deoxyribozymes

Abstract

The invention relates to a nucleic acid encoding the catalytic core of a DNAzyme, comprising SEQ ID NO. 1 (5'-G G C T A G C T A C A A C A-3'). Specific chemical modifications are introduced at positions 5, 6, and / or 14 of the core and within the substrate-binding arms to achieve functional improvements, such as: • Enhanced catalytic activity or reduced activity • Altered target association / dissociation and turnover • Modified RNA selectivity (increased or decreased) • Controlled antisense effects via RNase recruitment • Changed metal-ion dependence (e.g., Mg2*) • Improved cellular lifetime, reduced innate immune response, or conversely increased immune stimulation Modifications at the RNA-DNA interface reduce electrostatic repulsion, lowering metal-ion requirements. Additional arm modifications increase cellular stability and precision by limiting non-specific RNase H1 cleavage. The invention also provides methods to improve SNP selectivity using auxiliary mismatches in the binding arms.

Description

[0001] HEINRICH HEINE UNIVERSITY CH Kilger Anwaltspartnerschaft mbB Dusseldorf Fasanenstraße 29 Our Ref.: B407-0001W01 10719 Berlin

[0002] DEOXYRIBOZYMES BACKGROUND

[0003] Deoxyribozymes (DNAzymes, or Dz) are DNA sequences capable of catalyzing chemical reactions. The RNA-cleaving 10-23 DNAzyme was among the first DNAzymes to be evolved and possesses clinical and biotechnical applications as a biosensor and a knockdown agent. RNA cleavage may also be done with ribozymes such as the hammerhead ribozyme, or protein-based enzymes such as RNase Hl. DNAzymes do not require the recruitment of other macromolecular components to cleave RNA and can turnover, thus they have a distinct advantage over other knockdown methods (siRNA, CRISPR, ASOs). DNAzymes have a high haemolytic stability and thus advantages. Also, they are more cost effective than RNA.

[0004] These DNAzymes are short (15-40 nt) single stranded DNA molecules that catalyze chemical reactions. These catalytic DNAs are not found in nature, rather they are designed or selected from a random oligonucleotide library and enriched for a desired activity, allowing for the precise control of both the desired reaction type and condition.

[0005] DNAzymes generally consist of two substrate binding arms flanking a central catalytic core; See Fig.

[0006] 1.

[0007] The flanking binding arms can be altered both in length and in sequence identity, allowing high substrate specificity and limiting off-target effects.

[0008] DNAzymes are also capable of enzymatic turnover and do not require the recruitment of other macromolecular components to function. These qualities give DNAzymes the potential to be an efficient and specific knockdown agent that could be applied in a clinical or biotechnical setting. DNAzymes have been artificially selected to catalyze numerous reactions such as RNA cleavage, RNA / DNA ligation, DNA base excision, Diels-Alder, thymine dimer resolution, and numerous other activities. RNA cleavage is the most common reaction performed by DNAzymes. DNAzymes as therapeutics have grown in popularity. There have been clinical trials of the DNAzyme for use in cancer and asthma treatment by targeting the reduction of protein expression through catalyzed mRNA cleavage.

[0009] Recently, NMR and molecular dynamics yielded structural insights at high resolution. The substrate adopts a bent conformation at the scissile phosphate and the precatalytic complex comprises three magnesium ion binding sites that are linked to structural rearrangements and activity. There is alsoa 2.7 A crystal structure of the 10-23 DNAzyme-substrate complex captured in a dimeric precatalytic condition.

[0010] DNAzyme-based therapeutic strategies display properties such as high selectivity, straightforward design, relatively small size, independence of protein cofactors, absence of permanent effects on the genome, as well as fast production. Development of in vitro and dedicated cellular applications based on randomized optimization or systematic screenings has led to gradual progress over the last two decades. Rational design strategies can further promote the advances of the DNAzyme technology. However, to exploit this pathway, it is instrumental to adequately understand the basis of the DNA-mediated catalysis process, including the underlying molecular structure, dynamic processes, and transition states. One problem is the considerable drop in activity when changing from in vitro to cellular conditions. As such the DNAzymes are known to have insufficient bioactivity for most applications.

[0011] In many experimental setups there is also the need for reliable controls that, for example, maintain antisense capability but no catalytic capability.

[0012] In this context, it is beneficial to have DNAzyme variants with only minor modifications that strongly reduce catalytic activity but otherwise have minimal effects on structure and RNA binding properties.

[0013] Furthermore, it can be beneficial to have DNAzyme variants that show different degrees of reduced activity, for example, when combining modifications that strongly enhance life-time and / or when targeting RNAs that should not be eliminated completely.

[0014] A further complication in deploying DNAzymes in complex cellular or in vivo environments is the unintended recruitment of endogenous nucleases. When the substrate-binding arms of a DNAzyme hybridize with a target RNA, they form an RNA: DNA heteroduplex. If this heteroduplex achieves a sufficient length and adopts a specific structure, such as a canonical A-form helical structure, it can be recognized by endogenous RNases, such as RNase Hl. This recognition triggers less-specific cleavage of the RNA strand, essentially mimicking an antisense oligonucleotide (ASO) effect. This RNase Hl-mediated background cleavage severely limits the single-nucleotide precision of the DNAzyme, particularly when attempting to target single-point mutations where allele-specific selectivity is required.

[0015] Unser Zeichen: B407-0001W01 2While in many therapeutic applications the presence of DNAzymes should lead to minimal innate immune responses, other therapeutic applications may benefit from increased activation of the innate immune response.

[0016] The present invention addresses the need for optimization of the DNAzymes for in vitro and in vivo applications.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention provides the following benefits. It provides for DNAzymes with modulated precision. It provides for DNAzymes with increased or decrease activity. It provides for DNAzymes with improved target specificity. It provides for DNAzymes with increased cellular life-time. It provides for DNAzymes that modulate immune response via chemical modifications in the DNA-RNA binding interface. It provides for DNAzymes with more than one improved ability, such as but not limited to a better or, when desired, reduced selectivity, precision, cellular stability, activity and effects on the innate immune response.

[0019] In one aspect, the invention provides a DNAzyme with a targeted modification architecture designed to evade RNase Hl recruitment and reduce or prevent non-specific RNA cleavage, while maintaining high DNA-mediated catalytic activity. Multiple RNase evasion pattern have been identified, i.e. sequence position at which substitutions or modifications of the DNA nucleotides in the DNAzyme's substrate binding arms promote RNase Hl evasion. In one embodiment modified nucleotides are introduced at the identified sequence positions to increase DNAzyme precision in the presence of RNases. Systematic optimization also identified minimal modification pattern, referred to as REP-1 and REP-2. The asymmetry of the identified pattern in regard to the 5' and 3' substrate binding arms, is well in line with the asymmetry of these regions in the pre-catalytic DNAzyme: RNA complex. In one embodiment modified nucleotides are introduced at the identified sequence positions of REP-1 or REP-2 to enable effective RNase Hl evasion with no or minor impact on the DNAzyme-mediated cleavage rate. In a preferred embodiment the used modifications include 2'0Me modifications or 2'MOE modifications or RNA substitutions. These specific modification patterns (REP-1 and REP-2) are compatible with multiple DNAzymes targeting different RNA sequences, including but not limited to the 10-23 type DNAzymes targeting PrP (e.g., Dz839), EYFP (e.g., Dzl84), and KRAS (e.g., Dzl89). Specific nucleotide sequences embodying these RNase evasion patterns include SEQ. ID NOs: 284 to 296.

[0020] Unser Zeichen: B407-0001W01 3In another aspect, the present invention relates to the use of the aforementioned DNAzymes in therapeutic applications, characterized by potent in vivo reduction of the targeted RNA such as in the context of antiviral activity, immunological modulation, and / or treatment of diseases associated with occurrence of specific coding or non-coding RNA. When formulated in targeted lipid nanoparticles (LNPs), DNAzymes of this invention, including DNAzymes stabilized by suitable chemical modifications (e.g., 2'-0Me and / or LNA), significantly limit infection with chronic viral strains of Lymphocytic Choriomeningitis Virus (LCMV). Furthermore, the DNAzymes of the present invention demonstrate the ability to actively improve and rescue host immune function in vivo. The improvement of immune function achieved by the administration of the DNAzymes is characterized by the occurrence of one or more specific immunological modulations. In certain embodiments, the administration of the DNAzyme can either: (a) increase the production of pro-inflammatory cytokines, including but not limited to IFN-y and TNF-a, by CD8+ T cells; or (b) expand the populations of Short-lived effector cells (SLECs) and Memory precursor effector cells (MPECs); or (c) downregulate the surface expression of T-cell exhaustion markers, including TIM-3 and PD-1. It is explicitly contemplated that the DNAzymes may induce any one of these effects individually, any combination of two of these effects, or all three effects simultaneously.

[0021] Consequently, the administration of the DNAzymes effectively reduces systemic pathology and organ damage. In highly preferred, but optional, embodiments, this reduction in systemic pathology and organ damage is evidenced by lowered serum activity of physiological markers selected from Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and Lactate Dehydrogenase (LDH), though the reduction of organ damage is not strictly limited to the quantification or modulation of these specific markers.

[0022] Furthermore, it is explicitly contemplated that any of the DNAzymes disclosed herein, particularly those intended for therapeutic applications and medical interventions as defined in the present invention, may be formulated in targeted lipid nanoparticles (LNPs). The utility of LNP formulations is not restricted to variants exhibiting RNase evasion capabilities, but extends synergistically to all DNAzyme architectures described herein, including those deployed for antiviral activity, immunological modulation, and oncological interventions such as the suppression of tumor growths associated with targets like VGEF and TGF-1.

[0023] Therapeutic benefits related to the aforementioned DNAzymes are also demonstrated for the two cancer-associated targets VGEF and TGF-pi. Cellular and in vivo experiments demonstrate that the Unser Zeichen: B407-0001W01 4used chemical modifications consistently show the strongest therapeutic effects, including the suppression of tumor growths.

[0024] The invention relates to a nucleic acid polymer encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme),

[0025] wherein said catalytic core region comprises the following sequence SEQ ID NO. 1, wherein the number in parenthesis designates the position number,

[0026] 5'-G (1), G (2), C (3), T (4), A (5), G (6), C (7), T (8), A (9), C (10),

[0027] A (11), A (12), C (13), G (14), A (15)-3' (SEQ ID NO. 1),

[0028] and wherein said core region nucleic acid sequence comprises one or more of the following modifications, at position 5,

[0029] Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'

[0030] SEQ ID

[0031]

[0032] pos. / Modification NO.

[0033] 1

[0034] SEQ ID

[0035]

[0036] A*(5)=etheno-dA NO.

[0037] 2

[0038] SEQ ID

[0039] 5' G G C T N* G C T A C A A C G A 3' N*(5)=dNebularine NO.

[0040] 3

[0041] SEQ ID

[0042]

[0043] N*(5)=ANT NO.

[0044] 4

[0045] SEQ ID

[0046]

[0047] A*(5)=Me-Ph NO.

[0048] 5

[0049] at position 6,

[0050] Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'

[0051] Unser Zeichen: B407-0001WO1 5SEQI D5' G G C T A G C T A C A A C G A 3' Pos. / Modification NO.

[0052] 1 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=6-S-dG NO.

[0053] 6 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=6-Se-dG NO.

[0054] 7 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=8-oxo-dG NO.

[0055] 8 SEQ ID

[0056] 5' G G C T A N* C T A C A A C G A 3' G*(6)=Iso-dG NO.

[0057] 9 SEQ ID G*(6)=6-O-methyl- 5' G G C T A N* C T A C A A C G A 3'

[0058]

[0059] NO. dG 10

[0060] SEQ ID

[0061] 5' G G C T A N* C T A C A A C G A 3' G*(6)=7-deaza-dG NO.

[0062] 11

[0063] SEQ

[0064] O 5' G G C T A N* C T A C A A C G A 3' G*(6)=2'-OMe-dG NO.

[0065] 12

[0066] SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2'-MOE-dG NO.

[0067] 13 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=etheno-dA NO.

[0068] 14 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=ANT NO.

[0069] 15 SEQI DG*(6)=2-amino- 5' G G C T A N* C T A C A A C G A 3' NO. purine

[0070]

[0071] 16

[0072] Unser Zeichen: B407-0001W01 6SEQ

[0073] ID

[0074]

[0075] G T A N* C T A C A C A 3' G*(6)=2-amino-dA NO.

[0076] 17

[0077] SEQ ID

[0078] 5' G G C T A N* C T A A C A 3' G*(6)=dNebularine NO.

[0079]

[0080] 18

[0081] SEQ ID

[0082]

[0083] G G T A N* C T A A C G A 3' G*(6)=7MG NO.

[0084] 19

[0085] SEQ ID

[0086]

[0087] G G C T A N* C T A A C A 3' G*(6)=IOX NO.

[0088] 20

[0089] SEQ ID

[0090]

[0091] G T A N* C T A A C G A 3' G*(6)=6IP NO.

[0092] 21

[0093] SEQ ID

[0094]

[0095] G G C T A N* C T A C A A C A 3' G*(6)=3DS NO.

[0096] 22

[0097] SEQ ID G G T N* C T A C A C A 3' G*(6)=2DO NO.

[0098]

[0099] 23

[0100] SEQ ID

[0101]

[0102] G G C T A N* C T A A C A 3' G*(6)=PYR NO.

[0103] 24

[0104] SEQ ID

[0105]

[0106] G G T A N* C T A A C A 3' G*(6)=NIP NO.

[0107] 25

[0108] SEQ ID

[0109]

[0110] G G C T A N* C T A C A C A 3' G*(6)=FIP NO.

[0111] 26

[0112] SEQ ID

[0113]

[0114] G G T N* C T A A C A 3' G*(6)=3NA NO.

[0115] 27

[0116] SEQ ID

[0117]

[0118] G G C T A N* C T A A C A 3' G*(6)=7OP NO.

[0119] 28

[0120] Unser Zeichen: B407-0001W01 7SEQ

[0121] ID G T A N* C T A C A C G A 3' G*(6)=3DO NO.

