JP2002512794A - Nucleotide triphosphates and their incorporation into ribozymes - Google Patents

Abstract

  (57) [Summary] Disclosed are novel nucleotide triphosphates, synthetic methods, methods of incorporating these nucleotide triphosphates into oligonucleotides, and isolation of novel nucleic acid catalysts (eg, ribozymes).

Description

DETAILED DESCRIPTION OF THE INVENTION

RELATED APPLICATIONS This patent application is filed in US Ser. No. 09 / 186,675 to Beigelman et al., Filed Nov. 4, 1998;
USSN 60 / 083,727 by Elman et al. and November 6, 1997.
Claims based on US Ser. No. 60 / 064,866 filed by Beigelman et al., All of which are entitled "Nucleotide Triphosphates and Their Incorporation into Oligonucleotides." ing.
Each of these applications, including the figures, is hereby incorporated by reference in its entirety.

[0002] The present invention relates to novel nucleotide triphosphate (NTP); concerns a method for incorporating into and novel nucleotide triphosphate within oligonucleotides; nucleotide triphosphates synthesis of phosphoric acid. The invention further relates to the incorporation of these nucleotide triphosphates into nucleic acid molecules using a polymerase under some novel reaction conditions.

[0003] The following is a brief description of nucleotide triphosphates. This summary is not intended to be exhaustive, but is provided only to assist in understanding the following invention. This summary is not an admission that all of the work described below is prior art to the claimed invention.

[0004] The synthesis of nucleotide triphosphates and their incorporation into nucleic acids using polymerase enzymes has greatly helped advance nucleic acid research. Polymerase enzymes utilize nucleotide triphosphates as precursor molecules to assemble oligonucleotides. Each nucleotide is linked by a phosphodiester bond formed by nucleophilic attack of the last 3 'hydroxyl group of the oligonucleotide on the 5' triphosphate of the next nucleotide. Nucleotides are incorporated into the oligonucleotide one at a time in the 5 'to 3' direction. This process allows for the production and amplification of RNA from virtually any DNA or RNA template.

[0005] Most natural polymerase enzymes incorporate standard nucleotide triphosphates into nucleic acids. For example, DNA polymerases include dATP, dTTP, dCTP and d
GTP is incorporated into DNA, and RNA polymerases generally contain ATP, CTP, UTP
Incorporates TP and GTP into RNA. However, there are certain polymerases that can incorporate non-standard nucleotide triphosphates into nucleic acids (Jo).
yce, 1997, PNAS 94, 1619-1622, Huang et.
al. , Biochemistry 36, 8231-8242).

[0006] Before nucleosides can be incorporated into RNA transcripts using polymerase enzymes, they must be converted to nucleotide triphosphates that are recognizable by these enzymes. Phosphorylation of unprotected nucleosides with POCl3 and trialkyl phosphate has been shown to give nucleoside 5'-phosphorodichloridates (Yoshikawa et al., 1969, Bull. Chem. So.
c. (Japan) 42, 3505). Adenosine or 2'-deoxyadenosine 5'-triphosphate was synthesized by adding an additional step consisting of treatment with excess tri-n-butylammonium pyrophosphate in DMF, followed by hydrolysis (Ludwig). , 1981, Acta Biochim. Et Biop.
hys. Acad. Sci. Hung. 16, 131-133).

[0007] Non-standard nucleotide triphosphates are readily converted to RN by traditional RNA polymerases.
A Not transferred into the transcript. Mutations were introduced into RNA polymerase to facilitate the incorporation of deoxyribonucleotides into RNA (Sousa
& Padilla, 1995, EMBO J .; 14,4609-4621,
Bonner et al. , 1992, EMBO J .; 11,3767-37
75, Bonner et al. , 1994, J.A. Biol. Chem. 42
, 25120-25128, Aurup et al. , 1992, Bioch.
chemistry 31,9636-9641).

[0008] McGee et al. , International PCT Publication No. WO 95/35102, discloses 2'-NH2-N into RNA using bacteriophage T7 polymerase.
TP, 2'-F-NTP and 2'-deoxy-2'-benzyloxyamino U
The incorporation of TP is described.

[0009] Wieczorek et al. , 1994, Bioorganic &
Medicinal Chemistry Letters 4,987-99
4, describes the incorporation of 7-deaza-adenosine triphosphate into RNA transcripts using bacteriophage T7 RNA polymerase.

[0010] Lin et al. , 1994, Nucleic Acids Research.
rch 22,5229-5234 uses 2'-N into bacteriophage T7 RNA polymerase and RNA using a polyethylene glycol containing buffer.
Reports incorporation of H2-CTP and 2'-NH2-UTP. This article describes the use of polymerase synthetic RNA in in vitro selection of aptamers against human neutrophil elastase (HNE).

SUMMARY OF THE INVENTION The present invention relates to novel nucleotide triphosphate (NTP) molecules and their incorporation into nucleic acid molecules, including nucleic acid catalysts. The NTP of the present invention is different from other NTPs known in the art. The invention further relates to the incorporation of these nucleotide triphosphates into oligonucleotides using RNA polymerase; the invention further relates to novel transcription for incorporation of modified (non-standard) and unmodified NTP into nucleic acid molecules It also relates to the conditions. Furthermore, the invention relates to a method for the synthesis of a novel NTP.

In a first aspect, the invention features an NTP having the formula: triphosphate-OR (eg, Formula I below):

Embedded image Wherein R is any nucleoside; in particular nucleoside 2'-O-methyl-2,6-diaminopurine riboside; 2'-deoxy-2'-amino-2,6
-Diaminopurine riboside; 2 '-(N-alanyl) amino-2'-deoxy-uridine;2'-(N-phenylalanyl)amino-2'-deoxy-uridine;2'-deoxy-2'-( N-β-alanyl) amino; 2′-deoxy-2
2′-C-allyl uridine; 2′-O-amino-uridine; 2′-O-methylthiomethyl adenosine; 2′-O-methylthiomethyl cytidine; 2′-O-methylthiomethyl Guanosine; 2'-O-methylthiomethyluridine;2'-deoxy-2'-(N-histidyl)aminouridine;2'-deoxy-2'-amino-5-methylcytidine;2'-(
N-β-carboxamidine-β-alanyl) amino-2′-deoxy-uridine; 2′-deoxy-2 ′-(N-β-alanyl) -guanosine; 2′-O-amino-adenosine; 2 ′-( N-lysyl) amino-2'-deoxy-cytidine; 2
'-Deoxy-2'-(L-histidine) aminocytidine; and 5-imidazoleacetic acid 2'-deoxy-5'-uridine triphosphate. In a second aspect, the invention relates to pyrimidine nucleotide triphosphates (UTP, 2'-O-MTM-
(Eg UTP, dUTP, etc.). In the method, a phosphorylating reagent (such as phosphorous oxychloride, phospho-tris-triazolide, phospho-tris-triimidazolide, etc.), a trialkyl phosphate (phosphorus), under conditions suitable for the formation of pyrimidine monophosphate. A monophosphorylation step of contacting a pyrimidine nucleotide with a mixture having trimethyl phosphate (such as triethyl or trimethyl phosphate) and dimethylaminopyridine (DMAP); and a pyrophosphorylation reagent (pyrroline) under conditions suitable for the formation of pyrimidine triphosphate. (Such as tributylammonium acid) with pyrimidine monophosphate.

[0013] As used herein, the term "nucleotide" is art-recognized and includes natural bases (standard) and modified bases well known in the art. Such a base is generally located at the 1 'position of the sugar moiety.
Nucleotides generally contain base, sugar and phosphate groups. Nucleotides may be unmodified or modified at the sugar, phosphate and / or base moieties (also shown interchangeably as nucleotide analogs, modified nucleotides, unnatural nucleotides, non-standard nucleotides, etc. For example, Usman and
McSwigen, supra; Eckstein et al. , International PC
T Publication No. WO 92/07065; Usman et al. , International PCT
See Publication No. WO 93/15187; all are incorporated herein by reference). Limbach et al. , 1994, Nucleic Ac
ids Res. 22,2183, there are several examples of modified nucleobases known in the art. Some non-limiting examples of base modifications that can be introduced into nucleic acids without significantly affecting catalytic activity include inosine, purines,
Pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,
4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (for example, 5-methylcytidine)
, 5-alkyluridines (eg, ribothymidine), 5-halouridines (eg, 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (eg, 6-methyluridine) and others (Burgin et al.
, 1996, Biochemistry, 35, 14090). In this regard, "modified base" means a nucleotide base other than adenine, guanine, cytosine and uracil at position 1 'or equivalents thereof;
Such bases can be used in the catalytic core of an enzymatic nucleic acid molecule and / or in the substrate binding region of such a molecule. Such modified nucleotides include
Dideoxynucleotides that have pharmaceutical utility well known in the art, as well as utility in basic molecular biology methods such as sequencing, are included.

By “unmodified nucleoside” or “unmodified nucleotide” is meant one of the bases adenine, guanine, cytosine, uracil, linked to the 1 ′ carbon of β-D-ribo-furanose.

“Modified nucleoside” or “modified nucleotide” means a nucleotide base that contains a modification in the chemical structure of the base, sugar and / or phosphate of the unmodified nucleotide.

“Pyrimidine” means a nucleotide that includes a modified or unmodified derivative of a 6-membered pyrimidine ring. An example of a pyrimidine is a modified or unmodified uridine.

By “nucleotide triphosphate” or “NTP” is meant a nucleoside in which three inorganic phosphate groups are linked to the 5 ′ hydroxyl group of a modified or unmodified ribose or deoxyribose sugar. The 1 'position of the sugar can include a nucleobase or hydrogen. The triphosphate moiety may be modified to include chemical modifications that do not destroy the functionality of the group (ie, allow for incorporation into an RNA molecule).

In another preferred embodiment, the nucleotide triphosphate (NTP) of the invention is an RNA
It is incorporated into the oligonucleotide using a polymerase enzyme. RNA polymerases include, but are not limited to, mutant and wild-type bacteriophage T7, SP6 or T3 RNA polymerase. Applicants have also discovered that the NTPs of the present invention can be incorporated into oligonucleotides using certain DNA polymerases, such as Taq polymerase.

In yet another preferred embodiment, the present invention provides a method for producing a modified NT
It features a method for capturing P. The method comprises using a DNA template, RNA polymerase, NTP under conditions suitable for incorporation of the modified NTP into the oligonucleotide.
And incubating a mixture comprising a promoter of modified NTP uptake.

“Promoter of modified NTP incorporation” means a reagent that facilitates the incorporation of modified nucleotides into nucleic acid transcripts by RNA polymerase. Such reagents include, but are not limited to, methanol; LiCl; polyethylene glycol (PEG); diethyl ether; propanol; methylamine;

In another preferred embodiment, the modified nucleotide triphosphate is a nucleic acid molecule, such as an enzymatic nucleic acid, an antisense, a 2-5A antisense chimera, an oligonucleotide,
Triplex-forming oligonucleotides (TFO), aptamers, etc. (Stul et.
al. , 1995 Pharmaceutical Res. 12,465)
Can be incorporated by transfer.

“Antisense” refers to RNA-RNA or RNA-DNA or RNA-
PNA (protein nucleic acid; Egholm el al., 1993 Nature)
e 365,566) refers to non-enzymatic nucleic acid molecules that bind to target RNA by interaction and alter the activity of target RNA (for review, Stein and
Cheng, 1993 Science 261, 1004; Agrawal
et al. No. 5,591,721; Agrawal, US Pat. No. 5,652,356). Typically, an antisense molecule will be complementary to a target sequence along one contiguous sequence of the antisense molecule. However, in certain embodiments, the antisense molecule may bind to the substrate such that the substrate molecule forms a loop, and / or the antisense molecule may bind to the substrate such that the antisense molecule forms a loop. They may be combined. Thus, the antisense molecule is complementary to two (or more) non-contiguous substrate sequences or the two (or more) non-contiguous substrate sequence portions of the antisense molecule are complementary to the target sequence. Yes, or both.

“2-5A antisense chimera” means an antisense oligonucleotide comprising a 5 ′ phosphorylated 2′-5 ′ linked adenylate moiety. These chimeras bind to the target RNA in a sequence-specific manner and activate cellular 2-5A-dependent ribonuclease, which in turn cleaves the target RNA (Torrence et a).
l. 1993 Proc. Natl. Acad. Sci. USA 90,13
00).

“Triplex-forming oligonucleotide (TFO)” refers to a double-stranded D in a sequence-specific manner.
It refers to an oligonucleotide that can bind to NA to form a triple-stranded helix. The formation of such a triple-stranded helix has been shown to inhibit transcription of the target gene (Duval-Valentin et al., 1992 Proc.
. Natl. Acad. Sci. USA 89, 504).

As used herein, “oligonucleotide” means a molecule having two or more nucleotides. A polynucleotide may be single-stranded, double-stranded or multi-stranded, and may have modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.

“Nucleic acid catalysis” catalyzes (varies in rate and / or rate) various reactions, including the ability to repeatedly cleave another nucleic acid molecule in a nucleotide sequence-specific manner (endonuclease activity) A) a nucleic acid molecule. Such molecules with endonuclease activity have complementarity at the substrate binding region to a particular gene target and have enzymatic activity to specifically cleave RNA or DNA in that target. That is, a nucleic acid molecule having endonuclease activity cleaves RNA or DNA intramolecularly or intermolecularly, whereby the target R
NA or DNA molecules can be inactivated. This complementation allows for sufficient hybridization of the enzymatic RNA molecule to the target RNA or RNA, allowing cleavage to occur. 100% complementarity is preferred, but 50%
A low complementarity of -75% is also useful in the present invention. Nucleic acids may be modified at the base, sugar and / or phosphate groups. The term enzymatic nucleic acid is ribozyme, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotide, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease. Minizyme, leadzyme, oligozyme, finedelon or DN
Used interchangeably with phrases such as the A enzyme. All of these terms describe nucleic acid molecules having enzymatic activity. The particular enzymatic nucleic acid molecule described in the present application is not a limitation of the present invention and what is important in the enzymatic nucleic acid molecule of the present invention is that the specific nucleic acid molecule complementary to one or more target nucleic acid regions Having a specific substrate binding site, and having, within or around the substrate binding site, a nucleotide sequence that confers nucleic acid cleavage activity to the molecule (Cech et al., US Pat. No. 4,987,07).
No. 1, Cech et al. , 1988, 260 JAMA 3030).

