JP2003511016A - Design of High Affinity RNase H to Replenish Oligonucleotides - Google Patents

Abstract

  (57) [Summary] PROBLEM TO BE SOLVED: To provide a bicyclic DNA analog, such as LNA and LNA modification, which forms a high-affinity duplex with complementary RNA and is useful for designing an oligomer in which the duplex is a substrate for RNase H About the field of things. The oligonucleotide may consist partially or completely of an LNA analog having very high affinity and the ability to recruit RNaseH. This indicates that oxy-LNA may be used to construct new antisense molecules with increased biological activity. Also, oxy-LNA can be used to create DNA, RNA, or other analogs, such as thiol, to create high affinity RNase H replenishing antisense compounds without having to stick to any immobilized design. It may be used in combination with a non-oxy-LNA such as -LNA or amino-LNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of bicyclic DNA analogs useful for designing oligomers that form high affinity duplexes with complementary RNA, said duplexes being substrates for RNase H. The oligonucleotide may be partially or wholly composed of a bicyclic DNA analog.

[0002]

[Prior art]

The term "antisense" relates to the use of oligonucleotides as therapeutic agents. Briefly, antisense drugs act by binding to mRNA, thereby blocking or modulating its translation into protein. Therefore, antisense drugs may be used to directly block the synthesis of disease-causing proteins. It may, of course, also be used to block the synthesis of normal proteins if they are involved in and exacerbated pathophysiological processes. Furthermore, it should be emphasized that antisense drugs can be used to activate genes rather than suppress them. Illustratively, this can be achieved by blocking the synthesis of the native suppressor protein.

Mechanistically, hybridizing oligonucleotides either cause physical interference with the translation process or specifically degrade the mRNA portion of the mRNA / antisense oligonucleotide duplex. It is believed that its effect is elicited by supplementing the cellular enzyme (RNase H).

Not unexpectedly, oligonucleotides must satisfy a number of different requirements in order to be useful as antisense agents. Importantly, antisense nucleotides have high affinity and specificity for their target mR.
Both extracellular and internal endo- and exo-nucleases that must bind to NA, must have the ability to recruit RNase H, must be able to reach sites of intracellular activity. Must be stable to, non-toxic / minimum immunostimulatory, etc.

Natural DNA only exhibits a modest affinity for RNA, and is deficient in nuclease resistance for many other important properties. Therefore, great efforts have been made to identify new analogs with improved antisense properties. In particular, this study shows that the enhanced affinity for complementary nucleic acids and the natural DN
The focus has been on identifying new analogs that combine the ability of A to recruit RNase H. Both of these properties have been shown to correlate biological activity in a strongly practical way. Of the vast number of analogs revealed from this study, only a few retain the ability to recruit RNase H, and only a few provide a useful increase in affinity. Absent.
Unfortunately, those that provide a useful increase in affinity are unable to supplement RNase H.

In the face of these results, the field has been as a means to provide more potent antisense drugs as a mixed backbone oligonucleotide, ie, a double mechanism of action, 1) at the ribosome level. It redirects to an acting antisense molecule by high affinity mediated translational arrest, and 2) activation of RNase H. These molecules, called gab-mers, typically contain at least 6 contiguous low affinity phosphorothioates (RNase H).
Flanked by a series of high-affinity analogs (non-RNase H-replenishing analogs) that enhance the ability to promote translation arrest. While expected to outperform current phosphorothioate antisense drugs, the gab-mer is not considered an ideal antisense molecule. In particular, their weakness may be their requirement for an immobilized design and the presence of high and low affinity domains in the molecule, which may impair biological activity.

The enzyme RNase H selectively binds to a heterologous DNA / RNA duplex and degrades the RNA portion of the duplex. Homologous DNA / DNA and RNA / RNA duplexes (DNA / DNA duplexes that differ only at the 2 ′ position molecularly)
/ DNA: 2'-H / 2'-H; RNA / RNA: 2'-OH / 2'-OH, and DNA / RNA: 2'-H / 2'-OH) are not substrates for the enzyme. . This suggests that either the molecular composition at the 2'-position itself or the structural features it imposes on the helix are crucial for enzyme recognition. Consistent with this, all 2'-modified analogs previously reported to show increased affinity have lost the ability to recruit RNase H.

