇ／ｍＬ変異Ｔ７ポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）および０．３μＭテンプレートＤＮＡを用いて転写が行われて、ＡＲＣ４２１８ｄＣｍＤプールが生成された。

And then used as a template for in vitro transcription with T7 RNA polymerase (Y639L / H784A / K378R). 200mM HEPES, 40mM DTT, 2mM _{spermidine, 0.01% w / v TritonX-} 100,10% PEG-8000,8mM MgCl 2, 2.5mM MnCl 2, 1.5mM dCTP, 1.5mM mUTP, 1.5mM mGTP , 1.5 mM mATP, 1 mM GMP, 0.01 unit / μL inorganic pyrophosphatase, and ˜9 μg / mL mutant T7 polymerase (Y639L / H784A / K378R) and 0.3 μM template DNA to perform transcription, and ARC4218dCmD A pool was created.

Selection 100 μL final volume of selection buffer (1 × Dulbecco's PBS (DPBS)) with 330 pmol (2 × 10 ¹⁴ molecules) with 20 pmol of protein bound to HSA blocked hydrophobic plates (Maxisorp plates, Nunc, Rochester, NY). ) DCmD ARC4218 pool was incubated for 1 hour at room temperature, and the wells were washed 5 times with 200 μL DPBS to remove non-specific binding RNA was eluted and primer ARC4208 (SEQ ID NO: 30) was reverse transcribed with a ThermoScript RT-PCR ™ system (Invitrogen, Carlsbad, Calif.) According to the manufacturer's instructions. The cDNA was amplified by PCR with Taq polymerase (New England Biolabs, Beverly, Mass.) Using RC4219 (SEQ ID NO: 29) and ARC4208 (SEQ ID NO: 30) according to the manufacturer's instructions, as described above for pool preparation. The template was transcribed and purified by ethanol precipitation followed by a desalting step using a Micro Bio-spin column (Cat. # 732-6250, Bio-Rad Laboratories, Hercules, CA).

  All rounds were performed with h-vWF immobilized on a hydrophobic plate. The first three selection rounds were initiated by immobilizing 10 pmol h-vWF and 10 pmol human vWF Al domain on the surface of a Nunc Maxisorp hydrophobic plate in 100 μL 1 × DPBS for 30 minutes at room temperature. Starting at round 4, 20 pmol of h-vWF was immobilized on the hydrophobic plate for 2 rounds, followed by 20 pmol of human vWF Al domain immobilized on the hydrophobic plate for one round. The plate was washed three times with 200 μL DPBS and then incubated for 30 minutes with blocking buffer (1 × DPBS and 5% HSA). The supernatant was then removed and the wells were washed three times with 200 μL DPBS. Starting from round 2, the pool RNA was incubated in empty wells for 30 minutes at room temperature and then incubated for 30 minutes in wells previously blocked with 100 μL blocking buffer. From round 2 onwards, non-specific competitors were added to the positive selection step (0.1 mg / mL tRNA). In either case, pooled RNA bound to the immobilized target was added after reverse transcription (“RT”) mixture (3 ′ primer, SEQ ID NO: 30, and Thermoscript RT, Invitrogen, Carlsbad, Calif.) Followed by 65 ° C. Incubation for 1 hour was directly reverse transcribed in the selection plate. The resulting cDNA was used as a template for PCR (Taq polymerase, New England Biolabs, Beverly, Mass.) And transcription as described for Round 1. Table 7 shows the conditions for each round.

ｄＣｍＤアプタマー結合分析
プールのタンパク質結合親和性をモニタするために、選択の全体にわたりドットブロット結合アッセイが行われた。トレース^３２Ｐ―エンド標識したプールＲＮＡが、ｈ―ｖＷＦと組み合わされ、ＤＰＢＳバッファー中で３０μＬの最終体積で、室温で３０分間インキュベートされた。混合物がドットブロット装置に適用され（Ｍｉｎｉｆｏｌｄ―１ Ｄｏｔ Ｂｌｏｔ、Ａｃｒｙｌｉｃ、Ｓｃｈｌｅｉｃｈｅｒ ａｎｄ Ｓｃｈｕｅｌｌ、ニューハンプシャー州キーン）、（上から下へ）ニトロセルロース、ナイロン、およびゲルブロット膜とアセンブルされた。タンパク質に結合されたＲＮＡは、ニトロセルロースフィルタに捕らえられ；非タンパク結合ＲＮＡは、ナイロンフィルタに捕らえられる。ラウンド５でｈ―ｖＷＦ結合の濃縮の開始が見られた。ラウンド５およびラウンド６プールの解離定数（Ｋ_Ｄ）が決定された。簡単にいうと、プールＲＮＡｓがγ―^３２Ｐ ＡＴＰで５’末端標識され、ドットブロットアッセイおよび１Ｘ ＤＰＢＳ（ｗ／Ｃａ２＋およびＭｇ２＋）（Ｇｉｂｃｏ、Ｃａｔａｌｏｇ＃１４０４０、Ｉｎｖｉｔｒｏｇｅｎ、カリフォルニア州カールズバッド）のバッファー条件を用いてＫ_Ｄ値が決定された。式：ＲＮＡ結合画分＝振幅^＊（（（ＡｐｔＣｏｎｃ＋［ｈ―ｖＷＦ］＋ＫＤ）−ＳＱＲＴ（（ＡｐｔＣｏｎｃ＋［ｈ―ｖＷＦ］＋ＫＤ）２−４（ＡｐｔＣｏｎｃ^＊［ｈ―ｖＷＦ］）））／（２^＊ＡｐｔＣｏｎｃ））＋バックグラウンドにデータをあてはめてＫ_Ｄｓが推定された。ラウンド６プールは、約２ｎＭのＫ_Ｄを有した。プールは、Ａｌドメインに対する結合についてもテストされ、約４ｎＭのＫ_Ｄを有した。