[0122]

[0123] 29

[0124] SEQ ID

[0125] 5

[0126]

[0127] ' G G C T A N* C T A A C A 3' G*(6)=2DS NO.

[0128] 30

[0129] SEQ ID

[0130]

[0131] G G T A N* C T A A C G A 3' G*(6)=B0G NO.

[0132] 31

[0133] SEQ ID

[0134]

[0135] G G C T A N* C T A A C A 3' G*(6)=2TA NO.

[0136] 32

[0137] SEQ ID G T A N* C T A A C G A 3' G*(6)=P0P NO.

[0138]

[0139] 33

[0140] SEQ ID

[0141]

[0142] G G C T A N* C T A A C A 3' G*(6)=NTZ NO.

[0143] 34

[0144] SEQ ID

[0145]

[0146] G G T N* C T A A C A 3' G*(6)=MES NO.

[0147] 35

[0148] SEQ ID

[0149]

[0150] G G C T A N* C T A A C A 3' G*(6)=TTZ NO.

[0151] 36

[0152] SEQ ID

[0153]

[0154] G G T A N* C T A A C A 3' G*(6)=MER NO.

[0155] 37

[0156] SEQ ID G G C T A N* C T A C A C A 3' G*(6)=M2R NO.

[0157]

[0158] 38

[0159] SEQ ID

[0160]

[0161] G T N* C T A A C A 3' G*(6)=M2S NO.

[0162] 39

[0163] SEQ ID

[0164]

[0165] G G C T A N* C T A A C A 3' G*(6)=BTG NO.

[0166] 40

[0167] Unser Zeichen: B407-0001W01 8SEQ

[0168] ID G T A N* C T A C A C G A 3' G*(6)=6TP NO.

[0169]

[0170] 41

[0171] SEQ ID

[0172] 5

[0173]

[0174] ' G G C T A N* C T A A C A 3' G*(6)=PHY NO.

[0175] 42

[0176] SEQ ID

[0177]

[0178] G G T A N* C T A A C G A 3' G*(6)=3TA NO.

[0179] 43

[0180] SEQ ID

[0181]

[0182] G G C T A N* C T A A C A 3' G*(6)=MTG NO.

[0183] 44

[0184] SEQ ID

[0185]

[0186] G T A N* C T A A C A 3' G*(6)=AIP NO.

[0187] 45

[0188] SEQ ID G G C T A N* C T A C A A C A 3' G*(6)=D0P NO.

[0189]

[0190] 46

[0191] SEQ ID

[0192]

[0193] G G T N* C T A c A C G A 3' G*(6)=AEG NO.

[0194] 47

[0195] SEQ ID

[0196]

[0197] G G C T A N* C T A A C A 3' G*(6)=AMG NO.

[0198] 48

[0199] SEQ ID

[0200]

[0201] G G T A N* C T A A C A 3' G*(6)=IOX2 NO.

[0202] 49

[0203] SEQ ID

[0204]

[0205] G G C T A N* C T A C A C A 3' G*(6)=Me-Ph NO.

[0206] 50

[0207] SEQ ID G G T N* C T A A C A 3' G*(6)=6aG NO.

[0208]

[0209] 51

[0210] SEQ ID

[0211]

[0212] G G C T A N* C T A A C A 3' N*(6)=6-cG NO.

[0213] 52

[0214] Unser Zeichen: B407-0001W01 9SEQ

[0215] I D5' G G C T A N* C T A C A A C G A 3' N*(6)=7MP NO.

[0216] 53

[0217] at position 14,

[0218] Pos. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3 SEQ ID G G C T A G C T A C A A C G A 3' Pos. / Modification NO.

[0219] 1 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=6-S-dG NO.

[0220] 54 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=6-Se-dG NO.

[0221] 55 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=8-oxo-dG NO.

[0222] 56 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=Iso-dG NO.

[0223] 57 SEQ ID G G C T A G C T A C A A C N* A 3' ^(14)=6-O-methyl- NO. dG 58 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=7-deaza-dG NO.

[0224] 59 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=etheno-dA NO.

[0225] 60 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=ANT NO.

[0226] 61 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=2-amino-dA NO.

[0227] 62 SEQ ID G G C T A G C T A C A A C N* A 3' G*(14)=dNebularine NO.

[0228] 63 SEQ G G C T A G C T A C A A C N* A 3' G*(14)=7MG

[0229]

[0230] ID Unser Zeichen: B407-0001W01 10NO.

[0231] 64 SEQ ID

[0232] 5' G G C T A G C T A C A A C N* A 3' G*(14)=IOX NO.

[0233] 65 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=6IP NO.

[0234] 66 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=3DS NO.

[0235] 67 SEQ ID

[0236] 5' G G C T A G C T A C A A C N* A 3' G*(14)=2DO NO.

[0237] 68 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=PYR NO.

[0238] 69 SEQ ID

[0239] 5' G G C T A G C T A C A A C N* A 3' G*(14)=NIP NO.

[0240] 70 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=FIP NO.

[0241] 71 SEQ ID

[0242] 5' G G C T A G C T A C A A C N* A 3' G*(14)=3NA NO.

[0243] 72 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=7OP NO.

[0244] 73 SEQ ID

[0245] 5' G G C T A G C T A C A A C N* A 3' G*(14)=3DO NO.

[0246] 74 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=2DS NO.

[0247] 75 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=B0G NO.

[0248] 76 SEQ ID

[0249] 5' G G C T A G C T A C A A C N* A 3' G*(14)=2TA NO.

[0250] 77 S

[0251] |EDQ

[0252] 5' G G C T A G C T A C A A C N* A 3' G*(14)=P0P

[0253]

[0254] Unser Zeichen: B407-0001W01 11NO.

[0255] 78 SEQ ID

[0256] 5' G G C T A G C T A C A A C N* A 3' G*(14)=NTZ NO.

[0257] 79 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=MES NO.

[0258] 80 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=TTZ NO.

[0259] 81 SEQ ID

[0260] 5' G G C T A G C T A C A A C N* A 3' G*(14)=MER NO.

[0261] 82 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=M2R NO.

[0262] 83 SEQ ID

[0263] 5' G G C T A G C T A C A A C N* A 3' G*(14)=M2S NO.

[0264] 84 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=BTG NO.

[0265] 85 SEQ ID

[0266] 5' G G C T A G C T A C A A C N* A 3' G*(14)=6TP NO.

[0267] 86 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=PHY NO.

[0268] 87 SEQ ID

[0269] 5' G G C T A G C T A C A A C N* A 3' G*(14)=3TA NO.

[0270] 88 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=MTG NO.

[0271] 89 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=AIP NO.

[0272] 90 SEQ ID

[0273] 5' G G C T A G C T A C A A C N* A 3' G*(14)=D0P NO.

[0274] 91 S

[0275] |EDQ

[0276] 5' G G C T A G C T A C A A C N* A 3' G*(14)=AEG

[0277]

[0278] Unser Zeichen: B407-0001W01 12NO.

[0279] 92 SEQ ID

[0280] 5' G G C T A G C T A C A A C N* A 3' G*(14)=AMG NO.

[0281] 93 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=IOX2 NO.

[0282] 94 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=Me-Ph NO.

[0283] 95 SEQ ID

[0284] 5' G G C T A G C T A C A A C N* A 3' G*(14)=6aG NO.

[0285] 96 SEQI DG*(14)=Me-Ph-6-S- 5' G G C T A G C T A C A A C N* A 3' NO. dG 97 SEQ ID G*(14)=Me-Ph-6- 5' G G C T A G C T A C A A C N* A 3' NO. Se-dG 98 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=PSO-6-S-dG NO.

[0286] 99 SEQ ID G*(14)=PSO-6-Se- 5' G G C T A G C T A C A A C N* A 3' NO. dG 100 SEQI DG*(14)=2'-MOE-6-S- 5' G G C T A G C T A C A A C N* A 3' NO. dG 101 SEQ ID G*(14)=2'-MOE-6- 5' G G C T A G C T A C A A C N* A 3' NO. Se-dG 102 SEQ

[0287] G*(14)=Me-Ph-2'- ID 5' G G C T A G C T A C A A C N* A 3' NO. MOE-6-S-dG 103 SEQI DG*(14)=Me-Ph-2'- 5' G G C T A G C T A C A A C N* A 3'

[0288]

[0289] NO. MOE-6-Se-dG 104

[0290] SEQ ID

[0291] 5' G G C T A G C T A C A A C N* A 3' N*(14)=6-cG NO.

[0292] 105

[0293] Unser Zeichen: B407-0001W01 13SEQ

[0294] ID

[0295] 5' G G C T A G C T A C A A C N* A 3' N*(14)=7MP NO.

[0296] 106

[0297] See also figure 2.

[0298] Additional modifications at position 14 include:

[0299] Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'

[0300] SEQ ID

[0301] 5' G G C T A G C T A C A A C G A 3' Pos. / Modification NO.

[0302] 1

[0303] SEQID5' G G C T A G C T A C A A C N* A 3' G*(14)=2'OMe-6-S- NO. dG 304 SEQ ID

[0304] 5' G G C T A G C T A C A A C N* A rG*(14)=PSO-2'OMe-

[0305]

[0306] NO. 6-S-dG 305

[0307] It provides for DNAzymes with enhanced or higher catalytic activity. That means, in various alternative embodiments, the DNAzyme of the present invention has an enhanced or optimized ability to cleave nucleic acid substrates, preferably RNA substrates. In preferred embodiments, the enhanced or optimized ability of an enzymatic DNA molecule to cleave RNA substrates shows about a 103- to 106-fold improvement over the uncatalyzed rate. In more preferred embodiments, an enzymatic DNA molecule of the present invention is able to cleave RNA substrates at a rate that is about 10-fold improved over previous molecules. In even more preferred embodiments, the enhanced or optimized ability to cleave RNA substrates is expressed as a 102-fold improvement over the progenitor. One skilled in the art will appreciate that the enhanced or optimized ability of an enzymatic DNA molecule to cleave nucleic acid substrates may vary depending upon the applied reaction conditions and properties of the targeted RNA.

[0308] The enhanced or optimized ability of an enzymatic DNA molecule of the present invention to cleave an RNA substrate may be determined in a cleavage reaction with varying amounts of labeled or unlabeled RNA substrate in the presence of enzymatic DNA molecule. The ability to cleave the substrate is generally defined by the catalytic rate (kcat) divided by the Michaelis constant (KM). The symbol kcat represents the maximal velocity of an enzyme reaction when the substrate approaches a saturation value. KM represents the substrate concentration at which the reaction rate is one-half maximal. Since in the area of RNA-cleaving DNAzymes, KM and hence kcat will be Unser Zeichen: B407-0001W01 14dependent on, e.g. the lengths of the binding arms, the catalytic activity is often described by the so-called kobs value (observed rate constant under single turnover condition). In addition, the inventors have another parameter that reports on the turnover capability, i.e. t_90 (the time required to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNA over Dz). Herein, the term " DNAzymes" refers to catalytically active single-stranded, synthetic DNA molecules, which do not occur in nature. DNAzymes of the 10-23 family represent a distinct class of DNA molecules, which were developed in the 1990s.

[0309] In the context of the present invention, the term "10-23 family" refers to a general DNAzyme model (Sontoro & Joyce, Proc. Natl. Acad. Sci. U. S. A., 94 (1997) 4262-4266). DNAzymes of the 10-23 model - also referred to as "10-23 DNAzymes" have a catalytic domain of 15 deoxyribonucleotides, which are flanked by two substrate binding domains (see WO 2005 / 033314). Potential advantages of DNAzymes include relatively high stability and no reliance on intracellular enzymes. The 10-23 family also covers known deletions (such as deletion of T(8)) and extensions (such as addition of one additional nucleotide at the 3' end following A(15)).

[0310] Terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes the degree of error associated with measurement of the particular quantity). In addition, all ranges disclosed herein are inclusive of the endpoints and also any intermediate range points, whether explicitly stated or not, and the endpoints are independently combinable with each other.

[0311] The term "comprising" or "comprises" as used herein means "including, but not limited to". The term is intended to be open-ended, to specify the presence of any stated features, elements, integers, steps, or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. The term "comprising" or "comprises" thus includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" or comprises" as used throughout the application and in particular within the claims may be replaced by the term "consisting of" or "consisting essentially of".

[0312] Unser Zeichen: B407-0001W01 15The term "treatment or treating" as used herein refer to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0313] The term "nucleic acid" as used herein refers to a biopolymer comprising monomers of nucleotides, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and other polynucleotides of modified nucleotides and / or nucleotide derivatives, and can be either double stranded (ds) or single stranded (ss). In some embodiments, modified nucleotides contain one or more modified bases (e.g. unusual bases such as inosine, and functional modifications to the bases such as amino), modified backbones (e.g. peptide nucleic acid, PNA) and / or other chemically, enzymatically, or metabolically modified forms. Examples of modified nucleotides which can be used to generate the nucleic acids disclosed herein include xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 6-thio guanines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine or fluorophore and quencher conjugated nucleotides. Furthermore, modifications affecting the phosphodiester backbone linkage and / or the ribose moiety as described in "internucleotide linkage modification" and "ribose modification", respectively (vide infra), are considered and can be implemented either as individual modification or in combination on the same nucleotide. Alternatively, the nucleic acid molecules can be produced biologically using an expression vector.