[0008]

[Problems to be Solved by the Invention]

Locked Nucleic Acid (LNA) is a novel, bicyclic nucleic acid analog in which the 2'- and 4'positions of the furanose ring are O-methylene (oxy-LNA).
), S- methylene (thio-LNA), or NH ₂ - methylene component (amino -LN
Bound by A). This binding limits the structural freedom of the furanose ring, resulting in the very high degree of affinity increase previously reported for DNA analogues (WO99 / 14226).

Despite the fact that the modification of LNA involves the 2'-position, we have found that when oxy-LNA is used as a component of the oligonucleotide, the activity of RNase H was close. It has been found to be independent of the stretch of DNA or phosphorothioate bases. In fact, we have discovered that an oligonucleotide consisting entirely of oxy-LNA can recruit RNase H. Thus, oxy-LNA oligonucleotides constitute the first ever DNA analogue to exhibit such a long-needed combination of very high affinity and RNase H recruiting ability. . The implication of this is that oxy-LNA itself may be used to construct novel antisense molecules with enhanced biological activity. Alternatively, Oxy-LNA is standard DNA, RN
A, or other analogs, such as non-oxy-, such as thio-LNA or amino-LNA.
It may be used in combination with LNA to create high affinity, RNase H supplementing antisense compounds without sticking to any fixed design.

[0010]

[Means for Solving the Problems]

One "oxy-LNA monomer" is defined as a nucleotide monomer of formula Ia:

[0011]

[Chemical 1]

[0012] Where X is oxygen; B is a nucleobase; R^{1 *}, R^Two, R^Three, R⁵ , And R^{5 *}Is hydrogen; P is the internucleoside linkage to the subsequent monomer.
R ', or a 5'-terminal group,^{3 *}Is for the preceding monomer
Is an internucleoside bond or is a 3'-terminal group;^{2 *}And R ^{4 *} Are both -O-CH_Two-Indicates that oxygen is bound to the 2'-position.

The term “nucleobase” refers to the naturally occurring adenine (A), guanine (G
), Cytosine (C), thymine (T) and uracil (U), and xanthine,
Diaminopurine, 8-oxo-N ⁶ -methyladenine, 7-deazaxanthine,
^{7-deazaguanine,} N 4, ^{N 4} - ^{Etanoshitoshin,} N 6, ^{N 6} - ethano -2,
6-diaminopurine, ^{5-methylcytosine, 5- (C 3 -C 6)} - alkynyl cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine iso cytosine,
Non-naturally occurring nucleobases such as 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and Benner et al., US Pat. No. 5,432,272, and Susan M. Freier and Karl-Heinz Atlmann,
Covers the "non-naturally occurring" nucleobases described in "Nucleic Acid Research, 1997, 25, 4429-4443". The term "nucleobase" thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. It will be apparent to those skilled in the art that various nucleobases previously thought to be “non-naturally occurring” have subsequently been found to be natural.

A “non-oxy-LNA” monomer is not an oxy-LNA per se, but is an oligo, which, in combination with an oxy-LNA monomer, has the ability to bind sequence-specifically to a complementary nucleic acid. Is broadly defined as a nucleoside (ie, a glycoside of a heterocyclic base) that can be used to construct Examples of non-oxy-LNA monomers include 2'-deoxynucleotide (DNA) or nucleotide (RN
A), or analogs of these monomers that are not oxy-LNA, such as thio-LNA and amino-LNA as described by Wengel and coworkers (
Singh et al., "J. Org. Chem. 1998, 6, 6078-9" and Susan M. Freier and K.
arl-Heinz Atlmann, "Nucleic Acid Research, 1997, 25, 4429-4443").

It will be appreciated that incorporation of a non-oxy-LNA monomer into an oxy-LNA oligo may alter the RNase H recruitment properties of the oxy-LNA / non-oxy-LNA chimeric oligo. Therefore, depending on the number and type of non-oxy-LNA monomers used and the position of these monomers in the resulting oxy-LNA / non-oxy-LNA chimeric oligo, the chimera will have a corresponding total It may have increased, unchanged or diminished RNase H recruitment capacity when compared to Oxy-LNA oligos.