“Enzymatic portion” or “catalytic domain” refers to a portion / region of an enzymatic nucleic acid molecule that is essential for cleavage of a nucleic acid substrate.

By “substrate binding arm” or “substrate binding domain” is meant a portion / region of an enzymatic nucleic acid molecule that is complementary (ie, capable of base pairing) to a portion of a substrate. Generally, such complementarity is 100%, but may be less if desired.
For example, about 10 out of 14 may form a base pair. That is, these arms include sequences in the enzymatic nucleic acid molecule that are intended to bring the enzymatic nucleic acid molecule and target together by complementary base pairing interactions. The enzymatic nucleic acid molecules of the invention may be continuous or discontinuous, and have binding arms of various lengths. The length of the binding arm is preferably longer than or equal to 4 nucleotides;
-100 nucleotides; more particularly 14-24 nucleotides in length. If 2
When two binding arms are selected, the length of the binding arms is symmetric (ie, each binding arm is the same length; eg, 5 and 5 nucleotides, 6 and 6 nucleotides or 7 and 7 nucleotides in length) or Seems to be asymmetric (ie, the binding arms are of different lengths; for example, 6 and 3 nucleotides; 3 and 6 nucleotides; 4 and 5 nucleotides; 4 and 6 nucleotides; 4 and 7 nucleotides long). Designed to.

As used herein, “nucleic acid molecule” means a molecule having nucleotides. Nucleic acids may be single-stranded, double-stranded or multi-stranded, and may contain modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. An example of a nucleic acid molecule according to the present invention is a gene encoding a macromolecule such as a protein.

In a preferred embodiment of the invention, the nucleic acid molecule (eg, an antisense molecule, triplex DNA or an enzymatic nucleic acid molecule) is 13 to 100 nucleotides in length, and in certain embodiments (eg, a specific ribozyme 35), 36, 37 or 38 nucleotides in length. In certain embodiments, the nucleic acid molecule is 15-100, 17
-100, 20-100, 21-100, 23-100, 25-100, 27-
100, 30-100, 32-100, 35-100, 40-100, 50-1
00, 60-100, 70-100 or 80-100 nucleotides in length.
Instead of the upper limit of the chain length range specified above of 100 nucleotides, the upper limit of the chain length range may be, for example, 30, 40, 50, 60, 70 or 80 nucleotides. Thus, for any chain length range, the chain length range for a particular embodiment will have a lower limit as specified, and an upper limit as specified, higher than the lower limit. For example, in certain embodiments, the chain length range will be 35-50 nucleotides in length. All such ranges are expressly included. Also in certain aspects,
The nucleic acid molecule can have any of the lengths specified above (eg, 21 nucleotides in length).

“Complementarity” means that a nucleic acid can form a hydrogen bond with another RNA sequence by traditional Watson-Crick or other non-traditional forms. For the nucleic acid molecules of the invention, the free energy of binding of the nucleic acid molecule to its target or complementary sequence is sufficient to allow proper function of the nucleic acid to proceed (eg, enzymatic nucleic acid cleavage, Antisense or triple helix inhibition). Determination of the binding free energy of a nucleic acid molecule is well known in the art (eg, Turner et al.).
. , 1987, CSH Syrnp. Quant. Biol. LII pp. 1
23-133; Frier et al. , 1986, Proc. Nat. Ac
ad. Sci. USA 83: 9373-9377; Turner et al.
. 1987; Am. Chem. Soc. 109: 3783-3785).
Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., five out of ten, For 6, 7, 8, 9, 10, 50%, 60%, 7
0%, 80%, 90% and 100% complementarity). "Perfect complementarity" means that all the contiguous residues of the nucleic acid sequence will hydrogen bond with the same number of contiguous residues in the second nucleic acid sequence.

In yet another preferred embodiment, the modified nucleotide triphosphates of the present invention can be used for combinatorial chemistry or in vitro selection of nucleic acid molecules having novel functions. Modified oligonucleotides can be synthesized enzymatically to generate a library for screening.

In another preferred embodiment, the present invention relates to nucleic acids (eg, enzymatic nucleic acid molecules, antisense nucleic acids, 2-5A antisense chimeras, triplexes) isolated using the methods described in the present invention. It features technologies based on DNA, RNA-cleaving chemical groups (antisense nucleic acids) and their use for diagnosing, down-regulating or inhibiting gene expression.

“Inhibit” refers to the activity of the target gene or the mRN encoding the target gene.
A or equivalent level of RNA is a nucleic acid molecule of the invention (eg, an enzymatic nucleic acid molecule,
Antisense nucleic acid, 2-5A antisense chimera, triple stranded DNA, antisense nucleic acid containing an RNA-cleaving chemical group). In one embodiment, the inhibition by the enzymatic nucleic acid molecule is preferably
Below the levels observed in the presence of enzymatically weakened nucleic acid molecules that can bind to the same site on NA but cannot cleave the RNA. In another embodiment, inhibition by nucleic acid molecules (including enzymatic nucleic acids and antisense molecules) is suitably stronger than the inhibition observed in the presence of oligonucleotides having, for example, scrambled sequences or mismatches. In another embodiment, the inhibition of the target gene by the nucleic acid molecule of the present invention is stronger in the presence than in the absence of the nucleic acid molecule.

In yet another preferred embodiment, the invention features a method for incorporating a number of compounds of formula I.

In yet another aspect, the invention features a catalytically active nucleic acid molecule of Formula II.

[0037]

Embedded image In the above formula, X, Y and Z independently represent nucleotide or non-nucleotide linkers, which may be the same or different; indicates a hydrogen bond formation between two adjacent nucleotides; Y 'Is a nucleotide complementary to Y; Z' is a nucleotide complementary to Z; l is an integer equal to or greater than 3, preferably less than 20, more particularly 4, 5 , 6,7,8
, 9, 10, 11, 12 or 15; m is an integer greater than 1, preferably less than 10, more particularly 2, 3, 4, 5, 6 or 7; O is a large integer, preferably less than 10, more particularly 3, 4, 5, 6 or 7; o is an integer equal to or greater than 3, preferably less than 20, more particularly Is 3,4,5,6,7,8,9,10,11,12 or 15;
l and o can be the same length (l = o) or different lengths (l ≠
o); each X (l) and X (o) is independently a target nucleic acid sequence (target is RNA, DN
A or an RNA / DNA mixed polymer), and is an oligonucleotide long enough to have a stable interaction with it; W is a linker ≧ 2 nucleotides long or a non-nucleotide linker A, U, C and G represent nucleotides; G is a nucleotide, preferably 2′-O-methyl; C represents a nucleotide, preferably 2′-amino (eg, 2′-amino). '-NH
And _ represents a chemical bond (e.g., a phosphate ester bond, an amide bond, a phosphorothioate, a phosphorodithioate, or other bond known in the art).

[0038] The enzymatic nucleic acid molecule of Formula II may independently comprise a cap structure, which may or may not be present independently.

“Sufficient length” means an oligonucleotide equal to or greater than 3 nucleotides.

“Stably interacting” refers to the interaction of an oligonucleotide with a target nucleic acid (eg, by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions).

“Chimeric nucleic acid molecule” or “chimeric oligonucleotide” means that the molecule may consist of both modified or unmodified DNA or RNA.

“Cap structure” means a chemical modification incorporated at the end of an oligonucleotide. These terminal modifications will protect the nucleic acid from exonuclease degradation and will aid delivery and / or localization within the cell. The cap is at the 5 'end (
5'-cap) or at the 3 'end (3'-cap) or at both ends. In a non-limiting example: 5'-cap is an inverted abasic moiety, 4'5'-methylene nucleotide; 1- (beta-D-erythrofuranosyl)
Nucleotides, 4'-thionucleotides, carbocyclic nucleotides; 1,5-anhydrohexitol nucleotides; L-nucleotides; alpha-nucleotides; modified base nucleotides; phosphorodithioate bonds; threo-pentofuranosyl nucleotides; Cyclic 3'-4'-seconucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide portion; 3'-3'-inverted Abasic portion; 3'-2
3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexyl phosphate; aminohexyl phosphate; 3'-phosphate;'-Phosphorothioate;phosphorodithioate; or a bridged or unbridged methylphosphonate moiety (more particularly, Beigelman et al., International PCT Publication No. WO 97/26).
270, incorporated herein by reference). In yet another preferred embodiment, the 3'-cap is 4'5'-methylene nucleotide; 1- (beta-D-erythrofuranosyl) nucleotide; 4'-thionucleotide, carbocyclic nucleotide; phosphate 5 '-Aminoalkyl; 1,3-diamino-2-phosphate
Propyl; 3-aminopropyl phosphate; 6-aminohexyl phosphate;
2-aminododecyl; hydroxypropyl phosphate; 1,5-anhydrohexitol; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3 '
3,4'-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5
-Dihydroxypentyl nucleotides, 5'-5'-inverted nucleotide moieties; 5
5'-phosphoramidate;5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; cross-linked and / or non-cross-linked 5'-phosphoramidate Selected from the group consisting of phosphorothioates and / or phosphorodithioates, bridged or unbridged methylphosphonates and 5'-mercapto moieties (more particularly, Beaucage and Iyer,
1993, Tetrahedron 49, 1925; incorporated herein by reference). The term "non-nucleotide" refers to any group or compound that can be incorporated into a nucleic acid strand in place of one or more nucleotide units, including sugar and / or phosphate substitutions and remaining It is possible that any base presents an enzymatic activity. The group or compound is adenosine, guanine,
It is abasic in that it does not contain commonly recognized nucleotide bases such as cytosine, uracil or thymine. As used herein, the term "basic"
Or, "an abasic nucleotide" includes a sugar moiety that lacks a base or has another chemical group at the 1 'position in place of a base.

In the context of a 2′-modified nucleotide as described in the present invention, “amino” means 2′-NH 2 or 2′-O-NH 2, which may be modified It is not necessary. Groups so modified are, for example, each Eckstein
net et al. No. 5,672,695 and Matulic-A.
damic et al. , WO 98/28317, which are hereby incorporated by reference in their entirety.

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG . 1 shows a scheme for NTP synthesis using a nucleoside substrate.

FIG. 2 shows a scheme for the in vitro selection method. A pool of nucleic acid molecules having a random core region and one or more regions of defined sequence is generated. These nucleic acid molecules are bound to a column containing immobilized oligonucleotides having a defined sequence, wherein the defined sequence is complementary to a region of the defined sequence of nucleic acid molecules in the pool. Immobilized oligonucleotide in column (target)
Is isolated and converted to complementary DNA (cDNA), followed by N
A new nucleic acid pool is formed by transcription using TP.

FIG. 3 shows the scheme of the two-column in vitro selection method. A pool of nucleic acid molecules having a random core and two flanking regions of the defined sequence (region A and region B) is generated. This pool is passed through a column containing immobilized oligonucleotides having regions A 'and B', each of which is complementary to regions A and B of the nucleic acid molecules in the pool. The column is brought to conditions sufficient for the immobilized oligonucleotide target to be easily cleaved. The molecule in the pool that cleaves the target (the active molecule) has the A 'region of the target bound to its A region, while the B region is free. The column is washed to isolate the active molecule with the bound target A 'region. This pool of active molecules will also contain certain molecules that are not active at cleaving the target (inactive molecules) but are dissociated from the column. To separate contaminating inert molecules from active molecules, the pool is passed through a second column containing immobilized oligonucleotides containing A 'sequences but no B' sequences. Inactive molecules bind to column 2, but active molecules do not bind to column 2 (since their A region is occupied by the A 'region of the target oligonucleotide from column 1). The column 2 is washed to isolate the active molecules and proceed to the next step as shown in FIG.

FIG. 4 is an illustration of a novel 48 nucleotide enzymatic nucleic acid motif, which was identified using the in vitro method described in the present invention. The molecules shown are exemplary only. The 5 'and 3' terminal nucleotides (referring to the nucleotides of the substrate binding arm, rather than just a single terminal nucleotide on the 5 'and 3' ends), are altered as long as these portions can base pair with the target substrate sequence Can be. In addition, the guanosine (G) shown at the cleavage site of the substrate can be changed to other nucleotides as long as the change does not eliminate the ability of the enzymatic nucleic acid molecule to cleave the target sequence. Substitutions in the nucleic acid molecule and / or substrate sequence can be readily tested, for example, as described herein.

FIG. 5 shows HCV used to demonstrate the efficacy of class I enzymatic nucleic acid molecule motifs
1 shows a scheme of a luciferase assay.

FIG. 6 is a graph showing a dose curve for an enzymatic nucleic acid molecule targeting site 146 on HCV RNA.

FIG. 7 is a bar graph showing that enzymatic nucleic acid molecules targeting four sites within HCV RNA can reduce RNA levels in cells.

FIG. 8 shows the secondary structure and cleavage rates of the characterized class II enzymatic nucleic acid motifs.