[0314] Herein, the term " DNA" refers to deoxyribonucleic acid composed of a single strand of monomeric units called nucleotides, wherein each nucleotide is composed of a nitrogen-containing nucleobase, a 2-deoxyribose sugar moiety, and a phosphate group, wherein the individual nucleotides are linked in the single-strand by a phosphate group linking the OH group in position 5' of a 2-deoxyribose sugar moiety to the OH group in 3' of a neighboring 2-deoxyribose sugar moiety. In particular embodiments, the nitrogen-containing nucleobases are independently selected from cytosine [C], guanine [G], adenine [A] and thymine [T], In particular embodiments, one or more of the nucleobases are non-canonical bases, in particular a non-canonical base selected from the list of: a

[0315] Unser Zeichen: B407-0001W01 16modified adenosine, in particular N6-carbamoyl-methyladenine or N6-methyadenine; a modified guanine, in particular 6-thio guanine, 6-Se guanine, 7-deazaguanine or 7- methylguanine; a modified cytosine, N4-methylcytosine, 5-carboxylcytosine, 5- formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine, or 5- methylcytosine; a modified thymidine, in particular a-glutamyl thymidine or a- putrescinyl thymine; a uracil or a modification thereof, in particular uracil, base J, 5- dihydroxypentauracil; or 5-hydroxymethyldeoxyuracil; deoxyarchaeosine and 2,6- diaminopurine.

[0316] A stretch or part of a single-strand of DNA may interact with a complementary stretch of RNA or DNA by interaction of complementary nucleobases to form a duplex, wherein cytosine and guanine, and adenine and thymine, and adenine and uracil, and occasionally guanine and uracil, and guanine and thymine are complementary to each other, respectively, by forming two (A / T, G / T in DNA or A / U, G / U in RNA) and three (G / C) hydrogen bonds between the nucleobases. A duplex may be formed by two single-strands of DNA or one single-stranded DNA and one single-stranded RNA that are fully complementary to each other, as in the case of genomic DNA, or by single-strands of DNA that are partially complementary to each other, including situations, where one single-strand of DNA is partially complementary to two or more other single-stranded DNA strands. A duplex may also be formed by two fully or partially self-complementary stretches of one single stranded DNA resulting in the formation of, e.g., a hairpin, a loop, a hybridization stem or higher order motives such as triplex DNA, pseudoknots or kissing hairpins.

[0317] Herein, the term 'RNA substrate' or 'RNA target' or 'target RNA' refers to a ribonucleic acid (RNA) that comprises a sequence able to form hydrogen bonds, preferentially Watson-Crick base pairing, with the binding arm region of the DNAzyme. This includes but is not limited to fully complementary sequences. As known to the art, the RNA substrate should contain a specific dinucleotide pair (purine -pyrimidine junction), including one or more unpaired nucleotides, required for DNAzyme catalysis (Fig. 1).

[0318] Herein, the term "catalytic core region" of DNAzyme refers to the following sequence SEQ. ID NO.

[0319] 1, wherein the number in parenthesis designates the position number, 5'-G (1), G (2), C (3), T (4), A (5), G (6), C (7), T (8), A (9), C (10), A (11), A (12), C (13), G (14), A (15)-3'. It includes the variations of the 10-23 family known to the art as well as all herein mentioned modifications.

[0320] Herein, "nucleotide" refers to a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate group, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar Unser Zeichen: B407-0001W01 17is a "nucleoside". When the nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose, it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a "base sequence" or "nucleotide sequence", and their grammatical equivalents, and is represented herein by a formula of which the left to right orientation is in the conventional direction of 5'-terminus to 3'-terminus, unless otherwise specified.

[0321] " Oligonucleotide or polynucleotide" generally refers to a polymer of single- or double-stranded nucleotides. As used herein, "oligonucleotide" and its grammatical equivalents will include the full range of nucleic acids. An oligonucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art.

[0322] The term "functional fragment" as used herein refers to a fragment of the nucleic acid that retains the functional property of the full-length nucleic acid, for example, the ability of the fragment to act as a DNAzyme for detecting a particular analyte, for example, specific metal ions. In some embodiments, modified nucleotides contain one or more modified bases (e.g. unusual bases such as inosine, and functional modifications to the bases such as amino), modified backbones (e.g. peptide nucleic acid, PNA) and / or other chemically, enzymatically, or metabolically modified forms.

[0323] The term "catalytic nucleic acid", "catalytic DNA", "deoxyribozyme", " DNA enzyme", " Dz" or " DNAzyme" as used herein can refer to a nucleic acid molecule or oligonucleotide sequence that can catalyze or initiate a reaction. DNAzymes can be single-stranded DNA and can include RNA, modified nucleotides and / or nucleotide derivatives. In some embodiments, the DNAzyme is " RNA-cleaving" and catalyzes the cleavage of a particular substrate, for example a nucleic acid sequence comprising one or more ribonucleotides, at a defined cleavage site. In some embodiments, the substrate is a target nucleic acid in a test sample or in vivo. In some embodiments, the DNAzyme cleaves a single ribonucleotide linkage. In some embodiments, the single ribonucleotide linkage is in a nucleic acid sequence wherein the remaining nucleotides are ribonucleotides. In some embodiments, the single ribonucleotide linkage is in a nucleic acid sequence wherein the remaining nucleotides are deoxyribonucleotides. In some embodiments, the DNAzyme cleaves a nucleic acid sequence at a single ribonucleotide linkage thereby producing a nucleic acid cleavage fragment.

[0324] The term "hybridizes", "hybridized" or "hybridization" as used herein refers to the sequence specific non-covalent binding interaction with a complementary, or partially complementary, nucleic acid sequence. When, for example, the 5'-end region of an aptamer hybridizes to the 3' -end region, it can form a duplex DNA element.

[0325] Unser Zeichen: B407-0001W01 18The term "wild-type" or "wt" or " WT" as used herein refers to the original 10-23 DNAzyme sequence comprising a catalytic core sequence according to SEQ. ID NO. 1 (Fig. 19), flanked by two substrate binding arms capable of hybridizing with the targeted RNA (Fig. 1).

[0326] Herein, "internucleotide linkage modification" is selected from the group consisting of a phosphorothioate, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkyl phosphotriester, a methyl phosphonate (Me-Ph), an alkyl phosphonate, a chiral phosphonate, a phosphinate, a phosphoramidate, an aminoalkylphosphoramidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a boranophosphate, a phosphodiester, a phosphonoacetate (PACE) and a peptide nucleic acid (PNA). Herein, "ribose modification" is selected from the group consisting of a 2' -OH (RNA), a 2'-O-Methyl (2'0Me), a 2'-O-Methoxyethyl (2'MOE), a 2'-F, a 2'-fluoroarabinonucleic acid (FANA), a Locked nucleic acid (LNA), and an (S)-constrained ethyl (cEt).

[0327] While many ribose modifications are affecting the 2' position, i.e. the position that distinguishes DNA from RNA, herein the respective modifications are e.g. referred to as 2'OMe-dG for a guanine that has a 2'-O-Methyl modification. Here the 'd' in 'dG' only highlights the DNA origin of the unmodified variant.

[0328] As used herein, the term "base pair" (bp) is generally used to describe a partnership of adenine (A) with thymine (T) or uracil (U), or of cytosine (C) with guanine (G), although it should be appreciated that less-common analogs of the bases A, T, C, and G may occasionally participate in base pairings. Nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration may also be referred to herein as "complementary bases".

[0329] The inventors have found that DNAzyme nucleotides that are in an interface region that is defined by the precatalytic complex structure include, G(1), G(2), G(6), C(13), G(14), and, in the arms: the five nucleotides flanking the core sequence on both sides are preferred for certain aspects of this invention. As used herein, the "interface" of the DNAzyme are these nucleotides designated above. U. S patent No. 6,110, 462 describes DNAzymes that contain modified nucleotides. U. S. patent No.

[0330] 6,673,611 describes deoxyribozymes with novel chemical 35 compositions. Some of these modified catalytic domains may be used to generate variants of the deoxyribozymes of the present invention. In the following the modifications are designated, sometimes with sugar sometimes only as the base. Figure 2 includes an overview of the used nomenclature and abbreviations.

[0331] Unser Zeichen: B407-0001W01 19Herein, an example of etheno-dA may be seen in Fig. 2A.

[0332] Herein, Nebularin is shown in Fig. 2B.

[0333] Herein, ANT is shown in Fig. 2C.

[0334] Herein, 8-Oxo-2'-deoxyguanosine (8-oxo-dG) is shown in Fig. 2D.

[0335] Herein, iso-dG is shown in Fig. 2E.

[0336] Herein, 6-O-methyl-dG is shown in Fig. 2F.

[0337] Herein 7-deaza-dG is shown in Fig. 2G.

[0338] Herein, 7MG is 7-Methylguanosine (m7G) is shown in Fig. 2H.

[0339] Herein, IOX is shown in Fig. 21.

[0340] Herein, 6IP is shown in Fig. 2J.

[0341] Herein, 3DS is shown in Fig. 2K.

[0342] Herein, 2DO is shown in Fig. 2L.

[0343] Herein, 6-cG is shown in Fig. 2M

[0344] Herein, Fig. 2N shows NIP.

[0345] Herein, Fig. 20 shows FIP.

[0346] Herein, Fig. 2P shows 3NA.

[0347] Herein, Fig. 2Q shows 70P

[0348] Herein 6-S-dG is shown in Fig. 2R. The inventors have found that this modification can be made at position 14 and at position 6 of the catalytic region. The modification 6-S-dG is very preferred at position 14 of the catalytic core region. It also shows effects and is claimed herein at position 6 of the catalytic core region.

[0349] Herein, 6-Se-dG is shown in Fig. 2S.

[0350] Herein, 2'-OMe-dG is shown in Fig. 2T.

[0351] Herein, 2'-O-MOE-dG is shown in Fig. 2U.

[0352] Unser Zeichen: B407-0001W01 20Herein, 2-amino-purine, It is shown in Fig. 2V.

[0353] Herein, 2-amino-dA, an adenine analog, which increases thermal stability of an A-T pairing, is shown in Fig. 2W.

[0354] Herein, PYR is shown in Fig. 2X.

[0355] Herein, 3DO is shown in Fig. 2Y.

[0356] Herein, Me-Ph-2'-MOE-6-S-dG is shown in Fig. 2Z and stands as an example of a combination of nucleobase modification 6-S-dG, sugar modification 2'MOE and Phosphate modification Me-Ph. Herein, the methyl-phosphonate modification Me-Ph is shown in Fig. 2AA were R1 can be a canonical nucleobase or modified nucleobases including all mentioned herein.

[0357] Herein, 6aG and its derivatives are shown in Fig. 2AB, where for 6aG R2=R3=H, and for derivatives of 6aG R2 and R3 can be either H, Me, Et or other organic side chains.

[0358] Herein, 2'MOE-6-S-dG is shown in Fig. 51C.

[0359] Herein, 2'0Me-6-S-dG is shown in Fig. 51D.

[0360] Herein, PSO-2'OMe-6-S-dG is shown in Fig. 51E.

[0361] Herein, the "flanking variable target recognition sequence" may also be referred to as the "flanking binding arm" or as used in Fig. 1 as "substrate binding arm".

[0362] DETAILED DESCRIPTION OF THE INVENTION

[0363] The invention relates to a nucleic acid polymer encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence SEQ ID NO. 1, wherein the number in parenthesis designates the position number,

[0364] 5'-G (1), G (2), C (3), T (4), A (5), G (6), C (7), T (8), A (9), C (10),

[0365] A (11), A (12), C (13), G (14), A (15)-3' (SEQ ID NO. 1),

[0366] and wherein said core region nucleic acid sequence comprises one or more of the following modifications, at position 5,

[0367] Unser Zeichen: B407-0001W01 21Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'

[0368] SEQ ID

[0369] 5' G G C T A G C T A C A A C G A 3' pos. / Modification NO.

[0370] 1

[0371] SEQ ID

[0372] 5' G G C T N* G C T A C A A C G A 3' A*(5)=etheno-dA NO.

[0373] 2

[0374] SEQ ID

[0375] 5' G G C T N* G C T A C A A C G A 3' N* (5)=dNebularine NO.

[0376] 3

[0377] SEQ ID

[0378] 5' G G C T N* G C T A C A A C G A N*(5)=ANT NO.

[0379]

[0380] 4

[0381] SEQ ID

[0382] 5' G G C T N* G C T A C A A C G A A*(5)=Me-Ph NO.

[0383]

[0384] 5

[0385] at position 6,

[0386] Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'

[0387] SEQ ID G G C T A G C T A C A A C G A 3' Pos. / Modification NO.

[0388] 1

[0389] SEQ ID

[0390] 5' G G C T A N* C T A C A A C G A 3' G*(6)=6-S-dG NO.

[0391] 6

[0392] SEQ ID

[0393] 5' G G C T A N* C T A C A A C G A 3' G*(6)=6-Se-dG NO.

[0394] 7

[0395] SEQ ID

[0396] 5' G G C T A N* C T A C A A C G A 3' G*(6)=8-oxo-dG NO.

[0397] 8

[0398] SEQ ID

[0399] 5' G G C T A N* C T A C A A C G A 3' G*(6)=Iso-dG NO.

[0400] 9

[0401] Unser Zeichen: B407-0001W01 22SEQI DG*(6)=6-O-methyl- 5' G G C T A N* C T A C A A C G A 3' NO. dG 10 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=7-deaza-dG NO.

[0402] 11 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2'-OMe-dG NO.

[0403] 12 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2'-MOE-dG NO.

[0404]

[0405] 13 SEQ ID

[0406] 5' G G C T A N* C T A C A A C G A 3' G*(6)=etheno-dA NO.

[0407] 14

[0408] SEQ ID

[0409] 5' G G C T A N* C T A C A A C G A 3' G*(6)=ANT NO.

[0410] 15

[0411] SEQ ID G*(6)=2-amino- 5' G G C T A N* C T A C A A C G A 3' NO. purine 16 SEQ JO 5' G G C T A N* C T A C A A C G A 3' G*(6)=2-amino-dA NO.

[0412] 17 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=dNebularine NO.

[0413] 18 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=7MG NO.

[0414] 19 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=IOX NO.

[0415] 20 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=6IP NO.

[0416]

[0417] 21

[0418] Unser Zeichen: B407-0001W01 23SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=3DS NO.

[0419] 22 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2DO NO.

[0420] 23 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=PYR NO.

[0421] 24 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=NIP NO.

[0422]

[0423] 25 SEQ ID

[0424] 5' G G C T A N* C T A C A A C G A 3' G*(6)=FIP NO.