As mentioned above, a wide variety of modifications to the deoxynucleotide backbone can be envisioned, and one large group of possible non-oxy-LNAs can be represented by Formula I:

[0017]

[Chemical 2]

Wherein X is —O—; B is selected from nucleobases; R ^{1 *} is hydrogen; P is a radical site for internucleoside linkage to subsequent monomers, or shows a 5'-terminal group, such internucleoside linkage or 5'-terminal group is optionally contain substituents R ^5, R ⁵ is included in the coupling between either hydrogen, or a nucleoside, R ^{3 *} Is a group P ^* showing an internucleoside bond to the preceding monomer, or is a 3 ′ terminal group; a non-group selected from the substituents R ² , R ^{2 *} , R ³ and R ^{4 *.} one or two pairs of pairs ^{substituent, -C (R a R b)} -, - C (R a) = C (R a) -, - C (R a)
= N -, - O -, - S -, - SO 2 -, - N (R a) -, and> C = Z, may indicate a biradical consisting of 1-4 groups / atoms selected from, Z is Selected from -O-, -S-, and -N (R ^a )-, R ^a and R ^b are each independently hydrogen, optionally substituted C _1-6 -alkyl, optionally substituted C _{2. -6} - alkenyl, _{hydroxy, C 1-6} - _{alkoxy, C 2-6} - alkenyloxy, _{carboxy, C 1-6} - alkoxy - _{carbonyl, C 1-6} - alkylcarbonyl, formyl, amino, mono- - and di ( C _1-6 - alkyl) amino, carbamoyl, mono- - and di _{(C 1-6} - alkyl) - amino - carbonyl, amino _{-C 1-6} - alkyl - aminocarbonyl, mono- - and di _{(C 1-6} - Alkyl) amino-C _1-6- Alkyl - _{aminocarbonyl, C 1-6} - alkyl - carbonyl amino, _{carbamide, C 1-6} - alkanoyloxy, _{sulphono, C 1-6} - alkylsulfonyloxy, nitro, azido, _{sulfanyl, C 1-6} - alkylthio, halogen , A photochemically active group, a thermochemically active group, a chelating group, a reporter group, and a ligand, the possible pairs of said unpaired substituents being (i) the atoms to which said unpaired substituents are attached. And (ii) forming a monocyclic unit with any intervening atoms; and further, each of the substituents R ² , R ^{2 *} , R ³ , R ^{4 *} present and not contained in the possible biradical is hydrogen, optionally substituted _{C 1-6} - alkyl, _{C 2-6} optionally substituted - alkenyl, _{hydroxy, C 1-6} - alkoxy, _{C 2 6} - alkenyloxy, _{carboxy, C 1-6} - alkoxy - _{carbonyl, C 1 -6} - alkylcarbonyl, formyl, amino, mono- - and di _{(C 1-6} - alkyl) amino, carbamoyl, mono- - and di (C _1-6 -alkyl) -amino-
Carbonyl, amino-C _1-6 -alkyl-aminocarbonyl, mono- and di (
C _1-6 - alkyl) amino _{-C 1-6} - alkyl - amino _{carbonyl, C 1-6} - alkyl - carbonyl amino, _{carbamide, C 1-6} - alkanoyloxy, _{sulphono, C 1-6} - alkylsulfonyloxy, Nitro, azido, sulfanyl, C _1-6 -alkylthio, halogen, photochemically active group, thermochemically active group,
A chelate group, a reporter group, and a ligand; and a basic salt and an acid addition salt thereof; with the proviso that the monomer is not oxy-LNA. )

Particularly preferred non-oxy-LNA monomers are 2'-deoxyribonucleotides, ribonucleotides, and analogs thereof, which are modified at the 2'-position of ribose and are 2'-O-. Methyl, 2'-fluoro, 2'-trifluoromethyl, 2'-O- (2-methoxyethyl), 2'-O-aminopropyl, 2
'-O-dimethylamino-oxyethyl, 2'-O-fluoroethyl, or 2'
-O-propenyl, etc., and analogs in which the modification comprises both the 2'and 3'positions of ribose, preferably such modifications in which the modification connects the 2'- and 3'-positions, Analogues as described by Wengel and coworkers (Nielsen et al.,
"J. Chem. Soc., Perkin Trans. 1, 1997, 3423-33" and WO99 / 1
In 4226), and a analogue the modification including both 2 'and 4' positions, preferably such analogs in which the modification to bind the 2'- and 4'-positions of the ribose, -CH ₂ -S- or _-CH 2 -NH-, or analogs such as those having a _-CH 2 -NMe- bridge (Singh et al, "J Org Chem 1998, 6, 6078 -... 9 " W in
See engel and co-workers. ). However, non-oxy-LNA monomers with a β-D-ribo structure are often the most relevant, and a more interesting example (and indeed applicable) of non-oxy-LNA is the natural β-D-ribo structure. It is a stereoisomer of the structure. Of particular interest are α-L-ribo, β-D-xylo, and α
-L-xylo structure (Beier et al., Science, 1999, 283, 699 and Eschenmoser,
Science, 1999, 284, 2118 see), in particular _{2'-4'-CH 2 -S -} , - CH 2
-NH _-, - CH 2 -O-, or _-CH 2 -NMe- bridge (Rajwansh
i et al., “Chem. Commun., 1999, 1395”, and Rajwanshi et al., “Chem. Commun.,
1999, "Wengel and co-workers).