Nucleotide Synthesis Dimethylaminopyridine (D) to phosphorylation protocols known in the art
The addition of MAP) can greatly increase the yield of nucleotide monophosphate while shortening the reaction time (FIG. 1). The synthesis of the nucleosides of the present invention has been described in several articles and in earlier applications of the applicant (Beigelman et al.).
al. , International PCT Publication No. WO 96/18736; Dudzcy et.
al. , International PCT Publication No. WO95111110; Usman et.
al. , International PCT Publication No. WO95 / 13378; Matulic-Ad
artic et al. , 1997, Tetrahedron Lett. 3
8, 203, Matulic-Adarnic et al. , 1997, Te
trahedron Lett. 38, 1669; all of which are incorporated herein). These nucleosides were dissolved in triethyl phosphate and cooled in an ice bath. Phosphorus oxychloride (POCl3) was added, followed by DMAP.
The reaction was then warmed to room temperature and left for 5 hours. This reaction forms nucleotide monophosphate, which can then be used to form nucleotide triphosphate. Tributylamine was added, followed by anhydrous acetonitrile and tributylammonium pyrophosphate. The reaction was quenched with TEAB and stirred at room temperature (about 20 ° C.) overnight. Triphosphate is purified using column purification and HPLC, the chemical structure is NMR
Confirmed by analysis. One skilled in the art will recognize that the reagents, temperatures, and purification methods of the reaction can be readily varied with alternatives and equivalents, while still providing the desired product.

[0053] Nucleotide triphosphates present invention provides a nucleotide triphosphate that can be used in a number of different functions.
The nucleotide triphosphates formed from the nucleosides of Table I are unique and differ in nature as compared to other nucleotide triphosphates known in the art. The incorporation of modified nucleotides into DNA or RNA oligonucleotides can alter the properties of the molecule. For example, modified nucleotides prevent nuclease binding and thus increase the chemical half-life of the molecule. This is particularly important if the molecule is to be used in cell culture or in vivo. It is known in the art that the introduction of modified nucleotides into these molecules can greatly increase the stability of the molecule and thereby its efficacy (Burgin et a
l. , 1996, Biochemistry 35, 14090-14097;
Usman et al. , 1996, Curr. Opin. Struct. B
iol. 6,527-533).

[0054] The modified nucleotides are incorporated using a wild-type or mutant polymerase. For example, mutant T7 polymerase has a modified nucleotide triphosphate, D
Used in the presence of NA template and a suitable buffer. One of skill in the art will recognize that other polymerases and their respective mutant variants can also be used to incorporate the NTPs of the present invention. The nucleic acid transcript is a radiolabeled nucleotide (α-32P
NTP). The radiolabeled NTP contains the same base as the modified triphosphate being tested. The effect of methanol, PEG and LiCl was tested by adding these compounds independently or in combination. Detection and quantification of nucleic acid transcripts is described in Molecular Dynamics Phos.
Performed using phorImager. Transcription efficiency was assessed by comparing modified nucleotide triphosphate incorporation with a total ribonucleotide incorporation control. Wild-type polymerase is available from the manufacturer (Boehringer Mannair).
n) was used for NTP incorporation using the buffer and instructions for use.

Transcription Conditions The rate of incorporation of modified nucleotide triphosphates into oligonucleotides can be enhanced by adding several different enhancers of modified NTP incorporation to conventional buffer conditions. Applicants have utilized methanol and LiCl to increase the rate of dNTP uptake using RNA polymerase. These modified NTP uptake promoters can be used in different combinations and ratios to optimize transcription. Optimum reaction conditions vary between nucleotide triphosphates and can be readily determined by standard experiments. Overall, however, it has been shown that the presence of a promoter of modified NTP incorporation, such as an inorganic compound such as methanol or lithium chloride, increases the average transfer rate.

Mechanism of Action of the Nucleic Acid Molecules of the Invention Antisense: Antisense molecules are modified or unmodified RNA, DNA or mixed polymer oligonucleotides that specifically bind to matching sequences and inhibit peptide synthesis. (Wu-Pong, Nov)
1994, BioPharm, 20-33). Antisense oligonucleotides bind to target RNA by Watson-Crick base pairing and block gene expression by sterically blocking or preventing ribosome translation of the bound sequence by activation of the RNase H enzyme. Antisense molecules also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev.
in Oncogenesis 7, 151-190).

In addition, binding of single-stranded DNA to RNA results in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). Until now, R
The only backbone-modified DNA that will serve as a substrate for Nase H is phosphorothioate and phosphorodithioate. Recently, 2'-arabino and 2'-fluoroarabino-containing oligos have also been reported to activate RNase H activity.

A number of antisense molecules have been reported that utilize a novel configuration of chemically modified nucleotides, secondary structures and / or RNase H substrate domains (Woolf et al., International PCT Publication No. WO98). /
13526; Thompson et al, filed April 20, 1998.
. , USSN 60 / 082,404; Ha filed September 21, 1998.
rtmann et al. USSN 60 / 101,174, all of which are incorporated herein in their entirety).

Triplex-forming oligonucleotide (TFO ): Single stranded DNA can be designed to bind to genomic DNA in a sequence-specific manner. TFOs include pyrimidine-rich oligonucleotides that bind DNA helices by Hoogsteen base pairing (Wu-Pong, supra). The resulting triple helix is DNA sense, D
It is composed of NA antisense and TFO and disrupts RNA synthesis by RNA polymerase. The TFO mechanism results in gene expression or cell death because binding is irreversible (Mukhopadhyay & Roth, supra).

[0060] 2-5A antisense chimeras : The 2-5A system is an R -sense found in higher vertebrates.
It is an interferon-mediated mechanism of NA degradation (Mitra et al., 19).
96, Proc Nat Acad Sci USA 93, 6780-678.
5). Two types of enzymes, 2-5A synthase and RNase L, are required for RNA cleavage. 2-5A synthase is 2'-5 'oligoadenylate (2-5A)
Requires double-stranded RNA to form 2-5A then acts as an allosteric effector for utilizing RNase L, which has the ability to cleave single-stranded RNA. The ability to form 2-5A with double-stranded RNA makes this system particularly useful for inhibiting viral replication.

The (2′-5 ′) oligoadenylate structure is covalently linked to an antisense molecule to form a chimeric oligonucleotide capable of RNA cleavage (Torre)
nce, supra). These molecules bind and activate the 2-5A-dependent RNase, and it is speculated that the oligonucleotide / enzyme complex then binds to the target RNA molecule, which can be degraded by the RNase enzyme.

[0062] Enzymatic nucleic acids : Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of the enzymatic nucleic acid, which is kept in close proximity to the enzymatic portion of the molecule that serves to cleave the target. Thus, the enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA through complementary base pairing, and acts to cleave the target RNA once bound to the correct site. Such strategic cleavage of the target RNA will destroy its ability to direct the synthesis of the encoded protein. After the enzymatic nucleic acid binds and cleaves its target, it is released from the RNA in search of another target and can repeatedly bind and cleave new targets.

The enzymatic properties of an enzymatic nucleic acid are a significant advantage, as the concentration of the enzymatic nucleic acid that affects therapeutic treatment is lower. This advantage reflects the ability of enzymatic nucleic acids to act enzymatically. Thus, one enzymatic nucleic acid molecule can cleave many molecules of the target RNA. In addition, enzymatic nucleic acid molecules are highly specific inhibitors, and the specificity of inhibition depends on the mechanism of target RNA cleavage as well as the mechanism of base pairing for binding to the target RNA. One mismatch or base substitution near the cleavage site can be selected to completely eliminate the catalytic activity of the enzymatic nucleic acid molecule.

A nucleic acid molecule having endonuclease enzyme activity can repeatedly cleave another RNA molecule in a nucleotide sequence-specific manner. Such enzymatic nucleic acid molecules can target virtually any RNA transcript and effective cleavage is achieved in vitro (Zaug et al., 324, Nature 429 1986).
Uhlenbeck, 1987 Nature 328,596; Kime
t al. , 84 Proc. Natl. Acad. Sci. USA 8788
, 1987; Dreyfus, 1988, Einstein Quart. J.
Bio. Med. Haseloff and Gerlach, 3;
34 Nature 585, 1988; Cech, 260 JAMA 303.
0, 1988; and Jefferies et al. , 17 Nuclei
c Acids Research 1371, 1989; Santoroe
t al. , 1997 supra).

Because of its sequence specificity, trans-cleaving enzymatic nucleic acid molecules are expected as therapeutics against human diseases (Usman & McSwigen, 1995 Ann).
. Rep. Med. Chem. 30, 285-294; Christoffer
sen and Marr, 1995J. Med. Chem. 38, 2023
-2037). Enzymatic nucleic acid molecules are specific RNs in the background of cellular RNA.
It can be designed to cleave the A target. Such cleavage renders the RNA non-functional,
The protein expressed from the RNA is extinguished. In this manner, the synthesis of proteins associated with the disease state is selectively inhibited.

Optimization of Nucleic Acid Catalytic Activity The catalytic activity of the enzymatic nucleic acid molecules described and identified using the method of the present invention is
It can be optimized as described by Rapper et al., supra, and using methods well known in the art. Details will not be repeated here, but altering the length of the binding arm of the enzymatic nucleic acid molecule or modifications that inhibit degradation by serum ribonuclease and / or enhance enzymatic activity (bases, sugars and / or phosphates) Chemically synthesizing an enzymatic nucleic acid molecule having (eg, Ecks
tein et al. , International Publication No. WO 92/07065; Perra
ult et al. , 1990 Nature 344, 565; Pieke
net et al. Usman, 1991 Science 253, 314;
and Cedergren, 1992 Trends in Biochem
. Sci. 17, 334; Usman et al. , International Publication No. WO93
No./15187; Rossi et al. , International Publication No. WO91 / 031
No. 62; Sproat, US Pat. No. 5,334,711; and Burg.
in et al. Which all describe various chemical modifications that can be made to the base, phosphate and / or sugar moieties of enzymatic nucleic acid molecules). Modifications that promote intracellular efficacy, and removal of bases from the stem-loop structure to reduce synthesis time and chemical requirements are desirable. (All of these references are incorporated herein by reference).

[0067] Sugar, base and phosphate modifications that can be introduced into nucleic acid molecules (eg, enzymatic nucleic acid molecules) without significantly affecting the catalyst and significantly promoting nuclease stability and potency are described. Some examples exist in the art. Enzymatic nucleic acid molecules have a nuclease resistance group, such as 2'-amino, 2'-C-allyl,
'-Fluoro, 2'-O-methyl, 2'-H, nucleotide base modifications (for review, Usman and Cedergren, 1992 TIBS 17,34.
Usman et al .; , 1994 Nucleic Acids Sym
p. Ser. 31, 163; Burgin et al. , 1996 Bioc
(see Chemistry 35, 14090) to enhance stability and / or promote catalytic activity. Sugar modifications of enzymatic nucleic acid molecules have been extensively described in the art (Eckstein et al.
al. , International PCT No. WO92 / 07065; PerTault e.
t al. Nature 1990, 344, 565-568; Pieken
et al. Science 1991, 253, 314-317; Usman
and Cedergren, Trends in Biochem. Sci
. 1992, 17, 334-339; Usman et al. International PCT
No. WO93 / 15187; Sproat, US Pat. No. 5,334,7.
No. 11 and Beigelman et al. , 1995J. Biol. C
hem. 270, 25702; all references are incorporated herein in their entirety). Such articles, without disturbing the catalyst,
General methods and strategies for determining positions for incorporating base and / or phosphate modifications and the like are described and incorporated herein by reference. In view of such teachings, similar modifications can be used to modify the nucleic acid catalysts of the present invention as described herein.

A nucleic acid catalyst having a chemical modification that maintains or enhances enzymatic activity is provided. Such nucleic acid molecules are generally more resistant to nucleases than unmodified nucleic acids. Thus, the activity is not significantly reduced in cells and / or in vivo. As exemplified herein, such enzymatic nucleic acid molecules are useful in cells and / or in vivo even if the overall activity is reduced by a factor of 10 (B
urgin et al. , 1996, Biochemistry, 35, 14.
090). Such enzymatic nucleic acid molecules are described herein as "maintaining" enzymatic activity.

[0069] Therapeutically delivered therapeutic nucleic acid molecules (eg, enzymatic and antisense nucleic acid molecules) optimally inhibit the translation of target RNA long enough so that the level of undesired proteins is reduced. Must be stable in cells up to. This time varies between hours and days, depending on the disease state. Clearly, these nucleic acid molecules must be resistant to nucleases to function as effective intracellular therapeutics. Improvements in the chemical synthesis of nucleic acid molecules described in the present invention and in the art have expanded the capabilities of modified nucleic acid molecules by introducing nucleotide modifications to promote nuclease stability as described above.

“Enhanced enzymatic activity” includes activities measured in cells and / or in vivo where the activity reflects both catalytic activity and enzymatic nucleic acid molecule stability. In the present invention, the product of these properties was not increased or significantly (less than a factor of 10) reduced in vivo as compared to the unmodified enzymatic nucleic acid molecule.

In yet another preferred embodiment, there is provided a nucleic acid catalyst having a chemical modification that maintains or enhances enzymatic activity. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, the activity will not be significantly reduced in cells and / or in vivo. As exemplified herein, such enzymatic nucleic acid molecules are useful in cells and / or in vivo even if the overall activity is reduced by a factor of 10 (Burgin et al., 1996, Bioch).
chemistry, 35, 14090). Such enzymatic nucleic acid molecules are described herein as "maintaining" the enzymatic activity of the enzymatic nucleic acid molecule of total RNA.

The use of these molecules will lead to better treatment of disease progression by conferring the potential of combination therapy (eg, multiple enzymatic nucleic acid molecules targeting different genes, known small molecules). Enzymatic nucleic acid molecules conjugated to molecular inhibitors, or enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs) and / or intermittent treatment in combination with other chemical or biological molecules). Treatment of a patient with a nucleic acid molecule will also include combinations of different types of nucleic acid molecules. Treatments have been devised that include a mixture of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs), antisense and / or 2-5A chimeric molecules against one or more targets to reduce the symptoms of the disease. Will be.