dCmD Aptamer Binding Analysis A dot blot binding assay was performed throughout the selection to monitor the protein binding affinity of the pool. Trace ³² P-endo labeled pool RNA was combined with h-vWF and incubated in DPBS buffer for 30 minutes at room temperature in a final volume of 30 μL. The mixture was applied to a dot blot apparatus (Minifold-1 Dot Blot, Acrylic, Schleicher and Schuell, Keene, NH) (from top to bottom) and assembled with nitrocellulose, nylon, and gel blot membranes. RNA bound to protein is captured on a nitrocellulose filter; non-protein bound RNA is captured on a nylon filter. In round 5, the onset of h-vWF binding enrichment was seen. The dissociation constant (K _D ) for round 5 and round 6 pools was determined. Briefly, pool RNAs are 5 ′ end labeled with γ- ³² P ATP, and buffer conditions of dot blot assay and 1 × DPBS (w / Ca2 + and Mg2 +) (Gibco, Catalog # 14040, Invitrogen, Carlsbad, Calif.) Are used. Used to determine the _KD value. Formula: RNA binding fraction = amplitude ^* (((AptConc + [h-vWF] + KD) -SQRT ((ApTConc + [h-vWF] + KD) 2-4 (ApConc ^* [h-vWF])))) / (2 ^* AptConc)) + background by applying the data _{K D} s is estimated. Round 6 pool had a _{K D} of about 2 nM. Pools are also tested for binding to Al domain, it had a _{K D} of about 4 nM.

Example 7 Aptamer Selection Using Y639L / H784A / K378R Mutant T7 RNA Polymerase Using a pool of 2′-OMe ATP, GTP, and UTP nucleotides and 2′-deoxy CTP nucleotides (hereinafter “dCmD”) Selection was made to identify aptamers to human IgE (hereinafter “h-IgE”). The selection strategy resulted in a high affinity aptamer specific for h-IgE.

  Human IgE was purchased from Athens Research & Technology (Cat. # 16-16-090705 Athens, GA). T7 RNA polymerase (Y639L / H784A / K378R) was expressed and purified as described above in Example 1. 2'-OMe ATP, GTP, and UTP nucleotides, and 2'-deoxy CTP nucleotides were purchased from TriLink BioTechnologies (San Diego, CA).

IgE aptamer selection Pool preparation Sequence

のＤＮＡテンプレートが、ＡＢＩ ＥＸＰＥＤＩＴＥ（商標）（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、カリフォルニア州フォスターシティ）ＤＮＡ合成装置用いて合成され、標準法により脱保護された。テンプレートは、プライマ

DNA templates were synthesized using an ABI EXPEDITE ™ (Applied Biosystems, Foster City, CA) DNA synthesizer and deprotected by standard methods. Template is a primer

により増幅されてから、Ｔ７ＲＮＡポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）によるｉｎ ｖｉｔｒｏ転写のテンプレートとして使用された。２００ｍＭ ＨＥＰＥＳ、４０ｍＭ ＤＴＴ、２．０ｍＭスペルミジン、０．０１％ｗ／ｖ ＴｒｉｔｏｎＸ―１００、１０％ＰＥＧ―８０００、８ｍＭ ＭｇＣｌ_２、２．５ｍＭ ＭｎＣｌ_２、１．５ｍＭ ｄＣＴＰ、１．５ｍＭ ｍＵＴＰ、１．５ｍＭ ｍＧＴＰ、１．５ｍＭ ｍＡＴＰ、１ｍＭ ＧＭＰ、０．０１単位／μＬ無機ピロホスファターゼ、および〜９μｇ／ｍＬ変異Ｔ７ポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）および０．３μＭテンプレートＤＮＡを用いて転写が行われ、ＡＲＣ４２１８ｄＣｍＤプールが生成された。

And then used as a template for in vitro transcription with T7 RNA polymerase (Y639L / H784A / K378R). 200 mM HEPES, 40 mM DTT, 2.0 mM spermidine, 0.01% w / v Triton X-100, 10% PEG-8000, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1.5 mM dCTP, 1.5 mM mUTP, 1. Transcription was performed using 5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01 unit / μL inorganic pyrophosphatase, and ˜9 μg / mL mutant T7 polymerase (Y639L / H784A / K378R) and 0.3 μM template DNA; An ARC4218dCmD pool was created.