[0020]   In this context, “oligo” is similar to “oligomer” and “origin” is similar.
The term "gonucleotide" refers to nucleosides linked by internucleoside linkages.
Means a continuous chain of leoside monomers (ie, a glycoside of a heterocyclic base)
. The bond between two consecutive monomers in the oligo is -CH._Two-, -O-
, -S-, -NR^H-,> C = O,> C = NR^H,> C = S, -Si (R ")_Two -, -SO-, -S (O)_Two-, -P (O)_Two-, PO (BH_Three)-, -P (O
, S)-, -P (S)_Two-, -PO (R ")-, -PO (OCH_Three)-, And
-PO (NHR^H)-Selected from 2 to 4, preferably 3 groups / atoms, R ^H Is hydrogen and C_1-4Selected from alkyl, R ″ is C_1-6-Alkyl and fu
Selected from phenyl. A representative example of such a bond is -CH._Two-CH_Two-CH_Two -, -CH_Two-CO-CH_Two-, -CH_Two-CHOH-CH_Two-, -O-CH_Two -O-, -O-CH_Two-CH_Two-, -O-CH_Two-CH = (paired with consecutive monomers
R when used as a bond⁵Including), -CH_Two-CH_Two-O-, -N
R^H-CH_Two-CH_Two-, -CH_Two-CH_Two-NR^H-, -CH_Two-NR^H-C
H_Two-, -O-CH_Two-CH_Two-NR^H-, -NR^H-CO-O-, -NR^H−
CO-NR^H-, -NR^H-CS-NR^H-, -NR^H-C (= NR^H) -NR ^H -, -NR^H-CO-CH_Two-NR^H-, -O-CO -O-, -O-CO-
CH_Two-O-, -O-CH_Two-CO-O-, -CH_Two-CO-NR^H-, -O-
CO-NR^H-, -NR^H-CO-CH_Two-, -O-CH_Two-CO-NR^H-,
-O-CH_Two-CH_Two-NR^H-, -CH = N-O-, -CH_Two-NR^H-O-
, -CH_Two-O-N = (R when used as a bond to consecutive monomers ⁵ Including), -CH_Two-O-NR^H-, -CO-NR^H-CH_Two-, -CH_Two−
NR^H-O-, -CH_Two-NR^H-CO-, -O-NR^H-CH_Two-, -ON
R^H-, -O-CH_Two-S-, -S-CH_Two-O-, -CH_Two-CH_Two-S-,
-O-CH_Two-CH_Two-S-, -S-CH_Two-CH = (for continuous monomers
R when used as a bond⁵Including), -S-CH_Two-CH_Two-, -SC
H_Two-CH_Two-O-, -S-CH_Two-CH_Two-S-, -CH_Two-S-CH_Two-,
-CH_Two-SO-CH_Two-, -CH_Two-SO_Two-CH_Two-, -O-SO-O-,
-OS (O)_Two-O-, -O-S (O)_Two-CH_Two-, -OS (O)_Two−
NR^H-, -NR^H-S- (O)_Two-CH_Two-, -OS (O)_Two-CH_Two-,
-OP (O)_Two-O-, -OP (O, S) -O-, -OP (S)_Two-O-
, -S-P (O)_Two-O-, -SP (O, S) -O-, -SP (S)_Two-O
-, -OP (O)_Two-S-, -OP (O, S) -S-, -OP (S)_Two−
S-, -SP (O)_Two-S-, -S-P (O, S) -S-, -S-P (S)_Two -S-, -O-PO (R ")-O-, -O-PO (OCH_Three) -O-, -O-
PO- (OCH_TwoCH_Three) -O-, -O-PO (OCH_TwoCH_TwoS-R) -O-
, -O-PO (BH_Three) -O-, -O-PO (NHR^N) -O-,-OP (O
)_Two-NR^H-, -NR^H-P (O)_Two-O-, -OP (O, NR^H) -O-
, -CH_Two-P (O)_Two-O-, -OP (O)_Two-CH_Two-And-O-Si (
 R ")_Two-O-; among them, -CH_TwoCO-NR^H-, -CH_Two-NR^H
-O-, -S-CH_Two-O-, -OP (O)_Two-O-, -OP (O, S)-
O-, -OP (S)_Two-O-, -NR^H-P (O)_Two-O-, -OP (O,
NR^H) -O-, -O-PO (R ")-O-, -O-PO (CH_Three) -O-,
And -O-PO (NHR^N) —O— and R^HIs hydrogen and C_1-4-Archi
Selected from Le, R "is C_1-6-Basically selected from alkyl and phenyl
Is preferable. Further representative examples can be found in Mesmaeker et al., "Current Opinion i
n Structural Biology 1995, 5, 343-355 "and Susan M. Freier and Kar.
l-Heinz Altmann, "Nucleic Acids Research, 1997, 25, 4429-4443"
It is shown. The left side of the internucleoside bond has a 5-membered ring as the substituent P at the 3'-position.
To the 5'-position of the preceding monomer.