Administration of Nucleotide Mono-, Di- or Tri-Phosphate and Nucleic Acid Molecules
It can be used alone or in combination with other pharmaceutical ingredients as a therapeutic agent. These molecules of the invention are described in Sullivan et al. , PCT WO94 / 02595
Alchtar et al .; , 1992, Trends Cell Bio
. , 2,139; and methods of delivering antisense oligonucleotide therapeutics, Ak
htar, 1995, incorporated herein by reference. The molecules of the present invention can be administered to cells by various methods known to those skilled in the art, for example, by encapsulation in liposomes, by iontophoresis, or by hydrogels, cyclodextrins, biodegradable nanocapsules and bioadhesive microspheres. Incorporation into other excipients such as fairs, but is not limited thereto. In some applications, enzymatic nucleic acid molecules are delivered directly to cells or tissues ex vivo, with or without the excipients described above. Alternatively, the modified nucleotide triphosphate, diphosphate or monophosphate / excipient combination is delivered locally by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (in the form of tablets or pills), topical, systemic, ocular, intraperitoneal and / or subarachnoid Delivery, including but not limited to. More detailed descriptions of delivery and administration can be found in Sullib, which is incorporated herein by reference.
an et al. , Supra, and Draper et al. , PCT W
O93 / 23569.

The molecules of the present invention can be used as a medicament. The medicament prevents, inhibits or treats the disease state of the patient (reduces some symptoms, preferably all symptoms).

The negatively charged nucleotide monophosphate, diphosphate or triphosphate of the invention is
Pharmaceutical compositions can be formed with (or without) stabilizers, buffers, and the like, and administered or introduced to a patient by any standard means. If the use of a liposome delivery mechanism is desired, standard protocols for liposome formation can be followed. The compositions of the present invention will also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; .

The present invention also includes pharmaceutically acceptable formulations of the described compounds. These formulations include salts of the above compounds, for example, ammonium, sodium, calcium,
Includes magnesium, lithium and potassium salts.

Pharmaceutical composition or formulation means a composition or formulation in a form suitable for administration (eg, systemic administration) to cells or a patient (preferably a human). Suitable forms, in part, depend upon the use or the route of administration, for example oral, transdermal or by injection. Such a form must not prevent the composition or formulation from reaching the target cells (ie, the cells to which the negatively charged polymer is desired to be delivered). For example, a pharmaceutical composition to be injected into the bloodstream should be soluble. Other factors are known in the art and include such considerations as toxicity and the form in which the composition or formulation prevents it from performing its effects.

“Systemic administration” means distribution throughout the body following systemic absorption in vivo or accumulation of the drug in the bloodstream. Routes of administration leading to systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these routes of administration may involve the desired negatively charged polymer (eg,
NTP) to accessible diseased tissues. The rate of entry of a drug into the circulation is related to molecular weight or size. The use of liposomes or other drug carriers containing the compounds of the present invention may be able to localize the drug to certain tissue types, such as, for example, tissues of the retinal endothelial system (RES). Also useful are liposome formulations that can facilitate the binding of drugs to cell surfaces, such as lymphocytes and macrophages. This method facilitates drug delivery to target cells by exploiting the immune recognition specificity of abnormal cells, such as cancer cells, by macrophages or lymphocytes.

The present invention also relates to surface-modified liposomes containing poly (ethylene glycol) lipids (P
Also characterized is the use of compositions comprising EG modifications or long-circulating liposomes or stealth liposomes). These formulations provide a method for increasing drug accumulation in target cells. This class of drug carriers is based on the monocyte phagocyte system (MPS or RE).
S) to opsonization and elimination, thereby allowing for longer blood circulation times and enhanced tissue exposure of the encapsulated drug (Lasic
et al. Chem. Rev .. 1995, 95, 2601-2627; Is
hiwata et al. Chem. Pharm. Bull. 1995, 4
3,1005-1011). Such liposomes have been shown to selectively accumulate in tumors, presumably by extravasation and entrapment in angiogenic target tissues (Lasic et al., Science 1995, 267, 1).
275-1276; Oku et al. , 1995, Biochim. Bio
phys. Acta, 1238, 86-90). Long-circulating liposomes, MPS
Promotes the pharmacokinetics and pharmacodynamics of the drug, especially when compared to normal cationic liposomes known to accumulate in the tissues of humans (Liu et al., JB
iol. Chem. 1995, 42, 24864-24870; Choi et.
al. , International PCT Publication No. WO 96/10391; Ansell et.
al. , International PCT Publication No. WO 96/10390; Hollande
t al. , International PCT Publication No. WO 96/10392; all of which are incorporated herein by reference). Long-circulating liposomes also greatly protect drugs from nuclease degradation compared to cationic liposomes, based on their ability to avoid accumulation in metabolically active MPS tissues such as liver and spleen It seems. All of these references are incorporated herein by reference.

The invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well-known in the pharmaceutical arts,
For example, Remington's Pharmac, which is incorporated herein.
electronic Sciences, Mack Publishing Co.
(Edited by AR Gennaro, 1985). For example, preservatives,
Stabilizers, dyes and aromatizers will be provided (supra, 1449). These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.

“Patient” means the organism or cell itself that is the donor or recipient of the explanted cell. "Patient" also means the organism to which the compound of the invention is administered. Preferably, the patient is a mammal (eg, a human), a primate or a rodent.

A pharmacologically effective amount is that dose required to prevent, inhibit the occurrence of, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state. The pharmacologically effective amount will be recognized by the type of disease, the composition used, the route of administration, the type of mammal being treated, the physiological characteristics of the particular mammal being considered, the concomitant drug therapy and those skilled in the art. It depends on other factors that will be affected. Generally, from 0.1 mg / kg to 100 mg, depending on the potency of the negatively charged polymer.
Amounts between mg / kg body weight / day are acceptable. In one embodiment, the invention provides an enzymatic nucleic acid molecule that can be delivered exogenously to a particular cell as needed.

The nucleic acid molecules of the invention are also administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of a number of compounds to treat an indication will increase the beneficial effects while reducing side effects.

[0084] Examples The following are non-limiting examples, the synthesis of nucleotide triphosphates of the present invention, capture, and analyze, and shows the activity of an enzymatic nucleic acid.

Applicants have synthesized pyrimidine nucleotide triphosphates using DMAP in the reaction. For pudding, standard protocols already reported in the art were used (Yoshikawa et al., Supra; Ludwig, supra). Described below are examples of the synthesis of pyrimidine nucleotide triphosphates and purine nucleotide triphosphates.

Example 1 Synthesis of Purine Nucleotide Triphosphate : 2'-O-methyl-guanosine -5'-triphosphate 2'-O-methylguanosine nucleoside (0.25 grams, 0.84 mmol
l) was dissolved in triethyl phosphate (5.0 ml) by heating to 100 ° C for 5 minutes. The resulting clear, colorless solution was cooled to 0 ° C. under an argon atmosphere using an ice bath. Then phosphorus oxychloride (1.8 eq, 0.141 ml) was added to the reaction mixture with vigorous stirring. The reaction was monitored by HPLC (using a sodium perchlorate gradient). After 5 hours at 0 ° C., tributylamine (0.6
5 ml) and then anhydrous acetonitrile (10.0 ml) and after 5 minutes (re-equilibrated to 0 ° C.) tributylammonium pyrophosphate (4.0 eq.
3 g) was added. After 15 minutes at 0 ° C., the reaction mixture was quenched with 20 ml of 2M TEAB (HPLC analysis under the above conditions indicated that monophosphate was consumed in 10 minutes) and then stirred at room temperature overnight. The mixture was co-evaporated (4x) with methanol under reduced pressure and then diluted with 50 ml of 0.05M TEAB. Perform DEAE Sephadex purification (0.05-0.6 M TEAB gradient) and pure triphosphate (0.52 g, 66.0% yield) (eluting around 0.3 M TEAB)
Was obtained; purity was confirmed by HPLC and NMR analysis.

[0087] Example 2: Synthesis of a pyrimidine nucleotide triphosphates: 2'-O- Mechiruchiomechi Le - uridine-5'-triphosphate 2'-O- methylthiomethyl - uridine nucleoside (0.27 g, 1.
0 mmol) was dissolved in triethyl phosphate (5.0 ml). The resulting clear, colorless solution was cooled to 0 ° C. under an argon atmosphere using an ice bath. Then phosphorus oxychloride (2.0 eq, 0.190 ml) was added to the reaction mixture with vigorous stirring. Add dimethylaminopyridine (DMAP, 0.2 eq, 25 mg)
The solution was warmed to room temperature and the reaction was monitored by HPLC (using a gradient of sodium perchlorate). After 5 hours at 20 ° C., tributylamine (1.0 ml
) Was added, followed by anhydrous acetonitrile (10.0 ml) and after 5 minutes tributylammonium pyrophosphate (4.0 eq, 1.8 g) was added. After 15 minutes at 20 ° C., the reaction mixture was quenched with 20 ml of 2M TEAB (HPLC analysis under the above conditions indicated that monophosphate was consumed in 10 minutes) and then stirred at room temperature overnight. . The mixture was co-evaporated (4x) with methanol under reduced pressure,
Dilute with 0.05 ml of TEAB. DEAE fast Sepharose purification (0.
05 to 1.0 M TEAB gradient) and confirmed by HPLC and NMR analysis showed pure triphosphate (0.40 g, 44% yield) (0.3 M T
(Eluted around EAB).

Example 3 Use of DMAP in Uridine 5'-Triphosphate Synthesis The reaction was carried out with a 20 mg aliquot of nucleoside dissolved in 1 ml of triethyl phosphate and 19 μl of phosphorus oxychloride. The reaction was performed at 40 minute intervals with H
It was automatically monitored by PLC and yield curves were generated for time up to 18 hours. The 5 ', 3', 2'-monophosphate (monophosphate elutes in this order) is purified using a reversed phase column and an ammonium acetate / sodium acetate buffer system (50 mM and 100 mM, respectively, pH 4.2). '-Triphosphate and starting nucleoside. The data is shown in Table 2. Under these conditions, the yield doubled and the reaction time to maximum yield was improved by a factor of 10 (reduced from 1200 minutes to 120 minutes to obtain a 90% yield). Selectivity for 5'-monophosphorylation was observed in all reactions. Subsequent triphosphorylation occurred with approximately the same yield.

Substance used for bacteriophage T7 RNA polymerase reaction Buffer 1: Prepare a 10X stock solution of buffer 1 by mixing reagents (400 mM Tris-Cl (pH 8.1), 200 mM MgCl ₂ , 100 mM
mM DTT, 50 mM spermidine, and 0.1% Triton X-100.
). Prior to the start of the polymerase reaction, methanol and LiCl are added to dilute the buffer, so that the final reaction conditions for buffer 1 consist of:
40 mM Tris pH (8.1), 20 mM MgCl ₂ , 10 mM DTT,
5 mM spermidine, 0.01% Triton X-100, 10% methanol, and 1 mM LiCl.

Buffer 2: Mix reagents to prepare a 10 × stock solution of Buffer 2 (400 mM Tris-Cl (pH 8.1), 200 mM MgCl ₂ , 100
mM DTT, 50 mM spermidine, and 0.1% Triton X-100.
). Prior to the start of the polymerase reaction, PEG, LiCl is added to dilute the buffer, so that the final reaction conditions of buffer 2 consist of: 40
mM Tris pH (8.1), 20 mM MgCl ₂ , 10 mM DTT, 5 m
M spermidine, 0.01% Triton X-100, 4% PEG, and 1
mM LiCl.

Buffer 3: Mix reagents to prepare a 10 × stock solution of Buffer 3 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl ₂ , 50 m
M DTT, 10 mM spermidine, and 0.02% Triton X-100.
). Prior to the start of the polymerase reaction, PEG was added to dilute the buffer,
The final conditions for the reaction of the buffer 3 is set to be the following: 40 mM Tris _{pH (8.0), 12mM MgCl 2} , 5mM DTT, 1mM spermidine, 0.002% Triton X-100, and 4% PEG.

Buffer 4: Mix reagents to prepare a 10 × stock solution of Buffer 4 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl ₂ , 50 m
M DTT, 10 mM spermidine, and 0.02% Triton X-100.
). Prior to the start of the polymerase reaction, PEG, methanol is added to dilute the buffer so that the final reaction conditions of buffer 4 consist of: 4
0 mM Tris pH (8.0), 12 mM MgCl ₂ , 5 mM DTT, 1 m
M spermidine, 0.002% Triton X-100, 10% methanol,
And 4% PEG.

Buffer 5: Mix reagents to prepare a 10 × stock solution of Buffer 5 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl ₂ , 50 m
M DTT, 10 mM spermidine, and 0.02% Triton X-100.
). Prior to the start of the polymerase reaction, PEG, LiCl is added to dilute the buffer, so that the final reaction conditions of buffer 5 consist of: 40
mM Tris pH (8.0), 12 mM MgCl ₂ , 5 mM DTT, 1 mM
Spermidine, 0.002% Triton X-100, 1 mM LiCl, and 4% PEG.

Buffer 6: Mix reagents to prepare a 10 × stock solution of Buffer 6 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl ₂ , 50 m
M DTT, 10 mM spermidine, and 0.02% Triton X-100.
). Prior to the start of the polymerase reaction, PEG, methanol is added to dilute the buffer, so that the final reaction conditions of buffer 6 consist of: 4
0 mM Tris pH (8.0), 12 mM MgCl ₂ , 5 mM DTT, 1 m
M spermidine, 0.002% Triton X-100, 10% methanol,
And 4% PEG.

Buffer 7: Mix reagents to prepare a 10 × stock solution of Buffer 7 (400 mM Tris-Cl (pH 8.0), 120 mM MgCl ₂ , 50 m
M DTT, 10 mM spermidine, and 0.02% Triton X-100.
). Prior to the start of the polymerase reaction, PEG, methanol, and LiCl are added to dilute the buffer so that the final reaction conditions for Buffer 7 consist of: 40 mM Tris pH (8.0), 12 mM MgCl _2, 5m
M DTT, 1 mM spermidine, 0.002% Triton X-100, 10
% Methanol, 4% PEG, and 1 mM LiCl.