[0425] 26

[0426] SEQ ID

[0427] 5' G G C T A N* C T A C A A C G A 3' G*(6)=3NA NO.

[0428] 27

[0429] SEQ ID

[0430] 5' G G C T A N* C T A C A A C G A 3' G*(6)=7OP NO.

[0431] 28

[0432] SEQ

[0433] O 5' G G C T A N* C T A C A A C G A 3' G*(6)=3DO NO.

[0434] 29

[0435] SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2DS NO.

[0436] 30 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=B0G NO.

[0437] 31 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2TA NO.

[0438] 32 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=P0P NO.

[0439]

[0440] 33

[0441] Unser Zeichen: B407-0001W01 24SEQ

[0442] ID G T A N* C T A C A C G A 3' G*(6)=NTZ NO.

[0443]

[0444] 34

[0445] SEQ ID

[0446] 5

[0447]

[0448] ' G G C T A N* C T A A C A 3' G*(6)=MES NO.

[0449] 35

[0450] SEQ ID

[0451]

[0452] G G T A N* C T A A C G A 3' G*(6)=TTZ NO.

[0453] 36

[0454] SEQ ID

[0455]

[0456] G G C T A N* C T A A C A 3' G*(6)=MER NO.

[0457] 37

[0458] SEQ ID G T A N* C T A A C G A 3' G*(6)=M2R NO.

[0459]

[0460] 38

[0461] SEQ ID

[0462]

[0463] G G C T A N* C T A C A C A 3' G*(6)=M2S NO.

[0464] 39

[0465] SEQ ID

[0466]

[0467] G G T N* C T A C A C A 3' G*(6)=BTG NO.

[0468] 40

[0469] SEQ ID

[0470]

[0471] G G C T A N* C T A A C A 3' G*(6)=6TP NO.

[0472] 41

[0473] SEQ ID

[0474]

[0475] G G T A N* C T A A C A 3' G*(6)=PHY NO.

[0476] 42

[0477] SEQ ID G G C T A N* C T A C A C A 3' G*(6)=3TA NO.

[0478]

[0479] 43

[0480] SEQ ID

[0481]

[0482] G T N* C T A A C A 3' G*(6)=MTG NO.

[0483] 44

[0484] SEQ ID

[0485]

[0486] G G C T A N* C T A A C A 3' G*(6)=AIP NO.

[0487] 45

[0488] Unser Zeichen: B407-0001W01 25SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=D0P NO.

[0489] 46 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=AEG NO.

[0490] 47 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=AMG NO.

[0491] 48 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=IOX2 NO.

[0492]

[0493] 49 SEQ ID

[0494] 5' G G C T A N* C T A C A A C G A 3' G*(6)=Me-Ph NO.

[0495] 50

[0496] SEQ ID

[0497] 5' G G C T A N* C T A C A A C G A 3' G*(6)=6aG NO.

[0498] 51

[0499] SEQ ID

[0500] 5' G G C T A N* C T A C A A C G A 3' N*(6)=6-cG NO.

[0501] 52

[0502] SEQ

[0503] O 5' G G C T A N* C T A C A A C G A 3' N*(6)=7MP NO.

[0504] 53

[0505] at position 14,

[0506] Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3' SEQ ID

[0507] 5' G G C T A G C T A C A A C G A 3' Pos. / Modification NO.

[0508] 1 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=6-S-dG NO.

[0509] 54 SEQ ID

[0510] 5' G G C T A G C T A C A A C N* A 3' G*(14)=6-Se-dG NO.

[0511]

[0512] 55SEQ5' G G C T A G C T A C A A C N* A 3' G*(14)=8-oxo-dG ID

[0513] Unser Zeichen: B407-0001W01 26NO.

[0514] 56 SEQ ID

[0515] 5' G G C T A G C T A C A A C N* A 3' G*(14)=Iso-dG NO.

[0516] 57 SEQ

[0517] G*(14)=6-O-methyl- JO 5' G G C T A G C T A C A A C N* NO. dG 58 S

[0518]

[0519] EQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=7-deaza-dG NO.

[0520] 59

[0521] SEQ ID

[0522] 5' G G C T A G C T A C A A C N* A 3' G*(14)=etheno-dA NO.

[0523] 60

[0524] SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=ANT NO.

[0525] 61

[0526] SEQ ID

[0527] 5' G G C T A G C T A C A A C N* A 3' G*(14)=2-amino-dA NO.

[0528] 62

[0529] SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=dNebularine NO.

[0530] 63

[0531] SEQ ID

[0532] 5' G G C T A G C T A C A A C N* A 3' G*(14)=7MG NO.

[0533] 64

[0534] SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=IOX NO.

[0535] 65

[0536] SEQ ID

[0537] 5' G G C T A G C T A C A A C N* A 3' G*(14)=6IP NO.

[0538] 66

[0539] SEQ

[0540] O 5' G G C T A G C T A C A A C N* A 3' G*(14)=3DS NO.

[0541] 67

[0542] SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=2DO NO.

[0543] 68

[0544] SEQ ID

[0545] 5' G G C T A G C T A C A A C N* A 3' G*(14)=PYR NO.

[0546] 69

[0547] S

[0548] |E

[0549] DQ

[0550] 5' G G C T A G C T A C A A C N* A 3' G*(14)=NIP Unser Zeichen: B407-0001W01 T1NO.

[0551] 70 SEQ ID

[0552] 5' G G C T A G C T A C A A C N* A 3' G*(14)=FIP NO.

[0553] 71 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=3NA NO.

[0554] 72 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=7OP NO.

[0555] 73 SEQ ID

[0556] 5' G G C T A G C T A C A A C N* A 3' G*(14)=3DO NO.

[0557] 74 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=2DS NO.

[0558] 75 SEQ ID

[0559] 5' G G C T A G C T A C A A C N* A 3' G*(14)=B0G NO.

[0560] 76 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=2TA NO.

[0561] 77 SEQ ID

[0562] 5' G G C T A G C T A C A A C N* A 3' G*(14)=P0P NO.

[0563] 78 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=NTZ NO.

[0564] 79 SEQ ID

[0565] 5' G G C T A G C T A C A A C N* A 3' G*(14)=MES NO.

[0566] 80 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=TTZ NO.

[0567] 81 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=MER NO.

[0568] 82 SEQ ID

[0569] 5' G G C T A G C T A C A A C N* A 3' G*(14)=M2R NO.

[0570] 83 S

[0571] |EDQ

[0572] 5' G G C T A G C T A C A A C N* A 3' G*(14)=M2S

[0573]

[0574] Unser Zeichen: B407-0001W01 28NO.

[0575] 84 SEQ ID

[0576] 5' G G C T A G C T A C A A C N* A 3' G*(14)=BTG NO.

[0577] 85 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=6TP NO.

[0578] 86 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=PHY NO.

[0579] 87 SEQ ID

[0580] 5' G G C T A G C T A C A A C N* A 3' G*(14)=3TA NO.

[0581] 88 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=MTG NO.

[0582] 89 SEQ ID

[0583] 5' G G C T A G C T A C A A C N* A 3' G*(14)=AIP NO.

[0584] 90 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=D0P NO.

[0585] 91 SEQ ID

[0586] 5' G G C T A G C T A C A A C N* A 3' G*(14)=AEG NO.

[0587] 92 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=AMG NO.

[0588] 93 SEQ ID

[0589] 5' G G C T A G C T A C A A C N* A 3' G*(14)=IOX2 NO.

[0590] 94 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=Me-Ph NO.

[0591] 95 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=6aG NO.

[0592] 96 SEQ ID G*(14)=Me-Ph-6-S- 5' G G C T A G C T A C A A C N* NO. dG 97 SEQ G*(14)=Me-Ph-6-

[0593]

[0594] |D5' G G C T A G C T A C A A C N*

[0595] Se-dG Unser Zeichen: B407-0001W01 29NO.

[0596] 98

[0597] SEQ ID

[0598] 5' G G C T A G C T A C A A C N* A 3' G*(14)=PSO-6-S-dG NO.

[0599] 99

[0600] SEQ

[0601] G*(14)=PSO-6-Se- O 5' G G C T A G C T A C A A C N* A 3'

[0602] NO. dG 100

[0603] SEQI DG*(14)=2'-MOE-6-S- 5' G G C T A G C T A C A A C N* A 3' NO. dG 101 SEQ ID G*(14)=2'-MOE-6- 5' G G C T A G C T A C A A C N* A 3' NO. Se-dG 102 SEQI DG*(14)=Me-Ph-2'- 5' G G C T A G C T A C A A C N* A 3'

[0604]

[0605] NO. MOE-6-S-dG 103

[0606] SEQ ID G*(14)=Me-Ph-2'- 5' G G C T A G C T A C A A C N* A 3'

[0607] NO. MOE-6-Se-dG 104

[0608] SEQI D5' G G C T A G C T A C A A C N* A 3' N*(14)=6-cG NO.

[0609] 105

[0610] SEQ ID

[0611] 5' G G C T A G C T A C A A C N* A 3' N*(14)=7MP NO.

[0612] 106

[0613] Additional modifications at position 14 include:

[0614] Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3' SEQI D5' G G C T A G C T A C A A C G A 31Pos. / Modification NO.

[0615] 1 SEQ ID

[0616] 5' G G C T A G C T A C A A C N* A 31 G*(14)=2'OMe-6-S-

[0617]

[0618] NO. dG 304

[0619] SEQ

[0620] O 5' G G C T A G C T A C A A CN, »,, G*(14)=PSO-2'OMe- NO. 6-S-dG 305

[0621] Unser Zeichen: B407-0001W01 30According to the invention each of the listed modifications can either stand on its own or be combined with an internucleotide linkage modification and / or ribose modification at the same nucleotide. This includes but is not limited to, for example at positionl4.

[0622] Me-Ph-6-S-dG (SEQ ID NO. 97 and 109),

[0623] Me-Ph-6-Se-dG (SEQ ID NO. 98 and 110),

[0624] PSO-6-S-dG (SEQ ID NO. 99),

[0625] PSO-6-Se-dG (SEQ ID NO. 100),

[0626] 2'MOE-6-S-dG (SEQ ID NO. 101 and 111),

[0627] 2'MOE-6-Se-dG (SEQ ID NO. 102 and 112),

[0628] Me-Ph-2'-MOE-6-S-dG (SEQ ID NO. 103),

[0629] Me-Ph-2'-MOE-6-Se-dG (SEQ ID NO. 104),

[0630] Additional examples for position 14 include:

[0631] 2'0Me-6S-dG (SEQ ID NO. 304),

[0632] PSO-2'OMe-6S-dG (SEQ ID NO. 305),

[0633] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence SEQ ID NO. 1, wherein the number in parenthesis designates the position number, 5'-G (1), G (2), C (3), T (4), A (5), G (6), C (7), T (8), A (9), C (10), A (11), A (12), C (13), G (14), A (15)-3' (SEQ ID NO. 1), and wherein said core region nucleic acid sequence and / or the binding arm sequence comprises one or more of the following modifications:

[0634] i) a modification that increases activity,

[0635] ii) a modification that reduces or abolishes activity,

[0636] iii) a modification that influences target association and / or dissociation resulting in a changed, reduced or improved catalytic turnover, iv) a modification that increases target RNA selectivity,

[0637] v) a modification that reduces target RNA selectivity,

[0638] vi) a modification that reduces the antisense effect via reduced RNase recruitment to increase the DNAzyme precision in (cellular) applications, Unser Zeichen: B407-0001WQ1 31vii) a modification that promotes the antisense effect via enhanced RNase recruitment to increase the overall effect of DNAzyme treatment in (cellular) applications

[0639] viii) a modification that increases affinity to, or reduces the need for, metal ions, including but not limited to Mg2+,

[0640] ix) a modification that increases cellular life-time,

[0641] x) a modification that decreases innate immune response, xi) a modification that increases innate immune response.

[0642] Herein, where functional language is used to define a modification, the terms are to be understood as follows. A 'modification that increases activity' refers to a modification that results in a statistically significant increase in the observed rate constant (kobs) or a statistically significant decrease in the time required to reach 90% maximal cleavage (t90), as measured by the FRET assay described herein, when compared to an otherwise otherwise identical but unmodified DNAzyme. 'Increased target RNA selectivity' refers to an increase in the ratio of the cleavage rate for the desired RNA target to the cleavage rate of an undesired but similar RNA, such as e.g. required in allele-specific gene silencing of single-nucleotide mutations. 'Increased cellular life-time' refers to the prolonged duration of a measurable biological effect, such as the reduction of a target RNA or viral titer in a cellular assay as described herein, beyond that observed for an unmodified DNAzyme.

[0643] The invention relates to a nucleic acid encoding at least one flanking variable target recognition sequence, wherein the target recognition sequence is between 5 and 30 nucleotides in length, and said target recognition sequence comprises one or more of the following modifications,

[0644] i. a modification that reduces or abolishes activity,

[0645] ii. a modification that influences target association and / or dissociation resulting in a changed, reduced or improved catalytic turnover,

[0646] ill. a modification that increases target RNA selectivity,

[0647] iv. a modification that reduces target RNA selectivity,

[0648] v. a modification that reduces the antisense effect via reduced RNase recruitment to increase the DNAzyme precision in (cellular) applications,

[0649] Unser Zeichen: B407-0001W01 32vi. a modification that promotes the antisense effect via enhanced RNase recruitment to increase the overall effect of DNAzyme treatment in (cellular) applications

[0650] vii. a modification that increases affinity to, or reduces the need for, metal ions, including but not limited to Mg2+,

[0651] viii. a modification that increases cellular life-time,

[0652] ix. a modification that decreases innate immune response,

[0653] x. a modification that increases innate immune response.

[0654] The inventors identified the nucleotide positions in the arms (flanking regions) that are essential for activity, e.g. positions where mismatches to the target RNA strongly reduce activity (Fig. 11). The respective positions are referred to as the 'selectivity hot spot region' or 'selectivity filter' (Fig.