The term “consecutive monomer” refers to a monomer that is adjacent in the 5′-terminal direction, and “preceding monomer” is a monomer that is adjacent in the 3′-terminal direction. It is about.

Monomers are classified as if they are Watson-Crick base pairing rules (eg, G is C,
A is a hydrogen bond according to T or A is according to U), or, for example, diaminopurine and T
, Inosine and C, pseudocytosine and G, and the like, including nucleobases capable of forming other hydrogen-bonding motifs, are said to be “complementary”.

When the modified oxy-LNA oligo contains at least two non-oxy-LNA monomers, these contain the same or different nucleobases at the 1'-position and are identical at all other positions. Well, or they may contain the same or different nucleobases at the 1'-position and not be identical at at least one other position.

Accordingly, the present invention is a method for degrading RNA in vivo (in a cell or organism) or in vitro, wherein a high affinity oligonucleotide is directed to a complementary RNA target sequence. A high affinity oligonucleotide that activates RNase H when hybridized with a high affinity oligonucleotide, wherein the high affinity oligonucleotide may consist exclusively of oxy-LNA monomers. Enter.

Alternatively, the high affinity oligonucleotide may consist of both oxy-LNA and non-oxy-LNA monomers, in which case up to 5 high affinity oligonucleotides, For example four, for example three, for example two, intervening non-oxy-LNA monomers are included anywhere in the given position of the oligonucleotide, for example said high affinity oligonucleotide is It consists of both -LNA and non-oxy-LNA, where no non-oxy-LNA monomers are located adjacent to each other.

The high affinity oligonucleotide may further comprise one or more segments of intervening non-oxy-LNA monomers. For example, a series of intervening non-oxy-LNA monomers are located in the center of the oligonucleotide, and
The segment consisting of the oxy-LNA monomer may be located on both sides. Alternatively, a series of contiguous non-oxy-LNA monomers may be located at one or both ends. Furthermore, the oxy-LNA segment may be mediated or interrupted by one or more non-oxy-LNA monomers. Further, the high affinity oligonucleotide may include more than one type of internucleoside linkage, eg, a mixture of phosphodiester and phosphorothioate linkages.

Accordingly, the resulting oxy-LNA monomer and / or non-oxy-LNA
High affinity oligos containing monomers can be represented by the general formula: 5 '-[XmYnXp] q-3' (In the formula, X is oxy-LNA, Y is non-oxy-LNA, m and p are integers from 0 to 30, and n is from 0 to 3. Q is an integer from 1 to 10 and m + n + p is multiplied by q, such as 8, for example 9, for example 10, for example 11
, For example 12, for example 13, for example 14, for example 15, for example 16, for example 17
, For example 18, for example 19, for example 20, for example 21, for example 22, for example 23
, Eg 24, eg 25, eg 26, eg 27, eg 28, eg 29
, Eg 35, eg 40, eg 45, eg 50, eg 60
, For example 70, for example 80, for example 90, 100, 6-100
Is within the range of. )

The present invention provides oligos that combine their high affinity and specificity for target RNA molecules with their ability to recruit RNase H, to the extent that oligos are useful as antisense therapeutics. The oligo may consist entirely of oxy-LNA monomers, or it may consist of both oxy and non-oxy-LNA monomers.