Example 4 Screening of Modified Nucleotide Triphosphates Using Mutant T7 RNA Polymerase Tested. Buffer 1-6 tested at 25 ° C was set as condition 1-6, and buffer 1-6 tested at 37 ° C was set as condition 7-12 (Table 3). Under each condition, the Y639F mutant T7 polymerase (Sousa
And Padilla, supra) (0.3-2 mg / 20 ml reaction solution), NTP (2 m each)
M), DNA template (10 pmol), inorganic pyrophosphatase (5 U /
ml) and α- ³² P NTP (0.8 mCi / pmol template) were combined and heated at the indicated temperature for 1-2 hours. The radiolabeled NTP used is different from the modified triphosphate tested. Samples were analyzed by polyacrylamide gel electrophoresis. PhosphorImager (Molecular Dynamics, Sunnyvale, CA)
Was used to quantify the amount of full-length transcript and compared to a total RNA control reaction. The data is shown in Table 4; the results of each reaction were reported as total ribonucleotide triphosphates (r
(NTP) expressed as percent of control. The control was a mutant T7 polymerase using a commercially available polymerase buffer (Boehringer Mannheim, Indianapolis IN).

Example 5 Incorporation of Modified NTP Using Wild-Type T7-RNA Polymerase Bacteriophage T7 RNA was obtained from Boehringer Mannheim at 0.4 U / μL.
Purchased at a concentration of Applicants have determined that the enzyme mixed with 50 μL of the reaction solution and 0.2 μC
i alpha - a a ³² P NTP those supplemented with commercially available buffer was used with nucleotide triphosphates by 2 mM. The template was a double-stranded PCR fragment, which was used in the previous screening. The reaction is performed at 37 ° C for 1
Conducted for hours. 10 μL of the sample was electrophoresed on 7.5% analytical PAGE, and
Bands were quantified using orImager. The results were calculated relative to the “all ribo” control (unmodified nucleotide triphosphate) and the results are shown in Table 5.

[0098] Example 6: Multiple modified nucleotide triphosphates combinations taken Write-modified nucleoside triphosphates to an oligonucleotide tested in the transfer protocol described in Example 4, two or more of these The rate of triphosphate uptake was measured. 2 '
- deoxy -2 '- (L-histidine) amino uridine (2'-his-NH ₂
-UTP) incorporation was tested using unmodified cytidine nucleotide triphosphate, γATP, and γGTP under reaction condition # 9. Data are expressed as percentage of incorporation of modified NTP relative to total γNTP control and are shown in Table 6a.

Two modified cytidines (2′-NH ₂ -CTP or 2′dCTP) are 2′-
It is taken at the same rate with his-NH ₂ -UTP. Then 2'-his-
The NH ₂ -UTP and 2'-NH ₂ -CTP using various unmodified and modified adenosine triphosphate was tested in the same buffer (Table 6b). 2'-his-
The best modified adenosine triphosphate for incorporation with both NH ₂ -UTP and 2'-NH ₂ -CTP was 2'-NH ₂ -DAPTP.

[0100] Example 7: modified nucleotides third reaction conditions for the phosphoric acid uptake optimization _{2'-his-NH 2 -UTP,} 2'-NH 2 -CTP, 2'-NH 2 -DAP
, And γGTP were tested under several reaction conditions (Table 7) using the incorporation protocol described in Example 9. The results show that
Of the buffer conditions tested, incorporation of these modified nucleotide triphosphates occurs in the presence of both methanol and LiCl.

[0102] Example 8: For the 2'-deoxy-2'-amino-modified to use the GTP and CTP novel enzymatic nucleic acid molecule motifs selected novel enzymatic nucleic acid molecule motifs selected enzymatic nucleic acid A pool of molecules,
It was designed to have two substrate binding arms (5 and 16 nucleotides in length) and a random region in between. The substrate has 5′-terminal biotin, 5 nucleotides complementary to the short binding arm of the pool, unpaired G (the desired cleavage site), and 16 nucleotides complementary to the long binding arm of the pool. . The substrate is bound to the column resin by the avidin-biotin complex. Figure 2 shows the general process of selection
Shown in The protocol described below represents one possible method that can be used for selecting enzymatic nucleic acid molecules and is provided as a non-limiting example of selecting enzymatic nucleic acid molecules using a combinatorial library.

Construction of the library : The oligonucleotides listed below were prepared using Operon Technologie
s (Alameda, CA). After gel purification of the template, it was passed through a Sep-Pak cartridge (Waters, Millford MA) according to the manufacturer's protocol. Primers (MST3, MST7c, MST3del) were used without purification.

Primers: MST3 (30 mer): 5′-CACTTAGCATTAACCCTCACTA
AAGGCCCGT-3 ′ MST7c (33-mer): 5′-TAATACGACTCACTATAGGAA
AGGTGTGCAACC-3 'MST3del (18-mer): 5'-ACCCTCACTAAAGGCCCGT-
3 'template: MSN60c (93mer): 5'-ACCCTCACTAAAGGCCCGT (N
) ₆₀ GGTTGCACACCTTTG-3 ′ MSN40c (73-mer): 5′-ACCCTCACTAAAGGCCCGT (N
) ₄₀ GGTTGCACACCTTTG-3 ′ MSN20c (53-mer): 5′-ACCCTCACTAAAGGCCCGT (N
) ₂₀ GGTTGCACACCTTG-3 ′ N60 library was constructed using MSN60c as a template and MST3 / MST7c as a primer. N40 and N20 libraries are MSN
It was constructed using 40c (or MSN20c) as a template and MST3del / MST7c as primer.

The single-stranded template was converted to double-stranded DNA by the following protocol: 5 nmol of template, 10 nmol of each primer were mixed in 10 ml of reaction solution using standard PCR buffer, dNTPs and taq DNA polymerase ( All reagents were obtained from Boerhinger Mannheim). The conditions of the synthesis cycle were 94 ° C for 4 minutes; (94 ° C for 1 minute; 42 ° C for 1 minute; 72 ° C for 2 minutes) x 4
72 ° C for 10 minutes. The product was checked on an agarose gel to confirm the length of each fragment (N60 = 123 bp, N40 = 91 bp, N20 = 71).
bp), followed by phenol / chloroform extraction and ethanol precipitation. 2
The concentration of the main chain product was 25 μM.

Transcription of the first pool was in a 1 ml volume containing: 500 pmol of double-stranded template (3 × 10 ¹⁴ molecules), 40 mM Tris-HCl (pH 8.0),
12 mM MgCl ₂ , 1 mM spermidine, 5 mM DTT, 0.002%
Triton X-100, 1 mM LiCl, 4% PEG8000, 10% methanol, 2 mM ATP (Pharmacia), 2 mM GTP (Pharmacia), 2 m
M 2'-deoxy-2'-amino-CTP (USB), 2 mM 2'-deoxy-2'-amino-UTP (USB), 5 U / ml inorganic pyrophosphatase (
Sigma), 5 U / μl T7 RNA polymerase (USB; optionally Y
The 639F mutant was used at 0.1 mg / ml (Sousa and Padilla, supra))
At 37 ° C. for 2 hours. The transcribed library was purified by denaturing PAGE (N
60 = 106 nucleotides, N40 = 74, N20 = 54), the obtained product was desalted using a Sep-Pak column, and then ethanol precipitation was performed.

Initial column selection : The following biotinylated substrate was synthesized using standard protocols (Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1).
990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids.
Res., 23, 2677-2684): 5'-biotin-C18 spacer-GCC GUG GGU UGC ACA CCU UUC C-C18
The spacer-thiol modifier C6S-S inverted abasic-3 'substrate was purified by denaturing PAGE and ethanol precipitation. 10 nmol of substrate was bound to a NeutrAvidin column using the following protocol: 400 μl U
ltraLink immobilized NeutrAvidin slurry (200 μl beads, Pierce, Rockford,
IL) was packed in a polystyrene column (Pierce). After washing the column twice with 1 ml of binding buffer (20 mM NaPO ₄ (pH 7.5), 150 mM NaCl), the cap was closed (ie the cap was placed on the bottom of the column to stop the effluent). 200 μl of substrate suspended in binding buffer was applied and incubated for 30 minutes at room temperature with occasional vortexing to ensure that the solution was uniformly bound and distributed to the resin. After the incubation, remove the cap and remove the column.
ml of binding buffer, then 1 ml of column buffer (50 mM Tris-
HCl (pH 8.5), 100 mM NaCl, 50 mM KCl). The column was now ready for use and the cap was closed. 1 nmol
Was loaded onto the column with 200 μl of column buffer. Incubate for 30 minutes at room temperature with occasional vortexing to allow the substrate to bind. After the incubation, the cap was removed and the column was washed twice with 1 ml of column buffer and the cap was closed. 200 μl elution buffer (50 m
M Tris-HCl (pH 8.5), 100 mM NaCl, 50 mM KCl
, 25 mM MgCl ₂ ) was applied to the column and incubated for 30 minutes at room temperature with occasional vortexing. The cap was removed and four 200 μl fractions were collected using elution buffer.

Second column (reverse selection) : A schematic of the events in the second column is shown generally in FIG. 3 and the substrate oligonucleotides used are shown below: 5'-GGU UGC ACA CCU UUC C- C18 spacer-biotin inverted abasic-3 'This column substrate was coupled to UltraLink NeutrAvidin resin as described above (40
pmol), and washed twice with elution buffer. The eluate from the first column purification was applied to the second column. By using this column, RNA that is non-specifically eluted from the first column will bind, while RNA having a catalytic event and the product bound to it will bind to the second column. Outflows through. Fractions were precipitated with ethanol using glycogen as carrier and rehydrated with sterile water for amplification.

Amplification : RNA and primer MST3 (10-100 pmol) were
Denatured at 90 ° C. for 3 minutes, then quenched on ice for 1 minute. The following reagents were added to the tubes (indicating the final concentration): 1X PCR buffer (Boerhinger Mannheim)
), 1 mM dNTP (for PCR; Boerhinger Mannheim), 2 U / μl
RNase inhibitor (Boerhinger Mannheim), 10 U / μl Superscript I
I reverse transcriptase (BRL). The reaction was incubated at 42 ° C for 1 hour,
For 5 minutes to destroy Superscript. The following reagents were then added to the tubes and the volume increased 5 fold for the PCR step (indicating final concentration / amount): MST7c primer (10-100 pmol, same amount as RT step), 1 ×
PCR buffer, taq DNA polymerase (0.025-0.05 U
/ Μl, Boerhinger Mannheim). The reaction was run in the following cycle: 94
° C, 4 minutes; (94 ° C, 30 seconds; 42-54 ° C, 30 seconds; 72 ° C, 1 minute)
x4-30 cycles; 72 ° C, 5 minutes; 30 ° C, 30 minutes. The number of cycles and the annealing temperature were determined for each round. If heteroduplex is observed,
The reaction was diluted 5-fold with fresh reagent and two more amplification cycles were allowed to proceed. The resulting product was analyzed for size on an agarose gel (N60 = 123
bp, N40 = 103 bp, N20 = 83 bp), followed by ethanol precipitation.

Transcription : The amplification product was transcribed using the above conditions with the following modifications: 10-20% of the amplification reaction was used as template and the reaction volume was 100
-500 μl, and the product sizes were slightly different (N60 = 106 nucleotides, N40 = 86, N20 = 66). A small amount of ³² P-GTP was added to the reaction for quantitative purposes.

Subsequent rounds : For the following rounds of selection, 20 pmol of input RNA and 40 pmol of a 22 nucleotide substrate were used on the column.

Pool activity : The activity of the pool was assayed under one turnover every 3 to 4 rounds. The activity assay conditions are as follows: 50 mM Tris-HCl (pH 8.5), 25 mM MgCl ₂ , 100 mM NaCl, 5 mM
0 mM KCl, traces of ³² P-labeled substrate, 10 nM RNA pool. The 2X pool mixed with buffer and the 2X substrate mixed with buffer were separately separated by 90%.
Incubated for 3 minutes at 37 ° C, then for 3 minutes at 37 ° C. Thereafter, an equal volume of 2X substrate was added to the tubes of the 2X pool (t = 0). Time points for the first assay were 4 and 24 hours: 5 μl was removed and 8 μl of cold STOP buffer (96%
Formamide, 20 mM EDTA, 0.05% bromophenyl blue / xylene cyanol). The sample was heated at 90 ° C. for 3 minutes and 20% sequencing
The gel was loaded. Quantification was performed using Molecular Dynamics Phosphorimager and ImageQuaNT software. The data is shown in Table 8.

Cloning a sample from the pool of oligonucleotides into a vector,
Sequencing was performed using standard protocols (Sambrook et al., Molecular Cloning: Experimental Manual, Cold Spring Harbor Laboratory Press). Enzymatic nucleic acid molecules were transcribed from representative numbers of these clones using the methods described in this application. To test individuals from each pool for RNA cleavage from N60 and N40, enzymatic nucleic acid molecules from the clones were treated with 2 mM MgCl ₂ ,
Incubated at 37 ° C. with 5/16 substrate mixed with pH 7.5, 10 mM KCl. The data in Table 10 shows that each of the enzymatic nucleic acid molecules isolated from the pool has activity.

Kinetic activity : The kinetic activity of the enzymatic nucleic acid molecule shown in Table 10 was determined by digesting the enzymatic nucleic acid molecule (10 nM) with a cleavage buffer (pH 8.5, 25 mM MgCl ₂ , 10 mM).
(0 mM NaCl, 50 mM KCl) by incubation at 37 ° C. with the substrate.

Magnesium dependency : The magnesium dependency in the fifteenth round of N20 was
The Cl ₂ was varied and the other conditions kept constant (50 mM Tris pH 8.0).
, 100 mM NaCl, 50 mM KCl, 1 revolution, 10 nM pool). The data are shown in Table 11, which shows that the activity increases with increasing magnesium concentration.