[0655] 11), and cover nucleotides in the DNAzyme arm from position -6 to +3, excluding the known requirements for the two nucleotides of the cleavage side (position -1 and 0). In a more preferred embodiment, the positions responsible for selectivity are -5 to +2, in an even more preferred embodiment positions -3, -2, 1, and 2. The inventors further found that targeting specific mismatches such as the stronger G-C pairings or mismatches that will lead to different structural features of the DNAzymes may extend the selectivity filer, in particular involving also the +4 and +5 position. Using this knowledge, it can be beneficial to increase or decrease the target association via chemical modifications in the selectivity filter. Using chemical modifications or specifically placing mismatches the selectivity of the DNAzyme can be modulated. One skilled in the art may recognize that chemical modification that enhance DNA-RNA hybridization may enhance target specificity and reduce off-target effects and that predominantly the nucleotides in the selectivity filter determine the DNAzyme's ability to targeting single-point mutations. 'Targeted mismatches' can for example occur in allele-specific single-point mutations that are associated with occurrence of two different target RNA variants, one carrying a single-point mutation and one without this mutation. In these cases, single-nucleotide selectivity is often desirable to reduce the RNA variant without a mismatch and not the variant containing the mismatch, or vice versa. In this regard placing the respective chemical modifications, including but not limited to the above-described ribose modifications and internucleotide linkage modifications, to the nucleotides in the selectivity filter will have strongly increasing or decreasing effects on selectivity that surpass their thermodynamic benefits (which are e.g. explored in antisense oligo nucleotide developments).

[0656] Unser Zeichen: B407-0001W01 33Note that when targeting single-point mutations the specific requirements for the dinucleotide pair of the cleavage site, so far, offer the highest selectivity (i.e. when the targeted single-nucleotide mutation is part of the cleavage site and the mutation results in a compatible cleavage site while the wild-type sequence does not (or vice versa)). However, this introduces strong restrictions to the accessible point mutations, leaving the largest part of therapeutically relevant single-point mutations inaccessible. In comparison to the dinucleotide pair of the cleavage site, the selectivity filter covers a much larger sequence space. Thus, targeting single-point mutations within the selectivity filter, preferably with the above-described chemical modifications suitable to further enhance DNA-RNA hybridization, greatly expands the applicability of the DNAzymes single-point mutation selectivity and is claimed in this invention.

[0657] In addition, the inventors have found and claim that also point mutations in the RNA target that are outside of the identified selectivity filter can be effectively targeted by using well-selected "auxiliary-mismatches". Auxiliary mismatches can be included in the DNAzyme design at positions that have no or little effect on cleavage activity of the targeted RNA. Suitable positions for these mismatches are referred to as auxiliary selectivity filter (Fig. 13). In contrast to the above described 'targeted mismatch', auxiliary mismatches will be present for all target RNA variants. While the auxiliary mismatches have no gain on selectivity on their own, their incorporation can boost selectivity of single-nucleotide mismatches at another position, i.e. the targeted mismatch. Here the targeted mismatch can be inside the selectivity filter but is preferentially outside of the selectivity filter. The inventors show that a single nucleotide mismatch outside of the selectivity filter, e.g., at position +7, cannot be selectively targeted by a DNAzyme with the respective compensating single nucleotide alteration at the +7 position (Fig. 14A). Similar observations are made for individual single-nucleotide mismatch at other positions outside of the selectivity filter, in particular when no stabilizing modifications are used. However, when designing DNAzymes that contain, in addition to the targeted mismatch (e.g. at position +7), an additional mismatch, i.e. an auxiliary mismatch at the edge or outside of the selectivity filter (e.g. at position +3), the desired single nucleotide selectivity can be effectively extended to the nucleotides outside of the selectivity filter (Fig. 14B). Note that, according to the provided data, the individual mismatches (position +3 or position +7) have no or little effect on the activity as compared to the fully matching sequence (Fig. 14A, B) and as such can both serve as auxiliary mismatch to target the other position, respectively. The inventors thus found new means to extend the single-nucleotide selectivity to all nucleotides of the DNAzymes arms and in one embodiment of this invention a DNAzyme design associated with increased target selectivity is claimed that comprises two or more mismatches,

[0658] Unser Zeichen: B407-0001W01 34where at least one mismatch is an auxiliary mismatch and at least one mismatch is the targeted mismatch.

[0659] In some applications reduced selectivity may also be beneficial to e.g. escape cancer or virus resistance strategies. Chemical modifications in the selectivity filter, such as phosphorothioate (PSO), or mismatch analogues to the auxiliary mismatches described above may be used to specifically decrease selectivity and e.g. increase tolerance for escape mutations.

[0660] One aspect that can generally reduce selectivity of DNAzyme cellular applications is the less selective antisense effect induced by the DNAzyme arms. The origin of the antisense effect is the hybridization of the DNAzyme arms with the target (or off-target) RNA. In many applications the formed DNA-RNA duplex will recruit RNase Hl, which then will lead to RNase Hl-mediated RNA cleavage. While correctly designed DNAzymes will not cleave an RNA that for example includes a single mismatch as described above (Figs. 11 and 12A), the DNAzymes often can still bind to the RNA and hence can induce RNase Hl-mediated cleavage (Fig. 12B, Fig 15A). The DNAzymes thus may have a single-nucleotide selectivity, however, in the presence of RNases this may not be fully exploited, since the RNase-mediated cleavage does not have this selectivity (Fig. 12B). Consequently, in many applications, such as RNA down regulation in a cellular context, the effective precision of the DNAzyme technology is not only determined by its inherent selectivity but may be limited by less selective RNase processes, often referred to as antisense effects. The inventors have identified ways to reduce this antisense effect via chemical and sequence modifications of the Dz arms enabling target-specific cleavage with ultimate precision even in the presence of RNases. Sequence modifications include nucleotide overhangs, such as dT overhangs often used in siRNA design, and specific mismatches, similar to the auxiliary mismatches described above, which weaken DNA-RNA interaction and thus reduce RNA recruitment. In one embodiment of the invention the same auxiliary mismatch is used to boost single-point selectivity and, simultaneously, reduce RNase recruitment, translating the increased selectivity to a realized precision in various (cellular) applications. Thus 'increased DNAzyme precision' refers to the realized increased targeted RNA selectivity in the presence of RNases. Chemical modifications to reduce antisense effects include but are not limited to the addition of steric blockers with the aim to sterically inhibit RNase binding, or a DNAzyme design affecting the RNase recognition motive which is based on formation of an A-form RNA-DNA double helix arrangements.

[0661] The inventors show and claim herein, that RNase recruitment can be inhibited by steric inhibition or modulation of double helix arrangements by including the following modifications or substitutions in one or more positions in the binding arm region:

[0662] Unser Zeichen: B407-0001W01 351. PEG spacer in various lengths or composition,

[0663] 2. hexaethylene glycol spacer in various lengths or composition,

[0664] 3. digoxigenin with or without linker,

[0665] 4. biotin with or without linker,

[0666] 5. fluorophore-spacer conjugates such as JOE-C12 spacer,

[0667] 6. Propynyl-dC as framework for the addition of suitable steric blocker via click chemistry, 7. Azid-dT on its own and as framework for the addition of suitable steric blocker via click chemistry,

[0668] 8. Dibenzocyclooctynes (DBCO)-dT on its own or in combination with suitable azid modification at a different position to establish an RNase-inhibiting inter-nucleotide bridge, 9. dSpacer (a basic site),

[0669] 10. RNA,

[0670] 11. LNA,

[0671] 12. 2'0Me,

[0672] 13. 2'MOE,

[0673] 14. FANA,

[0674] 15. auxiliary mismatches,

[0675] 16. overhang of unpaired nucleotides on one or both ends of the DNAzyme arms.

[0676] The overhang of unpaired nucleotides preferentially comprises 1 to 3 nucleotides that extend the substrate binding arms at the 3' and / or 5' end and do not have a base pairing partner in the target RNA.

[0677] One skilled in the art will recognize several suitable ways to include the steric blockers (1) - (5) in the above list.

[0678] In one embodiment the DNAzyme: RNA double helix arrangement is modulated by a single or repetitive pattern of two subsequent nucleotides in the arms interrupted by a series of 1-5 not or differently modified DNA positions. In another embodiment the DNAzyme: RNA double helix arrangement is modulated by a single or repetitive pattern of three subsequent nucleotides in the DNAzyme arms interrupted by a series of 1-5 not or differently modified DNA positions. In the preferred embodiment the DNAzyme: RNA double helix arrangement is modulated by a single or repetitive pattern of four subsequent nucleotides in the arms interrupted by a series of 1-5 not or differently modified DNA positions. In another embodiment the DNAzyme: RNA double helix

[0679] Unser Zeichen: B407-0001W01 36arrangement is modulated by a single or repetitive pattern of five subsequent nucleotides in the arms followed by a series of 1-5 not or differently modified DNA positions.

[0680] In another embodiment the DNAzyme comprises chemical modifications in the 5' substrate-binding arm and 3' substrate-binding arm systematically arranged to evade RNase Hl recruitment. This is characterized in that the DNAzyme comprises:

[0681] a. an " REP-1" modification pattern, wherein the modified nucleotides on the 5' arm are paired in two groups (positions -3 and -4, and positions -7 and -8 relative to the 5' -start of the catalytic core), and the 3' arm comprises modifications at the absolute 3'-terminal nucleotide and at the relative position located four nucleotides upstream of said 3'- terminal nucleotide (e.g., position +5 in a 9-nucleotide arm);

[0682] b. an " REP-2" modification pattern, comprising modifications on the 5' arm at positions -5, -6, and -2, with the 3' arm modified identically to REP-1; or

[0683] c. a hybrid modification pattern incorporating the architecture of REP-1 or REP-2 in conjunction with supplementary modifications in the substrate binding arms or the catalytic loop.

[0684] Modification in the arms can also increase target association and / or dissociation making catalytic turnover faster. One skilled in the art may recognize that for the design of the above-described modifications and pattern of modifications potential effects on the hybridization kinetics need to be considered and accompanied by e.g. shortening of the arm sequences or incorporation of PSO internucleotide linkage modifications.

[0685] In one aspect of the invention the modification or the modifications are selected to increase affinity or reduce the need for divalent metal ions, in particular Mg2+and Ca2+. In accordance with the present invention therefore it is preferred if position A(5) and / or G(6) are modified in order to achieve structural activation. In particular position 5 is identified to be important for Mg2+binding, which in turn can bring the catalytic core into an active conformation. For example, the inventors have found that according to the invention it is preferable for increased activity if Mg2+affinity enhancement is achieved. In this context, the higher Mg2+affinity associated with an adenine at position 5, will be reduced by a A5C mutation and abolished beyond detection in a A5G mutation. Position 6 is important since the inventors have shown that G(6) acts as Mg2+induced structural switch that is associated with the DNAzymes activation. The inventors show that specific chemical

[0686] Unser Zeichen: B407-0001W01 37modifications at position 6, including new nucleotide designs, can be used to either facilitate or inhibit DNAzyme activation.

[0687] In addition, position G(14) could be identified as central nucleotide in the catalysis reaction. Positioning of the guanine's functional groups and / or modulating its metal-ion interaction can promote or inhibit the cleavage reaction. The inventors show that the central role of G(14) is ideally suited to e.g., increase catalytic activity via specific chemical modifications.

[0688] Furthermore, the inventors found a crosstalk between position 6 and position 14, demonstrating that variants comprising modifications at both positions (position 6 and position 14) provide additional benefits such as reduced Mg2+dependency and / or increased stabilization of the inlineattack conformation, required for substrate cleavage.

[0689] The inventors have previously demonstrated that in a DNAzyme variant containing the A5C mutation, the cleavage activity can be increased by a 6-S-dG modification at position G(14). The increased activity was assumed to be associated with a reduction of Mg2+interaction in an inactive conformation of dG(14). Furthermore, a structural link between C(5) and G(14) was observed, rendering modifications at position 5 and 14 not independent. Consequently, it was unclear whether increasing Mg2+affinity at position 5 will be associated with similar activity increasing effects of the 6-S-dG modification. The inventors demonstrate and claim here that a 6-S-dG modification at position 14 in combination with an adenine at position 5, has a further increased activity as compared to the regular A(5) variant, as well as compared to the previously reported double modified (C(5), 6-S-dG(14)) variant.

[0690] Noteworthy, other activity-increasing modifications at position G(14), such as 2'MOE-dG14, have been found to be not transferable between DNAzyme variants that contain in the catalytic core sequence an adenine at position 5, i.e. a (A(5), 2'MOE-dG14) Dz variant or a cytosine at position 5, i.e. a (C(5), 2'MOE-dG14) Dz variant. In this regard, in vitro FRET activity measurements reveal an activity-lowering effect of the 2'MOE-dG14 modification as compared to unmodified dG14 when combined with a cytosine at position 5, e.g. reducing kObs more than 3-fold (Fig. 55). This further supports the non-trivial cross talk between position 5 and 14, highlighting the novelty of the herein first described (A(5), 6-S-dG(14)) Dz variant.

[0691] The catalytic enhancement conferred by the 6-Se-dG modification at position 14 is grounded in the isosteric and electronic relationship between sulfur and selenium chalcogens. Due to their position in group 16 of the periodic table, selenium shares and even surpasses the high electron polarizability and 'soft base' characteristics of sulfur, while exhibiting a slightly larger atomic radius. Unser Zeichen: B407-0001W01 38The substitution of the 06 oxygen with a selenium atom significantly alters the local hydrogen-bonding network and reduces the energetic penalty associated with adopting the catalytically active rotameric state. The necessity of specific electronic distributions is supported by Density Functional Theory (DFT) calculations of dipole moments across various heterocycles (as documented in Fig. 22). Dedicated Molecular Dynamics (MD) simulations demonstrate that, comparable to the 6-S-dG(14) variant, the 6-Se-dG modification at position 14 highly stabilizes the pre-catalytic transition state geometry, as evidenced by increased in-line attack (ILA) formation (Fig.