When both oxy-LNA and non-oxy-LNA monomers are present in the oligo, the RNase H recruitment properties of the chimeric oligo may be the same as or different from that of the corresponding oxy-LNA oligo. . Thus, in one aspect of the invention, non-oxy-LNA monomers do not alter the RNase H recruitment properties of oxy-LNA / non-oxy-LNA chimeric oligos as compared to the corresponding all-oxy-LNA oligos. Used in the method. In another aspect of the invention, non-oxy-L
NA monomers either increase or decrease the efficiency of promoting RNase H cleavage when hybridized to its complementary RNA target, as compared to the corresponding whole oxy-LNA oligo. Therefore, oxy-LNA oligo RNase H
Used to change replenishment characteristics.

When both oxy-LNA and non-oxy-LNA monomers are present in the oligo, including its complementary target RNA of the chimeric oligo and one or more Watson-Crick mismatches. Ability to discriminate between
The corresponding total oxy-LNA oligos may differ in their ability to discriminate between their paired and mismatched target RNAs. For example, one oxy-LNA
The ability of oligos to discriminate between complementary target RNAs and single base mispaired target RNAs was determined by the uniqueness of Applicant Danish's "oxy-LNA oligonucleotides, filed on the same date as the present application. Method of increasing the specificity of ox
y-LNA oligonucleotides) ”, as described in the patent application entitled
It can be enhanced by incorporating non-oxy-LNA monomers, such as DNA, RNA, thio-LNA, or amino-LNA, at mismatched positions or in close proximity. Therefore, in another aspect of the invention, the non-oxy-LNA monomer is compared to the corresponding all-oxy-LNA oligo,
It is used to construct oxy-LNA / non-oxy-LNA oligos that show enhanced specificity but exhibit unaltered RNase H recruitment properties. In another aspect of the invention, the non-oxy-LNA monomer is the corresponding all-oxy-LNA monomer.
It is used to construct oxy-LNA / non-oxy-LNA oligos that show increased specificity and altered RNase H recruitment properties compared to LNA oligos.

Furthermore, the oligonucleotide of the present invention comprises a protein, an amplicon, an enzyme,
It may be bound to a compound selected from polysaccharides, antibodies, haptens, and peptides.

[0032]

【Example】

Example 1 LNA-Containing Oligonucleotides Recruit RNase H Two 41-mer oligonucleotides that make up a linear double-stranded template for subsequent run-off transcription of T7 polymerase are described below. Used to obtain target RNA corresponding to the 15-mer oligonucleotide: DNA control ; 5'-gtgtccgagacgtttg-3 ' phosphothioate control ; 5'-gtgtccccagagacgttg-3' LNA gab-mer ; 5'-GTGTccgagaCGTG-3. '(LNA is uppercase, DNA is lowercase), and LNA mix-mer ; 5'-gTgTCCgAgACgTTg-3' (LNA is uppercase, DNA is lowercase)