[0115] Example 9: 2'-deoxy -2 '- (N-histidyl) amino UTP, 2'-off Ruoro -ATP, and 2'-deoxy-2'-amino CTP and GTP the novel to use Selection of Enzymatic Nucleic Acid Molecular Motifs The method described in Example 8 was followed by 2'-deoxy-2 '-(N-histidyl) amino UTP, 2'-fluoro-ATP, and 2'-deoxy-2'-amino CT
Repeated using P and GTP. However, in the first column Mg
Rather than trigger cleavage with Cl ₂ , an initial random modified pool of RNA was loaded on the substrate-resin in the following buffer: 5 mM NaOAc pH 5.2, 1,
M NaCl, 4 ° C. After washing the ampoule, set the resin at 22 ° C.,
mM HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 1
It was changed to mM CaCl ₂ and 1 mM MgCl ₂ . One selection of N60 oligonucleotides did not use divalent cations (MgCl ₂ , CaCl ₂ ). The resin was incubated for 10 minutes to react and the eluate was collected.

The enzymatic nucleic acid molecule pool has a 1-
Capable of cleaving 3%, background (in the absence of modified pool)
Was 0.2-0.4%.

Example 10: Synthesis of uridine 5-imidazoleacetic acid 2'- deoxy-5'-triphosphate triphosphate 5-dinitrophenylimidazoleacetic acid 2'-deoxyuridine nucleoside (80 mg) was added to 5 ml of triethylphosphoric acid under argon. Dissolve with stirring underneath,
The reaction mixture was cooled to 0 ° C. Phosphorous oxychloride (1.8 equivalents, 22 ml) at 0 ° C
Was added to the reaction mixture, and an additional 3 aliquots were added at room temperature over the course of 48 hours. The reaction mixture was then diluted with anhydrous MeCN (5 ml) and cooled to 0 ° C., after which tributylamine (0.65 ml) and tributylammonium pyrophosphate (4.0 eq, 0.24 g) were added. After 45 minutes, the reaction was quenched with 10 ml of aqueous methylamine for 4 hours. After co-evaporation with MeOH (3x), the material was purified by DEAE Sephadex followed by RP chromatography to give 15m
g of triphosphoric acid were obtained.

Example 11: Synthesis of 2 '-(N-lysyl) amino-2'-deoxy-cytidine triphosphate 2'-(N-lysyl) amino-2'-deoxy-cytidine (0.180 g, 0
. 22 mmol) was dissolved in triethyl phosphate (2.00 ml) under argon. The solution was cooled to 0 ° C. in an ice bath. Phosphorus oxychloride (99.999%, 3 eq, 0.0672 mL) was added to the solution and the reaction was stirred at 0 ° C. for 2 hours. Tributylammonium pyrophosphate (4 eq, 0.400 g) was dissolved in 3.42 mL of acetonitrile and tributylamine (0.165 mL). Acetonitrile (1 mL) was added to the monophosphate solution and then the pyrophosphate solution was added dropwise. The solution was clear. The reaction was allowed to warm to room temperature. After stirring for 45 minutes, methylamine (5 mL) was added and the reaction was stirred at room temperature for 2 hours. A two layer mixture resulted (there were small beads at the bottom of the flask). TLC (7: 1: 2 iPrO
H: NH ₄ OH: H ₂ O) showed the appearance of a triphosphate substance. The solution was concentrated, dissolved in water and loaded onto a freshly prepared DEAE Sephadex A-25 column. The column was washed with a gradient of TEAB buffer up to 0.6M and the product eluted in fractions 90-95. Fractions were analyzed by ion exchange HPLC. Each fraction showed one triphosphate peak (eluting at about 4.000 minutes). Coalesce fractions,
The buffer salts were removed by pumping down from methanol,
. 7 mg of the product was obtained.

Example 12: 2'-deoxy-2 '-(L-histidine) ^{aminocytidine} triphosphate 2'-[N-Fmoc, N ^imid -dinitrophenyl-histidyl] amino-2
'-Cytidine (0.310 g, 4.04 mmol) was dissolved in triethyl phosphate (3 ml) under argon. The solution was cooled to 0 ° C. Phosphorus oxychloride (1.8
(Equivalent, 0.068 mL) was added to the solution and stored in the freezer overnight. The next morning TLC (
10% MeOH / CH ₂ Cl ₂ ) showed a lot of starting material, so another equivalent of POCl ₃ was added. After 2 hours, TLC still showed starting material. Tributylamine (0.303 mL) and tributylammonium pyrophosphate (4 eq, 0.734 g) dissolved in 6.3 mL of acetonitrile (dropwise) were added to the monophosphate solution. The reaction was allowed to warm to room temperature. After stirring for 15 minutes, methylamine (10 mL) was added at room temperature and stirring was continued for 2 hours. TLC 7:
1: 2 iPrOH: NH ₄ OH: H ₂ O) showed the appearance of triphosphate material. The solution was concentrated, dissolved in water and loaded on a DEAE Sephadex A-25 column. The column was washed with a gradient of TEAB buffer up to 0.6M and the product eluted in fractions 170-179. Fractions were analyzed by ion exchange HPLC. Each fraction showed one triphosphate peak (eluted at -6.77 minutes). The fractions were combined and pumped down from methanol to remove buffer salts to give 17 mg of product.

[0120] Example 13: modified NTP (Class I motif) the initial screening applicants' novel enzymatic nucleic acid molecule motifs used pool 3x10 ¹⁴ of 2'-amino -dCTP / 2'-amino -dUTP It contained the individual sequence of the RNA. Applicants have optimized the transcription conditions to increase the amount of RNA product by including methanol and lithium chloride. 2'-amino-deoxynucleotides confer nuclease resistance without interfering with the reverse transcription and amplification steps of choice. Applicants have designed the pool to have two binding arms complementary to the substrate, separated by a random 40 nucleotide region. The 16-mer substrate has two domains, 5 and 10 nucleotides in length, which bind to the pool and are separated by unpaired guanosine. At the 5'-end of the substrate is biotin linked by a C18 linker. This allowed binding of the substrate to NeutrAvidin resin in column format. The desired reaction is to cleave at the unpaired G upon addition of the magnesium cofactor and then dissociate from the column due to the instability of the 5 base pair helix. The detailed protocol is as follows:

Preparation of Enzymatic Nucleic Acid Molecule Pool: To prepare the first pool of DNA, the following template oligonucleotides were loaded with taq polymerase and double-stranded D
Converted to NA. (Template = 5'-ACC CTC ACT AAA GGC CGT (N) ₄₀ GGT TGC
ACA CCT TTC-3 '; Primer 1 = 5'-CAC TTA GCA TTA ACC CTC ACT AAA GGC
CGT-3 '; Primer 2 = 5'-TAA TAC GAC TCA CTA TAG GAA AGG TGT GCA ACC-
3 '). All DNA oligonucleotides were synthesized by the operon method. The template oligo was used for denaturing PAGE and Sep-Pak chromatography columns (
Waters). RNA substrate oligos use standard solid-phase chemistry, denatured PA
Purified by GE followed by ethanol precipitation. Substrates for the in vitro cleavage assay were labeled at the 5'-end with gamma- ³² P-ATP and T4 polynucleotide kinase, denatured PAGE purification followed by ethanol precipitation.

5 nmol of template, 10 nmol of each primer, and 250 U of taq polymerase were added to 1 × PCR buffer (10 mM Tris-HCl (
pH 8.3), 1.5 mM MgCl ₂ , 50 mM KCl) and 0.2 mM
Were incubated in a 10 ml volume as follows with each dNTP: 94 ° C.
4 cycles; (94 ° C, 1 minute; 42 ° C, 1 minute; 72 ° C, 2 minutes); and 72 ° C, 10 minutes. The product was analyzed for size on a 2% Separide agarose gel, then extracted twice with a phenol buffer and then with chloroform-isoamyl alcohol, followed by ethanol precipitation. 500 pmol (3x1
The DNA of 0 ¹⁴ molecules) to produce a first RNA pool was transferred as follows. Template DNA was prepared using 40 mM Tris-HCl (pH 8.0), 12 mM
MgCl ₂ , 5 mM dithiothreitol (DTT), 1 mM spermidine,
0.002% Triton X-100, 1 mM LiCl, 4% PEG-800
0, 10% methanol, 2 mM ATP, 2 mM GTP, 2 mM 2′-amino-dCTP, 2 mM 2′-amino-dUTP, 5 U / ml inorganic pyrophosphatase, and 5 U / μl T7 RNA polymerase at room temperature, adding total volume Was 1 ml. The ³² P-GTP was included for detection - traces of alpha individual reaction. The transcription reaction was incubated at 37 ° C. for 2 hours, then an equal volume of STOP buffer (94% formamide, 20 mM EDTA, 0.05
% Bromophenol blue) was added. The obtained RNA was subjected to 6% denaturing PAGE.
Purified by gel, Sep-Pak chromatography, and ethanol precipitation.

First selection: 2 nmol of a 16-mer 5′-biotinylated substrate (5′-biotin-
C18 linker-GCC GUG GGU UGC ACA C-3 ') was added to the binding buffer (20m
Add 30 μl of UltraLink immobilized NeutrAvidin resin (400 μl slurry, Pierce) mixed with M NaPO ₄ (pH 7.5, 150 mM NaCl) at room temperature.
Allowed to bind for minutes. The obtained substrate column was mixed with 2 ml of the binding buffer and then 2 ml.
Column buffer (50 mM Tris-HCl (pH 8.5), 100 mM
NaCl, 50 mM KCl). Stop spill by capping, 1000
A mixture of pmol of the first pooled RNA in 200 μl of column buffer was added to the column and incubated at room temperature for 30 minutes. After removing the column cap and washing with 2 ml of column buffer, the flow was stopped by capping.
200 μl of elution buffer (= column buffer + 25 mM MgCl ₂ ) was added to the column and incubated at room temperature for 30 minutes. After removing the column cap and collecting the eluate, the column was washed three times with 200 μl of elution buffer. The eluate / wash was ethanol precipitated using glycogen as a carrier,
Rehydrated with μl of sterile H ₂ O. The eluted RNA was amplified by a standard reverse transcription / PCR amplification method. 5-31 μl of RNA together with 20 pmol of primer 1
After incubation at 90 ° C. for 3 minutes in a volume of 4 μl, the mixture was left on ice for 1 minute. The following reagents were added (indicating final concentrations): 1X PCR buffer, 1 mM each d
NTP, 2 U / μl RNase inhibitor, 10 U / μl SuperScrip
t II reverse transcriptase. The reaction was incubated at 42 ° C. for 1 hour, then at 95 ° C. for 5 minutes to inactivate reverse transcriptase. Then water and reagents for PCR (1 × PCR buffer, 20 pmol primer 2, and 2.5 U taq DNA polymerase) were added to increase the volume to 100 μl. Hybaid
The reaction was performed in a thermal cycler: 94 ° C, 4 minutes; (94 ° C, 30 seconds; 54 ° C,
30 seconds; 72 ° C., 1 minute) × 25; 72 ° C., 5 minutes. The product was analyzed by size on an agarose gel and ethanol precipitation was performed. Using 1/3 to 1/5 of the PCR DNA, the next generation of transcription was performed as described above in a volume of 100 μl. Successive rounds were performed using 20 pmol of RNA on a column containing 40 pmol of substrate.

Two column selection: At the eighth generation (G8), the column selection was changed to a two column format. 200 pmol of a 22-mer 5'-biotinylated substrate (5'-biotin-
C18 linker-GCC GUG GGU UGC ACA CCU UUC C-C18 linker-thiol modifier C6SS inverted abasic-3 ') was used for the selection column as described above. Elution was performed with 200 μl elution buffer and then washed with 1 ml elution buffer. 1200 μl of the eluate was passed by gravity through the product trap column. The product trap column was prepared as follows: 200 pmol
16-mer 5'-biotinylated "product"(5'-GGU UGC ACA CCU UUC C-C1
8 linker-biotin-3 ') was attached to the column as above and the column was equilibrated with elution buffer. The eluate from the product column was precipitated as described above. The product was amplified as described above, except that 100 pmol at 2.5 times more volume
Were used. 100 μl of the PCR reaction was used for the cycling process; the remaining fraction was amplified with the minimum number of cycles required for the product. After three rounds (G11), there was clear activity in a one-turn cleavage assay. By 13 th generation, 45% of the substrate was cleaved in 4 hours; pool of k _obs was 0.037 min ^-1 in 25 mM MgCl _2. Applicants have subcloned and sequenced the thirteenth generation; the pool was still very diverse. Because the applicant's purpose is an enzymatic nucleic acid molecule that works under physiological conditions, we decided to change the selection pressure rather than exhaustively analyze G13.

Reselection of the N40 pool was started with G12 DNA. A portion of G12 DNA was applied to hypermutagenesis PCR (Vartanian et al., 1996, Nucleic Acids Research
24, 2627-2631), a mutation was introduced at a frequency of 10% per site, and this was designated as N40H. At round 19, hypermutation was again introduced into a portion of the DNA to yield N40M and N40HM (4 parallel pools in total). The column substrate remained the same: the buffer was changed and the temperature of binding and elution was raised to 37 ° C. The column buffer was replaced with a physiological buffer (50 mM Tris-HCl (pH 7.5), 1
(40 mM KCl, 10 mM NaCl) and change the elution buffer to 1 mM.
It was replaced with Mg buffer (physiological buffer + 1mM MgCl _2).
The time to bind the pool to the column was ultimately reduced to 10 minutes, and the elution time was gradually reduced from 30 minutes to 20 seconds. Between rounds 18 and 23,
The k _obs of the N40 pool remained relatively constant at 0.035-0.04 min- ¹ . The 22nd generation from each of the 4 pools was cloned and sequenced.