[0692] 55). Specifically, the enhanced polarizability of the selenocarbonyl group restricts the conformational flexibility of the guanine base, locking the catalytic loop into an optimized orientation that facilitates the strictly required in-line attack geometry for RNA phosphodiester bond cleavage. Consequently, the 6-Se-dG(14) modification exerts a robust beneficial effect on catalytic activity equivalent to, or exceeding, that of the 6-S-dG(14) modification, validating its utility in highly active DNAzyme constructs.

[0693] In line with the above-described beneficial effects of 6-Se-dG, experimental FRET-based activity measurements confirm the activity enhancing effect of the 6-Se-dG modification at position 14 of the catalytic core (Fig. 49, SEQ. ID NO. 55).

[0694] Cellular Life-time

[0695] The inventors have identified modifications that increase cellular life-times. One skilled in the art will appreciate that a variety of modifications have been developed to increase cellular life-times of antisense oligonucleotides and that stabilizing effects of modifications have been explored for DNAzymes predominantly using (cell-free) nuclease degradation assays. While these assays report on nuclease resistance of the DNAzyme molecule, a direct connection to cellular life-times and in particular to the time-dependent capability of substrate cleavage are not well captured. In contrast, here the inventors provide a direct and unique link between the time the DNAzyme resides in a cellular context and its activity, e.g. determining modification-associated prolonged effects in a cellular context (Fig. 5). In one aspect of the invention, it is shown that a single 2'0Me modification at the 5' and 3' ends considerably extents the time of the observable target downregulation from 24 h to 72 h (Fig. 5D-I). This effect is not observed for LNA modification at these positions (Fig. 6).

[0696] In another highly preferred aspect of the invention, it is shown that a stabilizing architecture, termed the ELMO (Enhanced Locked-nucleic-acid and Methylated-2-'0xygen) pattern, comprising single LNA modifications flanking two inward-facing 2'0Me modifications at both the 5' and 3' Unser Zeichen: B407-0001W01 39extreme ends (i.e., 5'-LNA, 2'0Me, 2'0Me... 2'0Me, 2'0Me, LNA-3') considerably extends the antiviral efficacy in vivo (Fig. 44). Furthermore, this terminal stabilization pattern and comprising an unpaired nucleotide overhang on at least one LNA, and is structurally compatible with the effective RNase Hl evasion patterns (REP-1 and REP-2) detailed herein (Fig. 52).

[0697] The specific restriction to exactly two inward-facing 2'0Me modifications (the 2xO'Me ELMO pattern), as opposed to extended tracts of three or more 2'0Me modifications known in the art, provides an unexpected synergistic technical effect. Extended tracts of 2'0Me modifications overly stabilize the DNA: RNA heteroduplex, increasing the melting temperature (Tm) to a degree that restricts the dissociation of the cleaved RNA products. This thermodynamic trap leads to severe product inhibition and a consequently diminished catalytic turnover rate. By contrast, the 2xO'Me ELMO pattern optimizes the thermodynamic profile of the binding interface, permitting the use of elongated substrate-binding arms. These extended arms drastically increase target substrate specificity and reduce off-target binding, while the restricted 2xO'Me density ensures that the binding affinity remains within an optimal window to allow rapid product release and robust multiple-turnover enzymatic kinetics.

[0698] In some embodiments, in particular when combined with additional 2'0Me modifications such as present in the REP-1 or REP-2 design, it may also be thermodynamically beneficial to further reduce the used number of 2'0Me modifications in the ELMO pattern, for example going from the in total four 2'0Me modifications (i.e. 5'-LNA,2'OMe,2'OMe,...,2'OMe,2'OMe, LNA-3') to two 2'0Me modifications (i.e. 5'-LNA,2'OMe,...,2'OMe, LNA-3') or three 2'0Me modifications (i.e. either 5'-LNA,2'OMe,2'OMe,...,2'OMe, LNA-3', or 5'-LNA,2'OMe,...,2'OMe,2'OMe, LNA-3'), thus as used herein for SEQ ID NOs: 325 - 340.

[0699] Similarly to the excessive usage of 2'0Me modifications, the incorporation of LNA-modified nucleotides into the DNA-binding arms can overly stabilise the DNA: RNA heteroduplex. Thus, the ELMO pattern is designed with at least one of the LNA modifications, situated at either the absolute 3'-terminal nucleotide or the absolute 5'-terminal nucleotide, forming an overhang by constituting a mismatch to the respective RNA target sequence.

[0700] Moreover, the incorporation of an unpaired nucleotide overhang extending beyond the fully matching sequence of the target RNA introduces a structural distortion at the terminus of the heteroduplex. This overhang creates steric hindrance that further inhibits the recruitment and stable binding of endogenous nucleases, particularly RNase Hl, thereby operating synergistically

[0701] Unser Zeichen: B407-0001W01 40with the REP-1 and REP-2 architectures to maximize the intracellular lifetime and precision of the DNAzyme.

[0702] In another aspect of the invention the following modification are used, at one or more positions either individually or in combination, to prolong cellular-life times:

[0703] i) 2'M0e,

[0704] ii) 2'OME,

[0705] iii) FANA,

[0706] iv) LNA,

[0707] v) inverted nucleotides,

[0708] vi) L-DNA,

[0709] vii) PSO,

[0710] viii) phosphorodithioate,

[0711] ix) Me-Ph, and

[0712] x) PNA.

[0713] The inventors found that unmodified DNAzymes have a moderate effect on the innate immune response. Furthermore, the inventors found and claim the following ribose modifications and internucleotide linkage modifications to modulate the immune response when incorporated at one or more positions either individually or in combination in the RNA binding arms:

[0714] i) PSO,

[0715] ii) 2'M0e,

[0716] iii) 2'OME, and

[0717] iv) LNA.

[0718] In one embodiment of the invention the following combinations of modification within one DNAzyme have been found to be particularly beneficial to realize one or more desirable improvements:

[0719] 2'0Me(5'end),...6-S-dG(14),...2'0Me (3'end) (SEQ ID NO. 107),

[0720] 2'0Me(5'end),...6-Se-dG(14),...2'0Me (3'end) (SEQ ID NO. 108),

[0721] 2'0Me(5'end),... Me-Ph-6-S-dG(14),...2'0Me (3'end) (SEQ ID NO. 109),

[0722] Unser Zeichen: B407-0001W01 412'0Me(5'end),... Me-Ph-6-Se-dG(14),...2'0Me (3'end) (SEQ ID NO. 110), 2'0Me(5'end),...2'M0E-6-S-dG(14),...2'0Me (3'end) (SEQ ID NO. 111), and 2'0Me(5'end),...2'M0E-6-Se-dG(14),...2'OMe (3'end) (SEQ ID NO. 112).

[0723] Furthermore, the inventors have established that the cellular lifetime-enhancing ELMO pattern (comprising terminal LNAs and adjacent 2'0Me modifications) can be synergistically combined with the RNase Hl evasion patterns (REP-1 or REP-2) in a single unified molecule. This dual-architecture prevents endogenous nuclease degradation of the DNAzyme extremities while simultaneously inhibiting RNase Hl-mediated off-target cleavage of the internal RNA substrate heteroduplex. The herein reported data, including, for example, the effective combination of stabilizing modifications with RNase evasion patterns (Fig. 52) as well as the combination of stabilizing modifications in the binding arms with activity-increasing modifications in the catalytic core (Fig.

[0724] 48), support the general modular architecture of the DNAzyme system that may also comprise additional beneficial combinations, including but not limited to the combination of RNase evasion pattern REP-1 or REP-2 in the binding arms with activity-increasing modifications in the catalytic core.

[0725] Also, the inventors have found that modifications that stabilize the active structure can make the addition or presence of Mg2+at one or multiple binding sites unnecessary. The invention relates to such modifications as well.

[0726] The inventors claim and have found, that G(5) will strongly reduce Mg2+binding thereby making the DNAzyme inactive. This is a claimed and preferred modification to be used as a negative control. The inventors claim and a have found, that C(5) moderately reduce Mg2+binding activity. There are circumstances where this is needed.

[0727] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 2 and wherein the catalytic core region is characterized by a modification to etheno-dA at position 5.

[0728] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 3 and wherein the catalytic core region is characterized by a modification to dnebularine at position 5. Unser Zeichen: B407-0001W01 42The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 4 and wherein the catalytic core region is characterized by a modification to ANT at position 5.

[0729] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 5 and wherein the catalytic core region is characterized by a modification to Me-Ph at position 5.

[0730] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 6 and wherein the catalytic core region is characterized by a modification to 6-S-dG at position 6.

[0731] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 7 and wherein the catalytic core region is characterized by a modification to 6-Se-dG at position 6.

[0732] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 8 and wherein the catalytic core region is characterized by a modification to 8-oxo-dG at position 6.

[0733] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 9 and wherein the catalytic core region is characterized by a modification to Iso-dG at position 6.

[0734] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 10 and wherein the catalytic core region is characterized by a modification to 6-O-methyl-dG at position 6.

[0735] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 11 and wherein the catalytic core region is characterized by a modification to 7-deaza-dG at position 6.

[0736] Unser Zeichen: B407-0001WO1 43The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 12 and wherein the catalytic core region is characterized by a modification to 2'-0Me-dG at position 6.

[0737] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 13 and wherein the catalytic core region is characterized by a modification to 2'-MOE-dG at position 6.

[0738] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 14 and wherein the catalytic core region is characterized by a modification to etheno-dA at position 6.

[0739] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 15 and wherein the catalytic core region is characterized by a modification to ANT at position 6.

[0740] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 16 and wherein the catalytic core region is characterized by a modification to 2-amino-purine at position 6.

[0741] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 17 and wherein the catalytic core region is characterized by a modification to 2-amino-dA at position 6.

[0742] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 18 and wherein the catalytic core region is characterized by a modification to dnebularine at position 6.

[0743] Unser Zeichen: B407-0001WO1 44The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 19 and wherein the catalytic core region is characterized by a modification to 7MG at position 6.

[0744] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 20 and wherein the catalytic core region is characterized by a modification to IOX at position 6.

[0745] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 21 and wherein the catalytic core region is characterized by a modification to 6IP at position 6.

[0746] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 22 and wherein the catalytic core region is characterized by a modification to 3DS at position 6.

[0747] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 23 and wherein the catalytic core region is characterized by a modification to 2DO at position 6.

[0748] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 24 and wherein the catalytic core region is characterized by a modification to PYR at position 6.

[0749] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 25 and wherein the catalytic core region is characterized by a modification to NIP at position 6.

[0750] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 26 and wherein the catalytic core region is characterized by a modification to FIP at position 6.

[0751] Unser Zeichen: B407-0001WO1 45The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 27 and wherein the catalytic core region is characterized by a modification to 3NA at position 6.

[0752] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 28 and wherein the catalytic core region is characterized by a modification to 7OP at position 6.

[0753] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 29 and wherein the catalytic core region is characterized by a modification to 3DO at position 6.

[0754] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 30 and wherein the catalytic core region is characterized by a modification to 2DS at position 6.

[0755] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 31 and wherein the catalytic core region is characterized by a modification to BOG at position 6.

[0756] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 32 and wherein the catalytic core region is characterized by a modification to 2TA at position 6.

[0757] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 33 and wherein the catalytic core region is characterized by a modification to POP at position 6.

[0758] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 34 and wherein the catalytic core region is characterized by a modification to NTZ at position 6.

[0759] Unser Zeichen: B407-0001W01 46The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 35 and wherein the catalytic core region is characterized by a modification to MES at position 6.

[0760] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 36 and wherein the catalytic core region is characterized by a modification to TTZ at position 6.

[0761] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 37 and wherein the catalytic core region is characterized by a modification to MER at position 6.

[0762] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 38 and wherein the catalytic core region is characterized by a modification to M2R at position 6.

[0763] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 39 and wherein the catalytic core region is characterized by a modification to M2S at position 6.

[0764] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 40 and wherein the catalytic core region is characterized by a modification to BTG at position 6.

[0765] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 41 and wherein the catalytic core region is characterized by a modification to 6TP at position 6.

[0766] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 42 and wherein the catalytic core region is characterized by a modification to PHY thioguanine at position 6.

[0767] Unser Zeichen: B407-0001WO1 47The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 43 and wherein the catalytic core region is characterized by a modification to 3TA at position 6.

[0768] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 44 and wherein the catalytic core region is characterized by a modification to MTG at position 6.

[0769] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 45 and wherein the catalytic core region is characterized by a modification to AIP at position 6.

[0770] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 46 and wherein the catalytic core region is characterized by a modification to DOP at position 6.

[0771] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 47 and wherein the catalytic core region is characterized by a modification to AEG at position 6.

[0772] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 48 and wherein the catalytic core region is characterized by a modification to AMG at position 6.

[0773] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 49 and wherein the catalytic core region is characterized by a modification to IOX2 at position 6.

[0774] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 50 and wherein the catalytic core region is characterized by a modification to Me-Ph at position 6.

[0775] Unser Zeichen: B407-0001W01 48The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 51 and wherein the catalytic core region is characterized by a modification to 6aG at position 6.

[0776] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 52 and wherein the catalytic core region is characterized by a modification to 6-cG at position 6.

[0777] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 53 and wherein the catalytic core region is characterized by a modification to 7MP at position 6.

[0778] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 54 and wherein the catalytic core region is characterized by a modification to 6-S-dG at position 14.