At the 5'end of the sense template strand, a T7 polymerase recognition sequence and a transcription initiation site are included, followed by a DNA sequence encoding the target RNA sequence. These two complementary oligonucleotides were heated to 80 ° C. for 10 minutes to produce a linearized double-stranded template. Twenty μl of in vitro transcription reaction was performed with 500 μM of ATP, GTP, and CTP, 12 μM of UTP, and about 50 μCi, respectively.
Α- ³² P UTP, 1 × transcription buffer (Tris-HCl, pH 7.5), 1
Contains 0 mM dithiothreitol, 1% BSA, 20 U RNasin ribonuclease inhibitor, 0.2 μl template, and 250 units T7 RNA polymerase. The inclusion of RN acin inhibitors is the target RN from ribonuclease
This was to prevent the decomposition of A. The reaction was performed at 37 ° C. for 2 hours to produce the desired 24-mer ³² U-labeled RNA run-off transcript. For purification of target RNA, 1.5 μl (1.5 units) of DNase I was added to the RNA,
It was dissolved in a 15% polyacrylamide gel containing 7M urea and the exactly sized fragment was excised from the gel and placed in elution buffer (0.1%).
% SDS, 0.5 M ammonium acetate, 10 mM Mg-acetic acid) and further incubated overnight at room temperature. The target RNA sequence was then purified by ethanol precipitation and the supernatant was filtered through Millipore (0.45 m) and collected by ethanol precipitation. The pellet was diluted in TE-buffer and subsequently subjected to RNase H digestion assay. In the present text, the untreated substrate, namely the 24mer α
Reduction of ³² P UTP labeled target RNA sequences was assayed over time as follows. Reactions were carried out in a total volume of 110 μl and in nuclease-free 1 × buffer (added in the order listed) (20 mM Tris-HCl, pH 7.5).
, 40 mM KCl, 8 mM MgCl2, 0.03 mg / ml BSA), 10
It contained mM dithiothreitol, 4% glycerol, 100 nM oligonucleotide, 3 units of RN acin inhibitor, labeled target RNA strand, and 0.1 U of RNase H. Excess oligonucleotide was added to each reaction to ensure complete hybridization of the RNA target sequence. Two negative controls were also incubated, (1) without any oligonucleotide,
Alternatively, (2) was prepared in the same manner as above, except that RNase H was not added to the reaction mixture. All reactions were incubated at 37 ° C. 0, 1
At 0, 20, 40, and 60 minutes, 10 μl aliquots were taken and immediately ice-cold formamide loading buffer was added to quench the reaction, and −20.
Stored at ° C. The sample was heated to 85 ° C. for 5 minutes before running on a 15% polyacrylamide gel containing 7M urea. The gel was vacuum dried and exposed to autoradiographic film overnight, followed by densitometry using Easy Win imaging software (Hero Labs). Received the calculation by the meter. The volume density of untreated target RNA was calculated using background correction in each lane. The volume density for zero time was defined as the reference value for each incubation. Relative values for samples at other time points in the corresponding incubations were calculated based on these reference values.

FIG. 1 shows the results of the RNase H experiment in the examples. As expected, the control DNA and the phosphorothioate oligonucleotide were both R
Replenish Nase H very efficiently. Furthermore, as expected, LNA oligonucleotides containing a stretch of 6 DNA monomers in the center (LNA gab-mers) efficiently recruit RNase H. However, surprisingly, LN
A mix-mer (mix-me) containing only a single DNA monomer dispersed between A monomers.
rs) also supplements RNase H. We have found that when LNA is used as a component of the oligonucleotide, the activity of RNase H is a continuous stretch of D.
It was concluded that no NA or phosphorothioate bases were required.

[Brief description of drawings]

[Figure 1]   The results of this RNase H experiment are shown in FIG.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 47/48 A61K 48/00 48/00 A61P 43/00 105 A61P 43/00 105 C12N 15/00 ZNAA ( 81) Designated countries 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, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU. , ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA , ZWF F term (reference) 4B024 AA01 AA20 CA20 HA08 HA17 HA20 4C076 CC26 EE30 EE41 4C084 AA13 NA05 ZB212 4C086 AA02 AA03 EA16 MA01 MA04 NA05 ZB21

Claims (23)