Selection 330 pmol (2 × 10 ¹⁴ molecules) of dCmD ARC4218 pool was added to 100 μL final volume of selection buffer (1 × Dulbecco) with 20 pmol of protein bound to HSA blocked hydrophobic plates (Maxisorp plate, Nunc, Rochester, NY). Selection was initiated by incubating in PBS (DPBS) for 1 hour at room temperature. Wells were washed 5 times with 200 μL DPBS to remove non-specific binders. RNA was eluted and reverse transcribed with Primer ARC4208 (SEQ ID NO: 30) with a ThermoScript RT-PCR ™ system (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The cDNA was amplified by PCR using Taq polymerase (New England Biolabs, Beverly, Mass.) Using primers ARC4219 (SEQ ID NO: 29) and ARC4208 (SEQ ID NO: 30) according to the manufacturer's instructions. The template was transferred as described above for pool preparation. After the template was purified by ethanol precipitation, a desalting step was performed using a Micro Bio-spin column (Cat. # 732-6250, Bio-Rad Laboratories, Hercules, CA).

  All rounds were performed with h-IgE immobilized on a hydrophobic plate. Each selection round was initiated by immobilizing 20 pmol of h-IgE on the surface of a Nunc Maxisorp hydrophobic plate in 100 μL of 1 × DPBS for 30 minutes at room temperature. Plates were washed 3 times with 200 μL DPBS and then incubated with blocking buffer (1 × DPBS, and 5% HSA) for 30 minutes. The supernatant was removed and the wells were washed three times with 200 μL DPBS. Starting from round 2, the pool RNA was incubated for 30 minutes at room temperature in empty wells and then incubated for 30 minutes in wells previously blocked with 100 μL blocking buffer. From round 2 onwards, non-specific competitors were added to the positive selection step (0.1 mg / mL tRNA). In all cases, pooled RNA bound to immobilized h-IgE was added by reverse transcription (“RT”) mixture (3 ′ primer, SEQ ID NO: 30, and Thermoscript RT, Invitrogen, Carlsbad, Calif.) Followed by Of 1 hour incubation at 65 ° C was directly reverse transcribed in selective plates. The resulting cDNA was used as a template for PCR (Taq polymerase, New England Biolabs, Beverly, Mass.) And transcription as described per round 1. Table 8 shows the conditions for each round.

ｄＣｍＤアプタマー結合分析
プールのタンパク質結合親和性をモニタするために、選択の全体にわたりドットブロット結合アッセイが行われた。トレース^３２Ｐ―エンド標識したプールＲＮＡが、ｈ―ＩｇＥと組み合わされ、ＤＰＢＳバッファー中で３０μＬの最終体積で、室温で３０分間インキュベートされた。混合物がドットブロット装置に適用され（Ｍｉｎｉｆｏｌｄ―１ Ｄｏｔ Ｂｌｏｔ、Ａｃｒｙｌｉｃ、Ｓｃｈｌｅｉｃｈｅｒ ａｎｄ Ｓｃｈｕｅｌｌ、ニューハンプシャー州キーン）、（上から下へ）ニトロセルロース、ナイロン、およびゲルブロット膜とアセンブルされた。タンパク質に結合されたＲＮＡはニトロセルロースフィルタに捕らえられ；非タンパク結合ＲＮＡはナイロンフィルタに捕らえられる。ラウンド５でｈ―ＩｇＥ結合の濃縮の開始が見られた。ラウンド５および６プールの解離定数（Ｋ_Ｄ）が決定された。簡単にいうと、プールＲＮＡｓがγ―^３２Ｐ ＡＴＰで５’末端標識され、ドットブロットアッセイおよび１Ｘ ＤＰＢＳ（ｗ／Ｃａ２＋およびＭｇ２＋）（Ｇｉｂｃｏ、Ｃａｔａｌｏｇ＃１４０４０、Ｉｎｖｉｔｒｏｇｅｎ、カリフォルニア州カールズバッド）のバッファー条件を用いてＫ_Ｄ値が決定された。式：ＲＮＡ結合画分＝振幅^＊（（（ＡｐｔＣｏｎｃ＋［ｈ―ＩｇＥ］＋ＫＤ）−ＳＱＲＴ（（ＡｐｔＣｏｎｃ＋［ｈ―ＩｇＥ］＋ＫＤ）２―４（ＡｐｔＣｏｎｃ^＊［ｈ―ＩｇＥ］）））／（２^＊ＡｐｔＣｏｎｃ））＋バックグラウンドにデータをあてはめてＫＤｓが推定された。ラウンド６プールは、約８０ｎＭのＫ_Ｄを有した。