[0779] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 55 and wherein the catalytic core region is characterized by a modification to 6-Se-dG at position 14. The catalytic enhancement conferred by the 6-Se-dG modification at position 14 is grounded in the isosteric and electronic relationship between sulfur and selenium chalcogens. Due to their position in group 16 of the periodic table, selenium shares and even surpasses the high electron polarizability and 'soft base' characteristics of sulfur, while exhibiting a slightly larger atomic radius. The substitution of the 06 oxygen with a selenium atom significantly alters the local hydrogenbonding network and reduces the energetic penalty associated with adopting the catalytically active rotameric state. The necessity of specific electronic distributions is supported by Density Functional Theory (DFT) calculations of dipole moments across various heterocycles. Dedicated Molecular Dynamics (MD) simulations demonstrate that, comparable to the 6-S-dG(14) variant, the 6-Se-dG modification at position 14 highly stabilizes the pre-catalytic transition state geometry, as evidenced by increased in-line attack (ILA) formation. Specifically, the enhanced polarizability of the selenocarbonyl group restricts the conformational flexibility of the guanine base, locking the catalytic loop into an optimized orientation that facilitates the strictly required in-line attack geometry for RNA phosphodiester bond cleavage. Consequently, the 6-Se-dG(14) modification

[0780] Unser Zeichen: B407-0001WO1 49exerts a robust beneficial effect on catalytic activity equivalent to, or exceeding, that of the 6-S-dG(14) modification, validating its utility in highly active DNAzyme constructs.

[0781] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 56 and wherein the catalytic core region is characterized by a modification to 8-oxo-dG at position 14.

[0782] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 57 and wherein the catalytic core region is characterized by a modification to iso-dG at position 14.

[0783] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 58 and wherein the catalytic core region is characterized by a modification to 6-O-methyl-dG at position 14.

[0784] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 59 and wherein the catalytic core region is characterized by a modification to 7-deaza-dG at position 14.

[0785] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 60 and wherein the catalytic core region is characterized by a modification to etheno-dA at position 14.

[0786] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 61 and wherein the catalytic core region is characterized by a modification to ANT at position 14.

[0787] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 62 and wherein the catalytic core region is characterized by a modification to 2-amino-dA at position 14.

[0788] Unser Zeichen: B407-0001WO1 50The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 63 and wherein the catalytic core region is characterized by a modification to dnebularine at position 14.

[0789] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 64 and wherein the catalytic core region is characterized by a modification to 7MG at position 14.

[0790] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 65 and wherein the catalytic core region is characterized by a modification to IOX at position 14.

[0791] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 66 and wherein the catalytic core region is characterized by a modification to 6IP at position 14.

[0792] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 67 and wherein the catalytic core region is characterized by a modification to 3DS at position 14.

[0793] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 68 and wherein the catalytic core region is characterized by a modification to 2DO at position 14.

[0794] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 69 and wherein the catalytic core region is characterized by a modification to PYR thioguanine at position 14.

[0795] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 70 and wherein the catalytic core region is characterized by a modification to NIP at position 14. Unser Zeichen: B407-0001WO1 51The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 71 and wherein the catalytic core region is characterized by a modification to FIP at position 14.

[0796] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 72 and wherein the catalytic core region is characterized by a modification to 3NA at position 14.

[0797] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 73 and wherein the catalytic core region is characterized by a modification to 7OP at position 14.

[0798] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 74 and wherein the catalytic core region is characterized by a modification to 3DO at position 14.

[0799] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 75 and wherein the catalytic core region is characterized by a modification to 2DS at position 14.

[0800] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 76 and wherein the catalytic core region is characterized by a modification to BOG at position 14.

[0801] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 77 and wherein the catalytic core region is characterized by a modification to 2TA at position 14.

[0802] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 78 and wherein the catalytic core region is characterized by a modification to POP at position 14.

[0803] Unser Zeichen: B407-0001W01 52The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 79 and wherein the catalytic core region is characterized by a modification to NTZ at position 14.

[0804] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 80 and wherein the catalytic core region is characterized by a modification to MES at position 14.

[0805] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 81 and wherein the catalytic core region is characterized by a modification to TTZ at position 14.

[0806] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 82 and wherein the catalytic core region is characterized by a modification to MER at position 14.

[0807] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 83 and wherein the catalytic core region is characterized by a modification to M2R at position 14.

[0808] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 84 and wherein the catalytic core region is characterized by a modification to M2S at position 14.

[0809] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 85 and wherein the catalytic core region is characterized by a modification to BTG at position 14.

[0810] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 86 and wherein the catalytic core region is characterized by a modification to 6TP at position 14.

[0811] Unser Zeichen: B407-0001WO1 53The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 87 and wherein the catalytic core region is characterized by a modification to PHY at position 14.

[0812] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 88 and wherein the catalytic core region is characterized by a modification to 3TA at position 14.

[0813] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 89 and wherein the catalytic core region is characterized by a modification to MTG at position 14.

[0814] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 90 and wherein the catalytic core region is characterized by a modification to AIP at position 14.

[0815] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 91 and wherein the catalytic core region is characterized by a modification to DOP at position 14.

[0816] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 92 and wherein the catalytic core region is characterized by a modification to AEG at position 14.

[0817] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 93 and wherein the catalytic core region is characterized by a modification to AMG at position 14.

[0818] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 94 and wherein the catalytic core region is characterized by a modification to IOX2 at position 14.

[0819] Unser Zeichen: B407-0001W01 54The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 95 and wherein the catalytic core region is characterized by a modification to Me-Ph at position 14.

[0820] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 96 and wherein the catalytic core region is characterized by a modification to 6aG at position 14.

[0821] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 97 and wherein the catalytic core region is characterized by a modification to Me-Ph-6S-dG at position 14.

[0822] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 98 and wherein the catalytic core region is characterized by a modification to Me-Ph-6Se-dG at position 14.

[0823] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 99 and wherein the catalytic core region is characterized by a modification to PSO-6S-dG at position 14.

[0824] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 100 and wherein the catalytic core region is characterized by a modification to PSO-6Se-dG at position 14.

[0825] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 101 and wherein the catalytic core region is characterized by a modification to 2'-MOE-6-S-dG at position 14.

[0826] Unser Zeichen: B407-0001WO1 55The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 102 and wherein the catalytic core region is characterized by a modification to 2'-MOE-6-SE-dG at position 14.

[0827] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 103 and wherein the catalytic core region is characterized by a modification to Me-Ph-2'-MOE-6S-dG at position 14.

[0828] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 104 and wherein the catalytic core region is characterized by a modification to Me-Ph-2'-MOE-6Se-dG at position 14.

[0829] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 105 and wherein the catalytic core region is characterized by a modification to 6-cG at position 14.

[0830] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence, SEQ ID NO. 106 and wherein the catalytic core region is characterized by a modification to 7MP at position 14.

[0831] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), comprising the following sequence, SEQ ID NO. 107, that is flanked by a 5' substrate binding arm and a 3' substrate binding arm. The DNAzyme is characterized by a 2'0Me modification on the nucleotide at the 5' end of the binding arm, a modification to 6-s-dG at position 14 within the catalytic core, and a 2'0Me modification on the nucleotide at the 3' end of the binding arm.

[0832] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), comprising the following sequence, SEQ ID NO. 108, that is flanked by a 5' substrate binding arm and a 3' substrate binding arm. The DNAzyme is characterized by a 2'0Me modification on the nucleotide at the 5' end of the binding arm, a modification to 6-Se-dG(14) at position 14 within the catalytic core, and a 2'0Me modification on the nucleotide at the 3' end of the binding arm.

[0833] Unser Zeichen: B407-0001WO1 56The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), comprising the following sequence, SEQ ID NO. 109, that is flanked by a 5' substrate binding arm and a 3' substrate binding arm. The DNAzyme is characterized by a 2'0Me modification on the nucleotide at the 5' end of the binding arm, a modification to Me-Ph-6-S-dG at position 14 within the catalytic core, and a 2'0Me modification on the nucleotide at the 3' end of the binding arm.

[0834] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), comprising the following sequence, SEQ ID NO. 110, that is flanked by a 5' substrate binding arm and a 3' substrate binding arm. The DNAzyme is characterized by a 2'0Me modification on the nucleotide at the 5' end of the binding arm, a modification to Me-Ph-6-Se-dG at position 14 within the catalytic core, and a 2'0Me modification on the nucleotide at the 3' end of the binding arm.

[0835] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), comprising the following sequence, SEQ ID NO. 111, that is flanked by a 5' substrate binding arm and a 3' substrate binding arm. The DNAzyme is characterized by a 2'0Me modification on the nucleotide at the 5' end of the binding arm, a modification to 2'MOE-6-S-dG at position 14 within the catalytic core, and a 2'0Me modification on the nucleotide at the 3' end of the binding arm.

[0836] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme) characterized by the incorporation of highly optimized nucleotide analogues at position 14, specifically selected from 2'0Me-6-S-dG (SEQ ID NO. 304) and PSO-2'OMe-6-S-dG (SEQ ID NO.

[0837] 305). These specific modified variants exhibit strongly enhanced catalytic activity and constitute core elements of the present invention.

[0838] The invention relates to a nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), comprising the following sequence, SEQ ID NO. 112, that is flanked by a 5' substrate binding arm and a 3' substrate binding arm. The DNAzyme is characterized by a 2'0Me modification on the nucleotide at the 5' end of the binding arm, a modification to 2'MOE-6-Se-dG at position 14 within the catalytic core, and a 2'0Me modification on the nucleotide at the 3' end of the binding arm.

[0839] Unser Zeichen: B407-0001WO1 57The invention relates to structural combinations wherein a nucleobase modification at position 14 (selected from 6-S-dG or 6-Se-dG) is simultaneously combined with at least one additional modification involving an internucleotide linkage (selected from PSO or Me-Ph) and / or a ribose modification (selected from 2'MOE or 2'0Me). Specific combinatorial moieties substituting the wild-type G(14) include: (PSO, 2'0Me, 6-S)-dG; (PSO, 2'MOE, 6-S)-dG; (2'MOE, 6-S)-dG; (2'0Me, 6-S)-dG; (PSO, 2'0Me, 6-Se)-dG; (PSO, 2'MOE, 6-Se)-dG; (2'MOE, 6-Se)-dG; and (2'0Me, 6-Se)-dG.

[0840] The following modifications of the catalytic core region are disclaimed and not part of the invention:

[0841] a thymine at position 5,

[0842] a cytosine at position 5,

[0843] a 2'-0Me-dG at position 14,

[0844] a 2'-MOE-dG at position 14,

[0845] a 2-amino-purine at position 14, and

[0846] a 6-S-dG at position 14 when combined with cytosine at position 5

[0847] THE INTERFACE

[0848] The inventors have found that the DNAzymes according to the invention show close contacts between different regions of the DNA-backbone (DNA-DNA or DNA-RNA interface). Since the DNA or RNA backbone is negatively charged this has been found by the inventors to induce unfavorable electrostatic repulsion. The inventors have found that Mg2+binding can resolve this partially. However, removing the negative charge with suitable modifications is also a solution that the inventors have identified. The inventors thereby reduce Mg2+dependency.

[0849] The catalytic core regions according to the invention and designated in SEQ. ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 and 112 may comprise a chemical modification that can reduce the negative charge, involving one or more nucleotides of the interface such as Me-phosphonate modifications (Me-PH).

[0850] Unser Zeichen: B407-0001W01 58The charge-neutralizing Me-phosphonate (Me-Ph) internucleotide linkage modifications may be synergistically combined with the aforementioned novel nucleobase modifications at position 14. This yields uncharged, highly active variants, such as Me-Ph-2'OMe-6-S-dG and Me-Ph-2'MOE-6-Se-dG, that successfully mitigate unfavorable electrostatic repulsion in the DNA-RNA interface, thereby fundamentally reducing the DNAzyme's dependency on divalent metal ions for structural activation.

[0851] The catalytic core regions according to the invention and designated in SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 and 112 may comprise a chemical modification that can reduce the negative charge, involving one or more nucleotides of the interface such as H-Phosphonate DNA.

[0852] The catalytic core regions according to the invention and designated in SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 and 112 may comprise a chemical modification that can reduce the negative charge, involving one or more nucleotides of the interface such as morpholino (PMO).

[0853] PNAs are synthetic DNA analogs in which the phosphodiester backbone is replaced by repetitive units of N-(2-aminoethyl) glycine to which the purine and pyrimidine bases are attached via a methyl carbonyl linker. Hence, the catalytic core regions according to the invention and designated in SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 and 112 may comprise a PNA chemical modification involving one or more nucleotides of the interface.

[0854] Unser Zeichen: B407-0001WO1 59The catalytic core regions according to the invention and designated in SEQ. ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 and 112 may comprise a chemical modification that can reduce the negative charge, involving one or more nucleotides of the interface such as triazole-linked DNA.

[0855] The inventors have found that DNAzyme nucleotides that are in the respective interface region include, G(1), G(2), G(6), C(13), G(14), and, in the arms: the five nucleotides flanking the core sequence on both sides.

[0856] The invention relates to chemical modifications that can reduce negative charges. Claimed herein are modifications of the interface region that reduce negative charge. Preferable modifications of the interface region include (involving one or more nucleotides of the interface), Me-phosphonate Oligos (Me-PH), H-phosphonate DNA, Morpholino (PMO), PNA (peptide nucleic acid) and / or triazole-linked DNA.

[0857] Systematic screening of Me-Ph modifications across the DNAzyme sequence, as depicted in Fig. 53, reveals that strategic placement of Me-Ph internucleotide linkages adjacent to the catalytic core (specifically including the above defined "interface region") optimally minimizes electrostatic repulsion within the precatalytic complex. These specific placement pattern reduces the need for positively charged ions to counteract the otherwise present strong electrostatic repulsion of the DNA / RNA backbones, enabling increase activity even in environments characterized by severely limited magnesium availability.

[0858] The DNAzyme

[0859] The invention relates to an improved catalytic core region which may be part of one or two flanking regions known in the art. The invention also relates to the full DNAzyme.

[0860] The invention relates to a nucleic acid encoding a catalytic deoxyribozyme (DNAzyme) comprising, b. a catalytic core region according to the invention,

[0861] Unser Zeichen: B407-0001W01 60b. and, at least one flanking variable target recognition sequence, wherein the target recognition sequence is between 5 and 30 nucleotides in length, and

[0862] b. wherein said at least one flanking target recognition sequence can be either 5' or 3' of said catalytic core region.

[0863] If two flanking target recognition sequences are used one can be shorter for example 8 nucleotides in length with a lowest limit of 5.