[Claims] 1. A method for degrading RNA comprising providing a high affinity oligonucleotide that replenishes RNase H when hybridized to an RNA target sequence, wherein the high affinity oligonucleotide is exclusively oxy. -A method consisting of LNA monomers. 2. A method for RNA degradation comprising providing a high affinity oligonucleotide that replenishes RNase H when hybridized to an RNA target sequence, wherein the high affinity oligonucleotide is an oxy-nucleotide. LNA and non-oxy-L
A method consisting of both NA monomers, wherein up to 5 consecutive non-oxy-LNA monomers are present at any given position of said oligonucleotide. 3. A method for degrading RNA comprising providing a high affinity oligonucleotide that replenishes RNase H when hybridized to an RNA target sequence, the high affinity oligonucleotide being an oxy-nucleotide. LNA and non-oxy-L
A method consisting of both NA monomers, and none of the non-oxy-LNA monomers are located adjacent to each other. 4. Non-oxy-L in an oxy-LNA / non-oxy-LNA oligo.
4. The method of claim 2 or 3, wherein the presence of NA monomer does not alter the RNase H recruitment properties of the oligo as compared to the corresponding oxy-LNA oligo. 5. Non-oxy-L in an oxy-LNA / non-oxy-LNA oligo.
5. The method of claim 4, wherein the presence of NA monomer modifies the RNase H recruitment properties of the oligo as compared to the corresponding oxy-LNA oligo. 6. Non-oxy-L in an oxy-LNA / non-oxy-LNA oligo.
6. The method of claim 5, wherein the presence of NA monomer increases or decreases the RNase H recruiting ability of the oligo as compared to the corresponding oxy-LNA oligo. 7. Non-oxy-L in an oxy-LNA / non-oxy-LNA oligo.
The presence of the NA monomer discriminates between the target RNA complementary to the oligo and the target RNA containing one or more Watson-Crick mismatches as compared to the corresponding oxy-LNA oligo. 7. The method of any one of claims 2-6, which enhances its ability to do. 8. The oligonucleotide has the general formula: 5 ′-[XmYnXp] q-3 ′ (wherein X is oxy-LNA, Y is non-oxy-LNA, and m and p are from 0 to 30). Is an integer, n is an integer from 0 to 5, and q is 1 to 10.
Is an integer up to. ) Is shown, The method of any one of Claims 1-7 characterized by the above-mentioned. 9. The method according to any one of claims 2-8, wherein the non-oxy-LNA monomer is a deoxyribonucleotide. 10. The method of claim 9, wherein the deoxyribonucleotide is modified at the 2'-position of ribose. 11. The 2'-modification is hydroxyl, 2'-O-methyl, 2'-fluoro, 2'-trifluoromethyl, 2'-O- (2-methoxyethyl), 2 '.
-O-aminopropyl, 2'-O-dimethylamino-oxyethyl, 2'-O-
11. The method according to claim 10, which is fluoroethyl or 2'-O-propenyl. 12. The method according to claim 11, wherein the modification also includes a 3′-position, preferably a modification connecting the 2′-position and the 3′-position of the ribose. 13. The method according to claim 12, wherein the modification also includes a 4′-position, preferably a modification connecting the 2′-position and the 4′-position of the ribose. 14. The method of claim 13, wherein the modification is a —CH ₂ —S—, —CH ₂ —NH—, or —CH ₂ —NMe-bridge, selected from the group consisting of 2′-4 ′ bonds. the method of. 15. The nucleotide is α-D-ribo, β-D-xylo, or α-D.
The method according to any one of claims 9 to 14, which has an L-xylo structure. 16. All or some of the oxy-LNA monomers, or all or some of the non-oxy-LNA monomers, or all of both the oxy-LNA and non-oxy-LNA monomers. 16. A method according to any one of claims 9 to 15 wherein some or some contain 3'- or 5'-modifications which result in an internucleoside linkage other than the natural phosphodiester linkage. 17. The modification is -OP (O) _2- O-, -OP (O, S)-.
_{O -, - O-P (} S) 2 -O -, - NR H -P (O) 2 -O -, - O-P (O,
^{NR H) -O -, - O} -PO (R ") - O -, - O-PO (CH 3) -O-, and -O-PO ^(NHR N) selected from the group consisting of -O-, R ^H is hydrogen, and _{C 1-4} - is selected from alkyl, further R "is _{C 1-6} - is selected from alkyl and phenyl, a method according to claim 16. 18. Incorporation of at least one non-oxy-LNA monomer comprises:
The resulting oligo's affinity for complementary nucleic acid was determined by total oxy-LN
Compared with affinity A oligo, it varies from [Delta] T _m of less than ± 5 ° C., the method according to any one of claims 1 to 17. 19. The method of claim 18, wherein the affinity is altered by less than ± 10 ° C. 20. At least two non-oxy-LNA monomers are used, which contain nucleobases that are the same or different at the 1'-position and are identical at all other positions. The method according to 18 or 19. 21. At least two non-oxy-LNA monomers are used which contain nucleobases which are the same or different in the 1'-position and which are non-identical in at least one other position, 20. The method according to claim 18 or 19. 22. The oligomer according to any one of claims 1 to 21, which is used as a therapeutic compound, for example, an antisense compound. 23. The oligomer is a protein, an amplicon, an enzyme, a polysaccharide,
The compound according to claim 1, which is bound to a compound selected from an antibody, a hapten, and a peptide.
23. The oligomer according to any one of claims 23 to 23.