dCmD Aptamer Binding Analysis A dot blot binding assay was performed throughout the selection to monitor the protein binding affinity of the pool. Trace ³² P-endo labeled pool RNA was combined with h-IgE and incubated in DPBS buffer for 30 minutes at room temperature in a final volume of 30 μL. The mixture was applied to a dot blot apparatus (Minifold-1 Dot Blot, Acrylic, Schleicher and Schuell, Keene, NH) (from top to bottom) and assembled with nitrocellulose, nylon, and gel blot membranes. RNA bound to protein is captured on a nitrocellulose filter; non-protein bound RNA is captured on a nylon filter. Round 5 showed the onset of enrichment of h-IgE binding. The dissociation constants (K _D ) for the round 5 and 6 pools were determined. Briefly, pool RNAs are 5 ′ end labeled with γ- ³² P ATP, and buffer conditions of dot blot assay and 1 × DPBS (w / Ca2 + and Mg2 +) (Gibco, Catalog # 14040, Invitrogen, Carlsbad, Calif.) Are used. Used to determine the _KD value. Formula: RNA binding fraction = amplitude ^* (((AptConc + [h-IgE] + KD) -SQRT ((ApTConc + [h-IgE] + KD) 2-4 (ApTConc ^* [h-IgE])))) / (2 ^* Opts)) + data was applied to the background to estimate KDs. Round 6 pool had a _{K D} of approximately 80 nM.

Example 8 Transcription Introducing 2′-O-Methyl ATP, CTP, GTP and 2′-O-Methyl-5-Indolylmethylene UTP Nucleotides Using the In Vitro Transcription 2′-O-methyl A, C, G and 2′-O-methyl-5 indolylmethylene U are used to introduce the transcript. Modified uridines are shown below as nucleosides:

２００ｍＭ ＨＥＰＥＳ、４０ｍＭ ＤＴＴ、２ｍＭスペルミジン、０．０１％ｗ／ｖ ＴｒｉｔｏｎＸ―１００、１０％ ＰＥＧ―８０００、８ｍＭ ＭｇＣｌ_２、２．５ｍＭ ＭｎＣｌ_２、１．５ｍＭ ｍＣＴＰ、１．５ｍＭ ５―インドリル―ｍＵＴＰ、１．５ｍＭ ｍＧＴＰ、１．５ｍＭ ｍＡＴＰ、１ｍＭ ＧＭＰ、０．０１単位／μＬ無機ピロホスファターゼ、および〜９μｇ／ｍＬ変異Ｔ７ポリメラーゼ（Ｙ６３９Ｌ／Ｈ７８４Ａ／Ｋ３７８Ｒ）および０．３μＭテンプレートＤＮＡを用いて、３７℃で一晩転写が行われて、５’―末端に単一の２’―ヒドロキシＧＭＰを伴う２’―ＯＭｅ Ａ、２’―ＯＭｅ Ｇ、２’―ＯＭｅ Ｃおよび２’―ＯＭｅ―５―インドリルメチレンＵからなる転写産物が生成される（以下に「ＭＮＡ―Ｉ」）。各条件下の転写産物が、４００ｕＬの反応混合物を用いてＰＡＧＥ―ゲル分析によりアッセイされ、各条件での転写産物が、ＰＡＧＥ―ゲルのＵＶ―シャドウイングから視覚化される。

200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% w / v Triton X-100, 10% PEG-8000, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1.5 mM mCTP, 1.5 mM 5-indolyl-mUTP, 37 ° C. with 1.5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01 unit / μL inorganic pyrophosphatase, and ˜9 μg / mL mutant T7 polymerase (Y639L / H784A / K378R) and 0.3 μM template DNA 2'-OMe A, 2'-OMe G, 2'-OMe C and 2'-OMe-5-in with a single 2'-hydroxy GMP at the 5'-end A transcript consisting of drill methylene U is produced (hereinafter “MNA-I”). Transcripts under each condition are assayed by PAGE-gel analysis using 400 uL of reaction mixture, and transcripts at each condition are visualized from UV-shadowing of the PAGE-gel.

(Example 9) SELEX using Y639L / H784A / K378R mutant T7 RNA polymerase and 2′-O-methyl-5-indolylmethyleneuridine
SELEX against a protein target for therapeutic purposes is performed using a transcription template comprising multiple degenerate positions and the MNA-I transcription conditions described in Example 7 above.

330 pmol (2 × 10 ¹⁴ molecules) of MNA with 20 pmol of protein bound to HSA-blocked hydrophobic plates (Maxisorp plates, Nunc, Rochester, NY) in a final volume of 100 μL of selection buffer (1 × Dulbecco's PBS (DPBS)). The selection step is initiated by incubating the I transcription library for 1 hour at room temperature. To remove non-specific binders, the wells are washed 5 times with 200 μL DPBS. RNA is reverse transcribed in selected wells using ThermoScript RT-PCR ™ (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The resulting cDNA is amplified by PCR with Taq polymerase (New England Biolabs, Beverly, Mass.) Using the 5 'primer encoding the T7 RNA polymerase promoter according to the manufacturer's instructions. The resulting transfer template is transferred as described above under MNA-I conditions. The resulting transcript is purified by ethanol precipitation followed by a desalting step using a Micro Bio-spin column (Cat. # 732-6250, Bio-Rad Laboratories, Hercules, CA).