Cloning and Sequencing: Generations 13 and 22 were replaced with Novagen
Using the Perfectly Blunt Cloning kit (pT7Blue-3 vector) according to the protocol of the kit. Clones were screened for inserts by PCR amplification using vector-specific primers.
Positive clones were sequenced using the ABI Prism 7700 sequence detection system and vector specific primers. Sequences were aligned using MacVector software; two-dimensional folding was performed using Mulfold software (Zuker, 1989, Science 244, 48-52; Jaeger et al., 1989, Biochemistry 86, 7).
706-7710; Jaeger et al., 1989, edited by RF Doolittle, Methods in Enzymology, 183.
281-306). Individual clone transcription units were constructed by PCR amplification,
For this, 50 pmol of each of Primer 1 and Primer 2 was replaced with 1XPC
R buffer, 0.2 mM of each dNTP, and 2.5 U of ta
The following cycles were performed using q polymerase in a volume of 100 μl: 9
4 ° C, 4 minutes; (94 ° C, 30 seconds; 54 ° C, 30 seconds; 72 ° C, 1 minute) X2
0; 72 ° C, 5 minutes. The transcription unit was ethanol precipitated, rehydrated in 30 μl H ₂ O, and 10 μl was transferred in a 100 μl volume and purified as described above.

[0127] Thirty-six clones from each pool were sequenced and observed to be variants of the same consensus motif. A unique clone was prepared using 1 mM MgCl ₂
And assayed for activity under physiological conditions; 9 clones showed consensus sequences and were used in subsequent experiments. None of the mutations had a significant increase in activity; most of the mutations were in the region considered to be duplex, based on the proposed secondary structure. To make the motif shorter, Applicants have deleted the 3'-end 25 nucleotides necessary to bind to the primer for amplification. When the velocities of the full-length molecule and the molecule whose end was truncated were measured, both were 0.
. 04 min- ¹ ; thus, the size of the motif could be reduced from 86 to 61 nucleotides. The stem-loop structure as well as the base pairing of the substrate recognition arm were cleaved to make the molecule even shorter, resulting in a molecule of 48 nucleotides. In addition, many ribonucleotides were substituted with 2-O-methyl modified nucleotides to stabilize the molecule. An example of a new motif is shown in FIG. As will be recognized by those skilled in the art, the molecules are not limited to the chemical modifications shown in the figures, which show only one possible chemically modified molecule.

Kinetic analysis The reaction rate for one revolution was determined using trace amounts of 5'- ³² P-labeled substrate and 10-100.
Performed on a pool of 0 nM enzymatic nucleic acid molecules. 2X mixed with 1X buffer
The 2X pool / enzymatic nucleic acid molecule mixed with substrate and 1X buffer was separately incubated at 90 ° C for 3 minutes, then equilibrated to 37 ° C for 3 minutes. Equivalent 2
X substrate was added to the pool / enzymatic nucleic acid molecule at t ₀ and the reaction was incubated at 37 ° C. Each time point was quenched on ice in 1.2 volumes of STOP buffer. After heating the sample at 90 ° C. for 3 minutes, 15% sequencing
Separated by gel. Gels were imaged using a PhosphorImager and quantified using ImageQuant software (Molecular Dynamics). The curves most often fitted a double-exponential decay, but some curves required a straight-line fit.

Stability : Serum stability assays were performed as described previously (Beigelma
n et al., 1995, J. Biol. Chem. 270, 25702-25708). 1 μg of 5′- ³² P-labeled synthetic enzymatic nucleic acid molecule was added to 13 μl of unlabeled material and assayed for attenuation in human serum. Gels and quantification are as described in the reaction rate section.

Example 14: Inhibition of HCV using new motifs During HCV infection, the viral RNA undergoes enzymatic nucleic acid molecule cleavage in several processes, namely, envelope, translation, RNA replication, and packaging. Exists as a potential target. The target RNA may be more or less utilized by enzymatic nucleic acid molecule cleavage at any one of these stages. Although the association of the HCV initiation ribosome entry site (IRES) with the translation device is mimicked in the HCV 5'UTR / luciferase reporter system (Example 9), these other viral processes do not appear in the OST7 system. . None of the resulting RNA / protein complexes are associated with the target viral RNA. In addition, these processes will link in HCV-infected cells and will strongly affect the availability of target RNA.
Accordingly, Applicants have tested whether enzymatic nucleic acid molecules designed to cleave the 5'-UTR of HCV affect the viral replication system.

In recent years, Lu and Wimmer have characterized HCV-poliovirus chimeras,
It is a poliovirus in which the IRES is replaced by an IRES from HCV (Lu and Wimmer, 1996, Proc. Natl. Acad. Sci. USA. 93, 1412-1417).
Poliovirus (PV) is a positive-strand RNA virus, like HCV, but unlike HCV, it has no envelope and replicates efficiently in cultured cells.
HCV-PV chimeras express a stable small plaque phenotype compared to wild-type PV.

To test the ability of the novel enzymatic nucleic acid molecule motif to inhibit HCV RNA in cells, a dual reporter system utilizing both firefly and Renilla luciferase was used (FIG. 5). A number of enzymatic nucleic acid molecules with novel class I motifs have been designed and tested (Table XII). 5 'HCV UT
Novel enzymatic nucleic acid molecule motifs targeting the R region may inhibit translation of transcripts into luciferase upon cleavage. OST-7 cells were transformed with 10% fetal calf serum,
12% per well in black 96-well plates (Packard) in DMEM medium containing 1% penicillin / streptomycin and 1% L-glutamine.
, 500 cells and incubated at 37 ° C. overnight. 5'-HCV UT
Plasmids containing the T7 promoter expressing R and firefly luciferase (T7C1-341 (Wang et al., 1993, J. of Virol. 67, 3338-3344)) were combined with the pRLSV40 Renilla control plasmid (Promega) and then enzymatically. The reagent was mixed with the nucleic acid molecule and the cationic lipid to prepare a 5X concentration reagent (T7C1-
341 (4 μg / ml), pRLSV40 Renilla luciferase control (6 μg / ml), enzymatic nucleic acid molecule (250 nM), transfection reagent (28.5 μg / ml).

The complex mixture was incubated at 37 ° C. for 20 minutes. The medium was removed from the cells and 120 μl of Opti-mem medium was added to the wells, followed by 30 μl of 5X complex mixture. 150 μl of Opti-mem was added to the wells containing untreated cells. The complex mixture was incubated on OST-7 cells for 4 hours, lysed with passive lysis buffer (Promega), and fluoresced using a dual luciferase assay kit using the manufacturer's protocol (Promega). Was quantified. The data shown in FIG. 6 is a dose curve of an enzymatic nucleic acid molecule targeting site 146 of HCV RNA, expressed as a ratio between firefly and Renilla luciferase fluorescence. Enzymatic nucleic acid molecules are HCV at all enzymatic nucleic acid molecule concentrations
The amount of RNA could be reduced, resulting in an IC ₅₀ of about 5 nM. Other sites were also effective (FIG. 7), in particular enzymatic nucleic acid molecules targeting 133, 209, and 273 were able to reduce HCV RNA as compared to irrelevant (IRR) controls.

Example 15 Cleavage of Substrate Using Fully Modified Oligonucleotides The ability of all 2 'modified enzymatic nucleic acid molecules to cleave target RNA was tested, and which ribonucleotide sites were required. I checked if there was. Ribozyme constructed with 2'-O- methyl and 2'-amino (NH ₂₎ modified nucleotides (Table 12; Gene Name: no ribonucleic), it was performed as described kinetic analysis in Example 13. 100 nM enzymatic nucleic acid with trace amounts of substrate and 1 mM MgC
Mix under physiological conditions (37 ° C.) in the presence of l ₂ . The ribo-free nucleotides are compared to the enzymatic nucleic acid molecules shown in Table 12 (ribo) containing ribonucleotides and have a.
It has a _Krel of 13.

[0135] Example 16: screening the selection of novel enzymatic nucleic acid molecule motifs (Class II motif) began with a pool of modified RNA of> 10 ¹⁴ the following sequences: 5'-GGGA
GGAGGAAGUGCCU (N) ₃₅ UGCCGCGCUCGCUCCCAGUCC-3 '. RNA was generated enzymatically using mutant T7 Y639F RNA polymerase prepared by Rui Souza. The following modified NTPs were introduced: 2'-deoxy-2'-fluoro-adenine triphosphate, 2'-deoxy-2'-fluoro-uridine triphosphate or 2
'-Deoxy-2'-fluoro-5-[(N-imidazole-4-acetyl) propylamine] uridine triphosphate and 2'-deoxy-2'-amino-cytidine triphosphate; natural guanidine triphosphate using an acid to all selected, alpha - and to be able to label the RNA pool using the ³² P-GTP. The RNA pool was purified by denaturing gel electrophoresis (8% polyacrylamide 7M urea).

The following target RNA (Resin A) was synthesized, and Iodoacetyl Ultralink resin (Pierce) was synthesized.
According to the manufacturer's method: 5'-b-L-GGACUGGGAGCGAGCGCGGCGCAGGC
ACUGAAG-LSB3 ′; wherein b is biotin (Glenn Research catalog # 10-19)
53-nn), L is polyethylene glycol spacer (Glenn Research catalog # 10-1918-nn), S is thiol modifier C6 SS (Glenn Resea
rch catalog # 10-1936-nn), B is standard inverted deoxy abasic.

Buffer A (20 mM HEPES pH 7.4, 140 mM KCl, 1
The RNA pool was added to 100 μl of 5 μM Resin A mixed with 0 mM NaCl) and incubated at 22 ° C. for 5 minutes. The temperature was then raised to 37 ° C. for 10 minutes. The resin was washed with 5 ml of buffer A. Buffer B (20 mM
HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 1 mM
(MgCl ₂ , 1 mM CaCl ₂ ) was added to start the reaction. 20 in the first generation
The incubation was continued for 1 minute, and then decreased gradually to 1 minute in the last generation; a total of 13 generations. The reaction eluate was collected in 5M NaCl and a final concentration of 2M NaCl
I got Add 100 μl of 50% slurry Ultralink NeutraAvidin (Pierce
) Was added. Incubation for 20 minutes at 22 ° C. allowed the cleaved biotin product to bind to the avidin resin. The resin was then added to 5 ml of 20 mM HEPES pH
7.4, washed with 2M NaCl. 1.2 ml of denaturing wash 1 M NaCl
The desired RNA was removed with 10M urea at 94 ° C. for 10 minutes.
RNA was precipitated twice in 0.3 M sodium acetate to remove Cl ^- ions which inhibit reverse transcription. Reverse transcription and PCR amplification of a standard protocol were performed.
RNA was transcribed again with the modified NTP described above. After 13 generations, cloning and sequencing yielded 14 sequences that could cleave the target substrate. Six sequences were characterized and the secondary structure and cleavage kinetics were determined. The structure and kinetic data are shown in FIG. The sequences of the other eight enzymatic nucleic acid molecules are shown in Table XIII. The size, sequence, and chemical composition of these molecules can be modified as described in Example 13 above and well known to those skilled in the art.

Manipulation of Nucleic Acid Catalysts Sequence, chemical and structural variants of class I and class II enzymatic nucleic acid molecules
It can be manipulated and re-engineered using the methods set forth in this application and methods known in the art. For example, the size of class I and class II enzymatic nucleic acid molecules can be determined by methods known in the art (Zaug et al., 1986, Nature, 324, 429; Ruffner et al., 1990, Bio).
chem, 29, 10695; Beaudry et al., 1990, Biochem., 29, 6534; McCall et al., 1992, Pr.
Natl. Acad. Sci. USA, 89, 5710; Long et al., 1994, supra; Hendry et al., 1994,
BBA 1219, 405; Benseler et al., 1993, JACS, 115, 8483; Thompson et al., 1996, Nucl
Acids Res., 24, 4401; Michels et al., 1995, Biochem., 34, 2965; Been et al., 199.
2, Biochem., 31, 11843; Guo et al., 1995, EMBO. J., 14,368; Pan et al., 1994, Bioc.
Chem., 1992, Curr. Op. Struc. Bio., 2, 605; Sugiyama et al., 1
996, FEBS Lett., 392, 215; Beigelman et al., 1994, Bioorg. Med. Chem., 4, 171.
5; Santoro et al., 1997, PNAS 94, 4262; all of which are incorporated herein by reference in their entirety) to reduce or increase the total catalytic activity of the ribozyme to such an extent that it does not significantly decrease. it can.

One of skill in the art can use additional rounds of the in vitro selection strategy described herein and variations thereof to obtain additional nucleic acid catalysts, and such novel catalysts are also within the scope of the invention.

Applications The use of the NTPs described in this invention has several research and commercial applications. These modified nucleotide triphosphates can be used for the in vitro selection (development) of oligonucleotides with novel functions. Examples of in vitro selection protocols are incorporated herein as part of this specification (Joyce, 1989, Gene, 82, 83-87; B
eaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American
267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 2
61: 1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J.,
9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442).

In addition, these modified nucleotide triphosphates can be used to create combinatorial chemical libraries of modified oligonucleotides. There are several references to this technology (Brenner et al., 1992, PNAS 89, 5381-5383, Eaton,
1997, Curr. Opin. Chem. Biol., 1, 10-16), all of which are incorporated herein by reference.

Diagnostic Uses The enzymatic nucleic acid molecules of the invention may be used as a diagnostic tool to test for genetic drift and mutation in diseased cells, or to detect the presence of specific RNAs in cells. Due to the close relationship between the activity of the enzymatic nucleic acid molecule and the structure of the target RNA,
In any region of the molecule, mutations that alter base pairing and three-dimensional structure of the target RNA can be detected. By using multiple enzymatic nucleic acid molecules according to the present invention, nucleotide changes that are important for RNA structure and function in vitro and in cells and tissues can be mapped. Cleavage of the target RNA by enzymatic nucleic acid molecules can be used to inhibit gene expression and (essentially) reveal the role of certain gene products in disease progression. In this way, other gene targets may be revealed as important mediators of the disease. These experiments will allow for better treatment of disease progression by conferring the potential of combination therapy (eg, multiple enzymatic nucleic acid molecules targeting different genes, binding to known small molecule inhibitors) Radiation or intermittent therapy with combinations of enzymatic nucleic acid molecules, enzymatic nucleic acid molecules and / or other chemical or biological molecules. Other in vitro uses of the enzymatic nucleic acid molecules of the invention are well known to those of skill in the art and include the detection of the presence of mRNA associated with the relevant condition. Detection of such RNA is performed by treatment with an enzymatic nucleic acid molecule using standard methodology and then confirming the presence of a cleavage product.