  After 10 rounds of SELEX, the resulting transcript library is reverse transcribed, amplified by PCR, cloned and sequenced. Individual clones are transcribed under MNA-I conditions and assayed for their ability to bind and inhibit the target protein.

Example 10 Transcription Introducing 2′-OMe TTP, 2′-OMe ATP, 2′-OMe CTP, 2′-OMe GTP Nucleotide (mTmV Composition)
200 nM double-stranded transcription template with continuous degenerate region inside 40 nucleotides, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-OMe TTP, 2′- OMe ATP, 2′-OMe CTP, 2′-OMe GTP, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, 2.5 units / ml inorganic pyrophosphatase, pH 7.5 and K378R / Y639F A 400 ul transcript was prepared by / H784A mutant T7 polymerase. The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. A band was observed that co-migrated with the corresponding MNA transcript loaded in the adjacent well. Negative control transcripts incubated without 2′-OMe TTP produced no observable band.

Example 11 Transcription Introducing Deoxy TTP, 2′-OMe ATP, 2′-OMe CTP, 2′-OMe GTP Nucleotide (dTmV Composition)
200 nM double-stranded transcription template ARC3205, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-deoxy TTP, 2′-OMe ATP, 2′-OMe CTP, 2 '-OMe GTP, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, 2.5 units / ml inorganic pyrophosphatase, pH 7.5 and K378R / Y639F / H784A mutant T7 polymerase ready 400 ul transcription It was done. The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. A band was observed that co-migrated with the corresponding MNA transcript loaded in the adjacent well. Negative control transcripts incubated without 2′-deoxy TTP produced no observable band.

Example 12 Transcription Introducing 2′-OH TTP, 2′-OMe ATP, 2′-OMe CTP, 2′-OMe GTP Nucleotide (rTmV Composition)
200 nM, 40-nucleotide double-stranded transcription template with continuous degenerate region inside, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-OH TTP, 2′- OMe ATP, 2′-OMe CTP, 2′-OMe GTP, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, inorganic pyrophosphatase 2.5 units / ml, pH 7.5 and K378R / Y639F / A 400 ul transcript was prepared by H784A mutant T7 polymerase. The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. A band was observed that co-migrated with the corresponding MNA transcript loaded in the adjacent well. Negative control transcripts incubated without 2'-OH TTP produced no observable band.

Example 13 Transcription Introducing Deoxy UTP, 2′-OMe ATP, 2′-OMe CTP, 2′-OMe GTP Nucleotide (dUmV Composition)
200 nM double-stranded transcription template ARC3205, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-deoxy UTP, 2′-OMe ATP, 2′-OMe CTP, 2 '-OMe GTP, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, inorganic pyrophosphatase 2.5 units / ml, pH 7.5 and K378R / Y639F / H784A mutant T7 polymerase ready 400 ul transcription It was. The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. A band was observed that co-migrated with the corresponding MNA transcript loaded in the adjacent well. Negative control transcripts incubated without 2′-deoxy UTP produced no observable band.

Example 14 Transcription Introducing 2′-OH UTP, 2′-OMe ATP, 2′-OMe CTP, 2′-OMe GTP Nucleotide (rUmV Composition)
200 nM double-stranded transcription template ARC3205, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-OH UTP, 2′-OMe ATP, 2′-OMe CTP, 2 '-OMe GTP, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, inorganic pyrophosphatase 2.5 units / ml, pH 7.5 and K378R / Y639F / H784A mutant T7 polymerase ready 400 ul transcription It was. The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. A band was observed that co-migrated with the corresponding MNA transcript loaded in the adjacent well. Negative control transcripts incubated without 2′-OH UTP produced no observable band.

Example 15 Transcription Introducing 2′-OH GTP, Deoxy TTP, 2′-OMe ATP, 2′-OMe CTP Nucleotide (rGdTMM Composition)
200 nM double-stranded transcription template ARC3205, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-deoxy TTP, 2′-OMe ATP, 2′-OMe CTP, 2 '-OH GTP, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, inorganic pyrophosphatase 2.5 units / ml, pH 7.5 and K378R / Y639F / H784A mutant T7 polymerase prepares 400 ul transcription It was. The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. A band was observed that co-migrated with the corresponding MNA transcript loaded in the adjacent well. Negative control transcripts incubated without 2'-deoxy TTP produced no observable band.

Example 16 Transcription Introducing 2′-OMe QTP, 2′-OH GTP, 2′-OMe ATP, 2′-OMe CTP Nucleotide (mQrGMmM Composition)
200 nM 40 nucleotide double-stranded transcription template with continuous degenerate region inside, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-OMe QTP, 2′- OMe ATP, 2′-OMe CTP, 2′-OH GTP, 8 mM MgCl ₂ , 2.5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, inorganic pyrophosphatase 2.5 units / ml, pH 7.5 and K378R / Y639F / A 400 ul transcript was prepared by H784A mutant T7 polymerase. 2′-OMe QTP (mQ) is shown below:

溶液が３７℃で一晩インキュベートされ、沈殿され、変性ローディングバッファーに溶解され、変性ＰＡＧＥを用いて分析された後、ＵＶシャドウイングを用いて蛍光板上で可視化された。隣接するウェルにロードされた対応するＭＮＡ転写産物と共移動した明らかなバンドが観察された。２’―ＯＭｅ ＱＴＰを伴わずにインキュベートされた陰性対照転写物は、観察可能なバンドを生成しなかった。

The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. A clear band was observed that co-migrated with the corresponding MNA transcript loaded in the adjacent well. Negative control transcripts incubated without 2'-OMe QTP produced no observable band.

Example 17 Transcription to introduce 2′-OMe UTP, 2′-OMe ATP, 2′-OMe GTP, 2′-OMe CTP or 2′-OH NTPs using modified and wild type T7 polymerase (rGdTMM composition) object)
50 nM double-stranded transcription template with continuous degenerate region inside 30 nucleotides, 200 mM HEPES, 40 mM DTT, 2 mM spermidine, 0.01% Triton X-100, 1.5 mM each 2′-OMe UTP, 2′- OMe ATP, 2′-OMe CTP and 2′-OMe GTP, or 1.5 mM each of 2′-OH UTP, 2′-OH ATP, 2′-OH CTP and 2′-OH GTP, 8 mM MgCl ₂ , 2 5 mM MnCl ₂ , 1 mM GMP, 10% PEG 8000, inorganic pyrophosphatase 2.5 units / ml, pH 7.5 and the mutations shown below or wild type T7 polymerase (WT is K378R, F is K378R / Y639F, L is K378R / Y639L, FA is K378R / Y639F / H784A, LA is K 378R / Y639L / H784A), 400 ul transfer was prepared. The solution was incubated overnight at 37 ° C., precipitated, dissolved in denaturing loading buffer, analyzed using denaturing PAGE and visualized on a fluorescent plate using UV shadowing. As shown in FIG. 11, different transcription yields were observed.

  Although the invention has been described by way of explanation and examples, it will be appreciated by those skilled in the art that the invention may be practiced in various embodiments, and the foregoing description and examples are for illustrative purposes. There is no intention to limit the scope of the following claims.

Claims (54)