In certain instances, enzymatic nucleic acid molecules that can cleave only wild-type or mutant forms of the target RNA are used in the assays. The first enzymatic nucleic acid molecule is used to confirm the presence of wild-type RNA in the sample, and the second enzymatic nucleic acid molecule is
Used to identify A. Both wild-type and mutant RNA as reaction controls
Will be cleaved by both enzymatic nucleic acid molecules, revealing the relative efficiency of the enzymatic nucleic acid molecule in the reaction and not cleaving the "non-target" RNA species. The cleavage product from the synthetic substrate also serves to generate size markers for analysis of wild-type and mutant RNA in the sample population. Thus, each analysis consists of two enzymatic nucleic acid molecules,
It requires two substrates, and one unknown sample, which is combined with 6
Perform one reaction. The presence of cleavage products is confirmed using an RNase protection assay, allowing the full length and cleavage fragments of each RNA to be analyzed in one lane of a polyacrylamide gel. It is not necessary to quantify the results in order to gain insight into the expression of the mutant RNA in the target cells and the estimated risk of a desired phenotypic change. Expression of mRNA such that its protein product is involved in phenotypic development is sufficient to establish risk. If probes of comparable specific activity are used for both transcripts, a qualitative comparison of RNA levels is sufficient and the cost of initial diagnosis is reduced. Whether qualitatively or quantitatively comparing RNA levels, a higher ratio of mutant to wild type correlates with higher risk.

Further Uses The potential utility of the sequence-specific enzymatic nucleic acid molecules of the present invention has many similar applications for the study of RNA, in which DNA restriction nucleases are useful in the study of DNA. (Nathans et al., 1975 Ann. Rev. Biochem. 44: 273). For example, the pattern of restriction fragments can be used to establish a sequence relationship between two related RNAs, and large RNAs can be specifically cleaved into fragments of a useful size for study. The ability to manipulate the sequence specificity of an enzymatic nucleic acid molecule is ideal for cleaving RNA of unknown sequence. Applicants describe the use of nucleic acid molecules to down-regulate gene expression of target genes in bacteria, microorganisms, fungi, viruses, and eukaryotic systems, including plant or mammalian cells.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All references cited herein are hereby incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

As will be readily appreciated by those skilled in the art, the present invention is sufficient to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as representative of the presently preferred embodiments are exemplary and are not intended to limit the scope of the present invention. Modifications and other uses will occur to those skilled in the art, which are within the spirit of the invention and are defined in the appended claims.

As will be readily apparent to those skilled in the art, various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Accordingly, such further embodiments are within the scope of the present invention and the following claims.

The present invention, as suitably illustrated herein, may be practiced in the absence of elements, limitations (not specifically described herein). Thus, for example, in each example here,
Any of the terms "comprising,""consisting essentially of," and "consisting of." The terms and expressions used may be used in connection with the description. , And is not intended to be limiting, and is not intended to exclude equivalents or parts of the features shown and described in the use of such terms and phrases, and that various modifications may be understood. Thus, while this invention has been specifically disclosed in preferred embodiments, those skilled in the art may rely on any features, modifications, and variations of the concepts disclosed herein. It is to be understood that such modifications and variations are considered to be within the scope of the invention as defined in the specification and the appended claims.

Further, when describing features or aspects of the present invention in terms of Markush's group or other alternatives, the present invention will recognize, as will be appreciated by those skilled in the art, the individual members of Markush's group or other groups. Or from a member subgroup perspective.

Thus, further embodiments are within the scope of the present invention and the following claims.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Brief description of the drawings]

FIG. 1 shows a scheme for NTP synthesis using a nucleoside substrate.

FIG. 2 shows a scheme for the in vitro selection method.

FIG. 3 shows a scheme of a two-column in vitro selection method.

FIG. 4 is an illustration of a novel 48 nucleotide enzymatic nucleic acid motif.

FIG. 5 shows the scheme of the HCV luciferase assay used to demonstrate the efficacy of the class I enzymatic nucleic acid molecule motif.

FIG. 6 is a graph showing a dose curve of an enzymatic nucleic acid molecule targeting site 146 on HCV RNA.

FIG. 7 is a bar graph showing that enzymatic nucleic acid molecules targeting four sites within HCV RNA can reduce RNA levels in cells.

FIG. 8 shows the secondary structure and cleavage rates of the characterized class II enzymatic nucleic acid motifs.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Bewdley, Amber 80026, Bloomfield, Westlake Place 13068 (72) Inventor Carpeyski, Alexander 80026, Colorado, USA, Lafayette, Vernier Avenue 420 (72) Inventor Matrick-Adamic, Jensenka 80303, Colorado, United States Boulder, Forty Second Street, 760 South (72) Inventor Sweedler, David 80027, Louisiana, Colorado, United States Ville, St. Andrew's Lane 956 (72) Inventor Jinen, Shaun, 2378 Firth, Birch Street, Denver, Colorado, United States 80207 Firth Term (reference) 4B024 AA11 BA80 CA04 DA02 EA02 EA04 FA02 GA11 HA11 4B050 CC01 CC03 LL03 4B064 AF27 CA10 CA19 CC24 DA13

Claims (54)

[Claims] 1. Formula II: ## STR1 ## Wherein X, Y and Z independently represent nucleotides, which may be the same or different; l is an integer greater than or equal to 3; m is an integer greater than 1; n is an integer greater than 1; o is an integer greater than or equal to 3; Z 'is a nucleotide complementary to Z; Y' is a nucleotide complementary to Y; X (l ) And X (o) are each oligonucleotides of sufficient length to independently and stably interact with the target nucleic acid sequence;
W is a linker or ≧ 2 nucleotides; A, U, G and C represent nucleotides; C is 2′-amino; and _ represents a chemical bond. 2. When 1 is 4, 5, 6, 7, 8, 9, 10, 11, 12 and 15
The nucleic acid of claim 1, wherein the nucleic acid is selected from the group consisting of: 3. The nucleic acid of claim 1, wherein m is selected from the group consisting of 2, 3, 4, 5, 6, and 7. 4. The nucleic acid of claim 1, wherein n is selected from the group consisting of 2, 3, 4, 5, 6, and 7. 5. The nucleic acid of claim 1, wherein o is selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15. 6. The nucleic acid of claim 1, wherein l and o are the same length. 7. The nucleic acid according to claim 1, wherein 1 and o are of different lengths. 8. The nucleic acid of claim 1, wherein the target nucleic acid sequence is selected from the group consisting of RNA, DNA and mixed RNA / DNA polymers. 9. The nucleic acid of claim 1, wherein said chemical bond is selected from the group consisting of a phosphate bond, an amide bond, a phosphorothioate and a phosphorodithioate. 10. The nucleic acid of claim 1, wherein said C is selected from the group consisting of 2'-O-NH2. 11. A method of inhibiting gene expression in a cell, comprising the step of administering the enzymatic nucleic acid molecule according to claim 1 to the cell under conditions suitable for the inhibition. 12. A method for cleaving another RNA molecule, comprising contacting the enzymatic nucleic acid molecule of claim 1 with said another RNA molecule under conditions suitable for cleaving said another RNA molecule. Including methods. 13. The method according to claim 12, wherein the cleavage is performed in the presence of a divalent cation.
The method described in. 14. The method according to claim 12, wherein said divalent cation is Mg 2+. 15. The enzymatic nucleic acid molecule according to claim 1, wherein said enzymatic nucleic acid molecule is chemically synthesized. 16. The enzymatic nucleic acid molecule according to claim 1, wherein said enzymatic nucleic acid molecule comprises at least one ribonucleotide. 17. The enzymatic nucleic acid molecule according to claim 1, wherein said enzymatic nucleic acid molecule does not contain a ribonucleotide moiety. 18. The enzymatic nucleic acid molecule according to claim 1, wherein said enzymatic nucleic acid molecule comprises at least one 2-amino modification. 19. The enzymatic nucleic acid molecule according to claim 1, wherein said enzymatic nucleic acid molecule comprises at least three phosphorothioate modifications. 20. The method according to claim 19, wherein the phosphorothioate modification is 5 ′ of the enzymatic nucleic acid molecule.
20. The enzymatic nucleic acid molecule according to claim 19, which is terminal. 21. The enzymatic nucleic acid molecule according to claim 1, wherein said enzymatic nucleic acid molecule comprises a 5′-cap or 3′-cap or both 5′-cap and 3′-cap. 22. The enzymatic nucleic acid molecule according to claim 21, wherein said 5′-cap is a phosphorothioate modification. 23. The enzymatic nucleic acid molecule of claim 21, wherein said 3'-cap is an inverted abasic moiety. 24. Formula I: embedded image [Wherein R is independently 2′-O-methyl-2,6-diaminopurine riboside; 2′-deoxy-2′-amino-2,6-diaminopurine riboside; 2′-
(N-alanyl) amino-2′-deoxy-uridine; 2 ′-(N-phenylalanyl) amino-2′-deoxy-uridine; 2′-deoxy-2 ′-(N-β
-Alanyl) amino; 2'-deoxy-2 '-(lysyl) amino uridine; 2
2′-O-methylthiomethyl adenosine; 2′-O-methylthiomethyl cytidine; 2′-O-methylthiomethyl guanosine; 2′-O-methylthiomethyl -Uridine; 2'-deoxy-2 '-(N-histidyl) amino uridine; 2'-deoxy-2'-
Amino-5-methylcytidine; 2 '-(N-β-carboxamidine-β-alanyl) amino-2'-deoxy-uridine;2'-deoxy-2'-(N-β-
Alanyl) -guanosine; 2'-O-amino-adenosine; 2 '-(N-lysyl) amino-2'-deoxy-cytidine;2'-deoxy-2'-(L-histidine)aminocytidine; Imidazole acetic acid 2'-deoxy-5'-
Which is a nucleoside selected from the group consisting of uridine triphosphate. 25. A method for the incorporation of a compound of claim 24 into an oligonucleotide, wherein said compound is a nucleic acid template, RNA polymerase under conditions suitable for incorporation of said compound into said oligonucleotide. Contacting with a mixture comprising an enzyme and an enhancer of modified nucleotide triphosphate uptake. 26. The method according to claim 25, wherein said RNA polymerase is T7 RNA polymerase. 27. The method of claim 25, wherein said RNA polymerase is a mutant T7 RNA polymerase. 28. The method according to claim 25, wherein said RNA polymerase is SP6 RNA polymerase. 29. The method of claim 25, wherein said RNA polymerase is a mutant SP6 RNA polymerase. 30. The method of claim 25, wherein said RNA polymerase is T3 RNA polymerase. 31. The method of claim 25, wherein said RNA polymerase is a mutant T3 RNA polymerase. 32. The promoter for incorporation of a modified nucleotide triphosphate, wherein the promoter is Li
26. The method according to claim 25, wherein the method is selected from the group consisting of Cl, methanol, polyethylene glycol, diethyl ether, propanol, methylamine and ethanol. 33. A method for synthesizing a pyrimidine nucleotide triphosphate, comprising: (a) monophosphorylation, wherein the pyrimidine nucleoside is converted to a phosphorylating reagent, phosphate under conditions suitable for the formation of the pyrimidine nucleotide monophosphate. Contacting with a mixture comprising a trialkyl and dimethylaminopyridine; and (b) pyrophosphorylation, wherein said pyrimidine monophosphate and pyrroline from step (a) under conditions suitable for the formation of said pyrimidine nucleotide triphosphate. Contacting an oxidizing reagent. 34. The method of claim 33, wherein said pyrimidine nucleotide triphosphate is uridine triphosphate. 35. The uridine triphosphate has a 2′-sugar modification.
The method described in. 36. The method of claim 35, wherein said uridine triphosphate is 2'-O-methylthiomethyl uridine triphosphate. 37. The phosphorylating reagent is phosphorus oxychloride, phospho-tris-
34. The method of claim 33, wherein the method is selected from the group consisting of triazolide and phospho-tris-triimidazolide. 38. The method of claim 33, wherein said trialkyl phosphate is triethyl phosphate. 39. The method of claim 33, wherein said pyrophosphorylating reagent is tributylammonium pyrophosphate. 40. The method according to claim 25, wherein said oligonucleotide is RNA. 41. The oligonucleotide of claim 2, wherein the oligonucleotide is an enzymatic nucleic acid molecule.
5. The method according to 5. 42. The method of claim 25, wherein said oligonucleotide is an aptamer. 43. A kit for oligonucleotide synthesis comprising an RNA polymerase, a promoter for incorporation of modified nucleotide triphosphates and at least one compound of claim 24. 44. A kit for oligonucleotide synthesis comprising a DNA polymerase, a promoter for incorporation of modified nucleotide triphosphates and at least one compound of claim 24. 45. The RNA polymerase comprising a bacteriophage T7 RN
The kit according to claim 43, which is an A polymerase. 46. The RNA polymerase comprising bacteriophage SP6 R
The kit according to claim 43, which is NA polymerase. 47. The method wherein the RNA polymerase is bacteriophage T3 RN
The kit according to claim 43, which is an A polymerase. 48. The kit of claim 43, wherein said RNA polymerase is a mutant T7 RNA polymerase. 49. The kit of claim 43 or 44, wherein said kit comprises at least two compounds of claim 24. 50. A nucleic acid catalyst comprising a histidyl modification, wherein the nucleic acid catalyst is capable of catalyzing an endonuclease reaction in the absence of a metal ion cofactor. 51. The nucleic acid of claim 50, wherein said catalyst is capable of cleaving another nucleic acid molecule. 52. The nucleic acid of claim 51, wherein said another nucleic acid molecule is an RNA molecule. 53. The nucleic acid according to claim 51, wherein said another nucleic acid molecule is a DNA molecule. 54. The nucleic acid catalyst of claim 50, wherein said nucleic acid catalyst comprises at least one ribonucleotide.