A method of transcribing nucleic acids,
a) (i) 2′-OMe ATP, 2′-OMe GTP, 2′-OMe CTP, 2′-OMe TTP and 2′-OMe modified nucleotide triphosphates selected from 2′-OMe UTP (2 ′ A modified T7 RNA polymerase capable of introducing any of the 2′-modified NTPs compared to the ability of the corresponding unmodified RNA polymerase to introduce 2′-modified NTPs. -A modified T7 RNA polymerase with increased ability to introduce modified nucleotide triphosphates (2'-modified NTPs), (ii) at least one nucleic acid transcription template, and (iii) nucleotide triphosphates, Nucleotides in which the nucleotide triphosphate comprises guanidine triphosphate, all of which are 2′-OMe modified And a phosphoric acid, a step to prepare a transcription reaction mixture;
b) transcribing the transcription reaction mixture under conditions that result in single stranded nucleic acid, wherein all guanidine nucleotides after the first guanosine nucleotide in the resulting single stranded nucleic acid are 2′-OMe modified. A method comprising steps and   The method of claim 1, wherein the transcription reaction mixture further comprises magnesium ions, manganese ions, or both magnesium and manganese ions.   The method of claim 1, wherein the nucleic acid transcription template is at least partially double stranded.   The method of claim 1, wherein the nucleic acid transcription template is a complete duplex.   2. The method of claim 1, wherein the first guanosine nucleotide in the resulting single stranded nucleic acid is 2'-OMe modified.   The modified T7 RNA polymerase comprises amino acids modified at positions 639 and 784 relative to wild-type T7 polymerase, and when the modified amino acid at position 784 is alanine, the modified amino acid at position 639 is not phenylalanine. The method according to 1.   7. The method of claim 6, wherein the modified T7 RNA polymerase comprises leucine at position 639 and alanine at position 784.   4. The method of claim 3, wherein the double stranded oligonucleotide transcription template further comprises a leader sequence introduced into a fixed region at the 5 'end of the oligonucleotide transcription template.   9. The method of claim 8, wherein the leader sequence is an all purine leader sequence.   10. The method of claim 9, wherein the all purine leader sequence has a length selected from at least 8 nucleotides long, at least 10 nucleotides long; at least 12 nucleotides long; and at least 14 nucleotides long.   The method according to claim 2, wherein the reaction transfer conditions include both magnesium and manganese ions, and the concentration of magnesium ions is 3.0 to 3.5 times higher than the concentration of manganese ions.   Each NTP, 2′-modified NTP or 2′-OMe NTP is present at a concentration of about 0.5 mM, and the reaction transcription conditions include both magnesium and manganese ions, and the concentration of magnesium ions is about 5.0 mM. The method of claim 2, wherein the concentration of manganese ions is about 1.5 mM.   Each NTP, 2′-modified NTP or 2′-OMe NTP is present at a concentration of about 1.0 mM, and the reaction transcription conditions include both magnesium and manganese ions, and the concentration of magnesium ions is about 6.5 mM. The method of claim 2, wherein the manganese ion concentration is about 2.0 mM.   Each NTP, 2′-modified NTP, or 2′-OMe NTP is present at a concentration of about 2.0 mM, and the reaction transcription conditions include both magnesium and manganese ions, and the concentration of magnesium ions is about 9.6 mM. The method of claim 2, wherein the concentration of manganese ions is about 2.9 mM.   The method of claim 1, wherein the transcription reaction mixture further comprises substituted guanosine or guanosine.   16. The method of claim 15, wherein the substituted guanosine or guanosine is GMP.   The method of claim 1, wherein the transcription reaction mixture further comprises a polyalkylene glycol.   The method according to claim 17, wherein the polyalkylene glycol is polyethylene glycol.   The method of claim 1, wherein the transcription reaction mixture further comprises inorganic pyrophosphatase.   The transcription reaction mixture comprises 2′-deoxycytidine triphosphate (2′-deoxy CTP), 2′-O-Me adenosine triphosphate (2′-OMe ATP), 2′-OMe guanosine triphosphate GTP ( 2'-OMe GTP) and a mixture of 2'-OMe uridine triphosphate (2'-OMe UTP) or 2'-OMe thymidine nucleotide triphosphate (2'-OMe TTP). The method described.   The transcription reaction mixture comprises 2′-deoxythymidine triphosphate (2′-deoxy TTP) or 2′-deoxyuridine triphosphate (2′-deoxy UTP) and 2′-O-Me adenosine triphosphate (2 2. The composition of claim 1, further comprising a mixture of '-OMe ATP), 2'-OMe guanosine triphosphate GTP (2'-OMe GTP), and 2'-OMe cytidine triphosphate (2'-OMe CTP). the method of.   The transcription reaction mixture comprises 2′-OH thymidine triphosphate (2′-OH TTP) or 2′-OH uridine triphosphate (2′-OH UTP) and 2′-O-Me adenosine triphosphate (2 2. The composition of claim 1, further comprising a mixture of '-OMe ATP), 2'-OMe guanosine triphosphate GTP (2'-OMe GTP), and 2'-OMe cytidine triphosphate (2'-OMe CTP). the method of.   The method of claim 1, wherein the transcription reaction mixture further comprises a side chain modified nucleobase.   The nucleobase is uridine 5- and 6-position, thymidine 5- and 6-position, cytidine 5- and 6-position and exocyclic amine, adenosine 2-, 7- and 8-position and exocyclic amine 24. The method of claim 23, wherein the method is modified at a position selected from the group consisting of 7- and 8-positions of guanosine and an exocyclic amine.   25. The method of claim 24, wherein the modified nucleobase is uracil, cytosine or thymidine.   24. The method of claim 23, wherein the side chain is a hydrophobic aromatic side chain.   27. The method of claim 26, wherein the side chain is benzyl or indolyl. A method of identifying an aptamer, wherein the aptamer comprises a 2'-OMe modified nucleotide;
a) (i) 2′-OMe ATP, 2′-OMe GTP, 2′-OMe CTP, 2′-OMe TTP and 2′-OMe modified nucleotide triphosphates selected from 2′-OMe UTP (2 ′ A modified T7 RNA polymerase capable of introducing any of the 2′-modified NTPs compared to the ability of the corresponding unmodified RNA polymerase to introduce 2′-modified NTPs. -A modified T7 RNA polymerase with increased ability to introduce modified nucleotide triphosphates (2'-modified NTPs), (ii) at least one nucleic acid transcription template, and (iii) nucleotide triphosphates, Nucleotides in which the nucleotide triphosphate comprises guanidine triphosphate, all of which are 2′-OMe modified And a phosphoric acid, a step to prepare a transcription reaction mixture;
b) the transcription under conditions where the modified T7 RNA polymerase introduces at least one modified 2′-OMe nucleotide triphosphate (2′-OMe NTP) into the nucleic acid molecule of the candidate mixture of single-stranded nucleic acids. Preparing a candidate mixture of the single stranded nucleic acids by transcribing a reaction mixture, wherein all guanidine nucleotides after the first guanosine nucleotide of the single stranded nucleic acid molecule of the candidate mixture are 2 ′ -OMe modified, step;
c) contacting the candidate mixture with the target molecule;
d) separating the nucleic acid with increased affinity for a target molecule compared to the affinity of the candidate mixture from the candidate mixture;
e) amplifying said increased affinity nucleic acid to yield a mixture enriched in aptamers, thereby selectively excluding one of said guanidine nucleotides of the aptamer, all 2′-OMe The aptamer to the target molecule comprising a modified guanidine nucleotide is identified;
Method.   30. The method of claim 28, wherein the transcription reaction mixture further comprises magnesium ions, manganese ions, or both magnesium and manganese ions.   30. The method of claim 28, wherein the nucleic acid transcription template is at least partially double stranded.   30. The method of claim 28, wherein the nucleic acid transcription template is a complete duplex.   29. The method of claim 28, wherein the first guanosine nucleotide in the resulting single stranded nucleic acid is 2'-OMe modified.   The modified T7 RNA polymerase comprises amino acids modified at positions 639 and 784 relative to wild-type T7 polymerase, and when the modified amino acid at position 784 is alanine, the modified amino acid at position 639 is not phenylalanine. 28. The method according to 28.   34. The method of claim 33, wherein the modified T7 RNA polymerase comprises leucine at position 639 and alanine at position 784.   32. The method of claim 30, wherein the double stranded oligonucleotide transcription template further comprises a leader sequence introduced into a fixed region at the 5 'end of the oligonucleotide transcription template.   36. The method of claim 35, wherein the leader sequence is an all purine leader sequence.   37. The method of claim 36, wherein said all purine leader sequence has a length selected from at least 8 nucleotides long, at least 10 nucleotides long; at least 12 nucleotides long; and at least 14 nucleotides long.   30. The method of claim 29, wherein the reaction transfer conditions include both magnesium and manganese ions, and the magnesium ion concentration is 3.0-3.5 times higher than the manganese ion concentration.   Each NTP, 2′-modified NTP or 2′-OMe NTP is present at a concentration of about 0.5 mM, and the reaction transcription conditions include both magnesium and manganese ions, and the concentration of magnesium ions is about 5.0 mM. 30. The method of claim 29, wherein the manganese ion concentration is about 1.5 mM.   Each NTP, 2′-modified NTP or 2′-OMe NTP is present at a concentration of about 1.0 mM, and the reaction transcription conditions include both magnesium and manganese ions, and the concentration of magnesium ions is about 6.5 mM. 30. The method of claim 29, wherein the manganese ion concentration is about 2.0 mM.   Each NTP, 2′-modified NTP, or 2′-OMe NTP is present at a concentration of about 2.0 mM, and the reaction transcription conditions include both magnesium and manganese ions, and the concentration of magnesium ions is about 9.6 mM. 30. The method of claim 29, wherein the manganese ion concentration is about 2.9 mM.   30. The method of claim 28, wherein the transcription reaction mixture further comprises substituted guanosine or guanosine.   43. The method of claim 42, wherein the substituted guanosine or guanosine is GMP.   30. The method of claim 28, wherein the transcription reaction mixture further comprises a polyalkylene glycol.   45. The method of claim 44, wherein the polyalkylene glycol is polyethylene glycol.   30. The method of claim 28, wherein the transcription reaction mixture further comprises inorganic pyrophosphatase.   The transcription reaction mixture comprises 2′-deoxycytidine triphosphate (2′-deoxy CTP), 2′-O-Me adenosine triphosphate (2′-OMe ATP), 2′-OMe guanosine triphosphate GTP ( 28. further comprising a mixture of 2′-OMe GTP) and 2′-OMe uridine triphosphate (2′-OMe UTP) or 2′-OMe thymidine nucleotide triphosphate (2′-OMe TTP). The method described.   The transcription reaction mixture comprises 2′-deoxythymidine triphosphate (2′-deoxy TTP) or 2′-deoxyuridine triphosphate (2′-deoxy UTP) and 2′-O-Me adenosine triphosphate (2 29. The method of claim 28, further comprising a mixture of '-OMe ATP), 2'-OMe guanosine triphosphate GTP (2'-OMe GTP), and 2'-OMe cytidine triphosphate (2'-OMe CTP). the method of.   The transcription reaction mixture comprises 2′-OH thymidine triphosphate (2′-OH TTP) or 2′-OH uridine triphosphate (2′-OH UTP) and 2′-O-Me adenosine triphosphate (2 29. The method of claim 28, further comprising a mixture of '-OMe ATP), 2'-OMe guanosine triphosphate GTP (2'-OMe GTP), and 2'-OMe cytidine triphosphate (2'-OMe CTP). the method of.   30. The method of claim 28, wherein the transcription reaction mixture further comprises nucleobases modified with side chains.   The nucleobase is uridine 5- and 6-position, thymidine 5- and 6-position, cytidine 5- and 6-position and exocyclic amine, adenosine 2-, 7- and 8-position and exocyclic amine 51. The method of claim 50, modified at a position selected from the group consisting of 7- and 8-positions of guanosine and an exocyclic amine.   52. The method of claim 51, wherein the modified nucleobase is uracil, cytosine or thymidine.   51. The method of claim 50, wherein the side chain is a hydrophobic aromatic side chain.   54. The method of claim 53, wherein the side chain is benzyl or indolyl.

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