[0864] One may also consider DNAzyme operating at lower temperatures as normally present for therapeutic applications and which may only require one arm with 6-7 base parings in total. Herein, claimed therefore are "low temperature" DNAzymes wherein one flanking arm is between 6 and 20 nucleotides in length, 6 and 15 nucleotides in length, or 6 and 9 nucleotides in length.

[0865] The invention relates to "low temperature" DNAzymes wherein one flanking arm is under 20 nucleotides in length, under 15 nucleotides in length or under 9 nucleotides in length, wherein the region is at least 6 nucleotides in length.

[0866] The invention relates to a nucleic acid encoding a catalytic deoxyribozyme (DNAzyme) according to the invention, comprising,

[0867] a. two flanking variable target recognition sequences, wherein the target recognition sequences are between 5 and 30 nucleotides in length, and

[0868] b. wherein said recognition sequences are 5' and 3' of said catalytic core region.

[0869] The flanking regions preferably comprise nucleotide modifications as well. The invention therefore encompasses a catalytic deoxyribozyme (DNAzyme) according to the invention, comprising, a. two flanking variable target recognition sequences, wherein the target recognition sequences are preferably between 8 and 30 nucleotides in length, and

[0870] b. wherein said recognition sequences are 5' and 3' of said catalytic core region, wherein one or both variable target recognition sequences comprise a nucleotide modification in the first 20 nucleotides, more preferably the first 10 nucleotides more preferably the first 5 nucleotides adjacent to the catalytic core region.

[0871] As indicated above, it is preferable if the modification of the flanking region is adjacently close to the catalytic region. The flanking region may have, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or up to and over 30 modifications. The nucleotides of the flanking regions may have modifications of one or multiple kinds. That means for example

[0872] Unser Zeichen: B407-0001W01 61that a nucleotide " G" may be modified to 2'-OMe-dG at a given position and modified to something else at another position. It also includes multiple modifications at the same nucleotide such as e.g. dG(2'OMe, PSO, 6-S). The flanking region is the region that is adjacent to the catalytic core region and adjacent to said core region on the 5'- and 3'- side. The numbering above relates to the nucleotides 1 to 30 in each flanking region, counting starting from the core region; See Fig. 1. The flanking regions may comprise numerous modifications, but preferred compounds for modifications are, the ribose modifications and internucleotide linkage modifications described above including LNA (locked nucleic acid), inverted NA, TNA, 2'-0Me, 2'-MOE, FANA, 2'-F, RNA (i.e., replace DNA with RNA analogue) or, e.g. PSO (phosphorothioate).

[0873] The invention relates to nucleic acid, wherein the nucleic acid is selected from the group consisting of SEQ ID NO. 107 to 112 as shown below, wherein the flanking variable regions of the core regions of are modified by 2'-0Me on 3'- and 5' -ends,

[0874] Pos. 5' 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3' SEQ (5'=2'OMe),ID(G*(14)=6-S- 5' G G C T A G C T A C A A C N* A 3' NO. dG), (3'=2'OMe) 107 SEQ (5'=2'OMe),ID(G*(14)=6-Se- 5' G G C T A G C T A C A A C N* A 3'

[0875]

[0876] NO. dG), (3'=2'OMe) 108

[0877] SEQ (5'=2'OMe), ID (G*(14)=Me-Ph- 5' G G C T A G C T A C A A C N* A 3'

[0878] NO. 6-S-dG),

[0879] 109 (3'=2'OMe)

[0880] SEQ (5'=2'OMe),

[0881] (G*(14)=Me-Ph-ID5' G G C T A G C T A C A A C N* A 3'

[0882] NO. 6-Se-dG),

[0883] 110 (3'=2'OMe)

[0884] SEQ (5'=2'OMe),ID(G*(14)=2'MOE- 5' G G C T A G C T A C A A C N* A 3'

[0885] NO. 6-S-dG),

[0886] 111 (3'=2'OMe)

[0887] SEQ (5'=2'OMe), ID (G*(14)=2'MOE- 5' G G C T A G C T A C A A C N* A 3'

[0888] NO. 6-Se-dG),

[0889] 112 (3'=2'OMe)

[0890] For instance, to achieve the required increased lifetime and nuclease resistance, the stabilizing ELMO architecture, comprising terminal LNA modifications and adjacent inward-facing 2'0Me modifications, can be incorporated into the flanking regions. Furthermore, these flanking region Unser Zeichen: B407-0001W01 62stabilizations can be advantageously combined with the catalytic core variants described herein, particularly the highly active G(14) substitutions combined with internucleotide linkage (PSO or Me-Ph) or ribose (2'MOE or 2'0Me) modifications. This yields a unified DNAzyme architecture with comprehensively enhanced cellular stability, activity, and optimized turnover kinetics.

[0891] For the purposes of the present invention, the ELMO architecture is defined as a terminal stabilization motif comprising on each terminal end exactly one terminal or penultimate Locked Nucleic Acid (LNA) residue positioned adjacent to at least one but not more than two sequential 2'-O-methyl (2'0Me) modified residues directed inward toward the catalytic core. The ELMO architecture optionally, but preferably, includes an unpaired nucleotide overhang consisting of one or both terminal LNA nucleotides extending beyond the complementary target-binding sequence. The LNA overhang provides a beneficial combination of nuclease resistance and thermodynamic features including catalytic turnover, and serves to further physically disrupt RNase recognition clefts.

[0892] The inventors have modified the flanking regions in order to achieve one or more of the following preferable activities / effects, i) increasing lifetime (increasing stability, nuclease resistance), ii) increasing selectivity (specifically stabilizing base pairing with target RNA), iii) precision (reduce less specific RNase-mediated (antisense) effects), iv) decreasing selectivity (avoiding resistances), v) modulating immune response, and / or vi) modulate substrate turnover rates.

[0893] Ideally and according to the invention the substrate comprises a suitable cleavage site that comprises RNA, DNA modified with at least one RNA nucleotide, modified RNA, nucleotide analogs, or composites thereof.

[0894] Substrate specificity, as used herein, may refer to the specificity of the DNAzyme as described herein for a particular substrate, such as one comprising ribonucleotides only, deoxyribonucleotides only, or a composite of both. Substrate molecules may also contain nucleotide analogs. In various embodiments, an enzymatic nucleic acid molecule of the present invention may preferentially bind to a particular region of a hybrid or non-hybrid substrate.

[0895] The term or parameter identified herein as "substrate specificity" may also include sequence specificity; i.e., an enzymatic nucleic acid molecule of the present invention may "recognize" and bind to a nucleic acid substrate having a particular nucleic acid sequence. For example, if the substrate recognition domains of an enzymatic nucleic acid molecule of the present invention will Unser Zeichen: B407-0001W01 63only bind to substrate molecules having a series of one or two ribonucleotides (e.g., rA) in a row, then the enzymatic nucleic acid molecule will tend not to recognize or bind nucleic acid substrate molecules lacking such a sequence.

[0896] According to the invention the catalytic region and or the entire DNAzyme according to the invention promotes cleavage of a phosphoester bond via catalyzing a transesterification reaction at said cleavage site.

[0897] The catalytic DNAzyme molecule according to the invention may be single-stranded.

[0898] The invention also relates to a composition wherein said substrate nucleic acid is attached to said catalytic DNA molecule.

[0899] According to the invention said catalytic DNA molecule includes a conserved nucleotide core comprising one or more conserved nucleotide sequences.

[0900] In one disclosed embodiment, an enzymatic DNA molecule of the present invention comprises a conserved core flanked by two substrate binding (or recognition) domains or sequences that interact with the substrate through base-pairing interactions. In various embodiments, the conserved core comprises one or more conserved domains or sequences. In another variation, an enzymatic DNA molecule further comprises a "spacer" region (or sequence) between the regions (or sequences) involved in base pairing. In still another variation, the conserved core is "interrupted" at various intervals by one or more less-conserved variable or "spacer" nucleotides.

[0901] Compositions with DNAzymes

[0902] The invention also contemplates compositions containing one or more types or populations of DNAzymes of the present invention; e.g., different types or populations may recognize and cleave different nucleotide sequences. Compositions may further include a ribonucleic acid-containing substrate. Compositions according to the present invention may further comprise lead ion, magnesium ion, calcium ion, zinc ion, or other divalent or monovalent cations, as discussed herein.

[0903] Preferably, the enzymatic DNA molecule is present at a concentration of about 0.001 pM to about 1 mM. Typically, the DNAzyme is present at a concentration ratio of enzymatic DNA molecule to substrate from about 10:1 to about 1:1000. More preferably, the enzymatic DNA molecule is present in the composition at a concentration of about 0.01 pM to about 10 pM. Even more preferably, compositions contain the enzymatic DNA molecule at a concentration of about 0.02 pM Unser Zeichen: B407-0001W01 64to about 0.5 pM. Preferably, the substrate is present in the composition at a concentration of about 0.5 nM to about 1000 nM. One skilled in the art will understand that there are many sources of nucleic acid-containing substrates including naturally-occurring and synthetic sources.

[0904] Herein "present at a concentration" refers to the final reaction environment. That means in vitro or in vivo where the catalytic cleavage takes place.

[0905] Methods of Using DNAzymes

[0906] The methods of using DNAzymes as disclosed herein are legion. As discussed previously, molecules capable of cleaving the bonds linking neighboring nucleic acids (e.g., phosphoester bonds) have numerous uses encompassing a wide variety of applications. For example, DNAzymes having the within-disclosed capabilities, structures, and / or functions are useful in pharmaceutical applications (e.g. down-regulation of unwanted RNA), medical products (e.g., for wound debridement, clot dissolution, etc.), crop science applications, biotechnological and academic applications (e.g. tailored gene-silencing tools), as well as in household items (e.g., detergents, dental hygiene products, meat tenderizers). Industrial utility of the within-disclosed compounds, compositions and methods is also contemplated and well within the scope of the present invention.

[0907] The present invention also describes useful methods for cleaving any single-stranded, looped, partially or fully double-stranded nucleic acid; the majority of these methods employ the novel enzymatically active nucleic acid molecules of the present invention. In various embodiments, the single-stranded nucleic acid segment or portion of the substrate (or the entire substrate itself) comprises DNA, modified DNA, RNA, modified RNA, or composites thereof. The nucleic acid substrate must only be single-stranded or otherwise accessible at or near the substrate cleavage sequence so that an enzymatic nucleic acid molecule of the present invention can hybridize to the substrate cleavage sequence by virtue of the enzyme's recognition sequence.

[0908] A nucleic acid substrate that can be cleaved by a method of this invention may be chemically synthesized or enzymatically produced, or it may be isolated from various sources such as phages, viruses, prokaryotic cells, or eukaryotic cells, including animal cells, plant cells, eukaryotic cells, yeast cells and bacterial cells.

[0909] The present invention is capable of cleaving specific nucleic acid substrates. Here, a non-perfect match between target substrate sequence and target specific arm sequence can lead to an inability of cleavage. Thus, the invention shows a high degree of specificity and selectivity.

[0910] Unser Zeichen: B407-0001W01 65The invention also relates to a nucleic acid catalytic core or DNAzyme, wherein the modified nucleotide confers enhanced catalytic activity and / or improved stability in vivo relative to the corresponding unmodified 10-23 deoxyribozyme.

[0911] The invention also relates to a nucleic acid catalytic core or DNAzyme, wherein said nucleic acid has a 10- to 100-fold increased cleavage rate (k_obs) toward an RNA substrate under physiologically relevant conditions compared to an unmodified 10-23 deoxyribozyme.

[0912] The invention also relates to a nucleic acid catalytic core or DNAzyme for use as a medicament. The invention also relates to a nucleic acid catalytic core or DNAzyme for use as a pharmaceutical composition with an acceptable carrier.

[0913] The invention also relates to a nucleic acid catalytic core or DNAzyme, for use in treating a disease or disorder associated with the expression of a target RNA, such as an mRNA or non-coding RNA, wherein the deoxyribozyme is designed to cleave said target RNA in vivo and ameliorate the disease.

[0914] The invention also relates to a method of cleaving a target RNA in a sample or in a subject, the method comprising contacting the sample or administering to the subject an effective amount of the nucleic acid according to the invention under conditions suitable for cleavage of said target RNA.

[0915] In another aspect, the invention provides a kit for use in gene-silencing or addressing other needs for targeted RNA elimination, in laboratory applications, such as for example found in the area of molecular biology, cell biology, structural biology and pre-clinical studies. The kit comprising a nucleic acid according to the method of cleaving a target RNA in a sample or in a subject, optionally with buffers and reagents suitable for performing an RNA cleavage reaction, and instructions for use.

[0916] EXAMPLES

[0917] In silico assisted DNAzyme design and assessment

[0918] Our molecular dynamics (MD) simulations revealed key aspects in the 10-23 DNAzyme mechanism, where cleavage occurs only when the angle towards the 02' of rGO and both the phosphate and an Unser Zeichen: B407-0001W01 66oxygen of rll-1 reaches >120°. Furthermore, our MD simulations found that the distance between the 02' of rGO and N2 of G(14) should be less than 4 A. This cleavage formation, also described as an in-line attack (ILA), allows to focus on improving DNAzymes for various applications.

[0919] As some natural nucleobases failed to increase the ILA, the inventors attempted the use of modified, non-natural nucleobases. The inventors aimed to increase the activity by I) decreasing the distance between 02' of rGO and N2 of G(14) and II) increasing the formation of the previously described attack angle of the ILA as well as III) increasing the angle itself to reach an ideal configuration of 180° as often as possible, MD simulations of modified nucleobases were performed (see below for a description of the Methods) to identify novel nucleobases that could potentially lead to an improved ILA.

[0920] The identification of hitherto unknown non-natural nucleobases (Fig. 16).

[0921] Overall, single modifications yielded an increase in ILA formation of up to 15±2.1%. A total of 16 single modifications, were tested, of which 3 achieved an increase compared to the 6-S-dG14 variant (Seq-ID 54) and 10 an increase compared to the C(5)-variant. Furthermore, double modifications showed an increase of up to 37±3.6% by combining 6IP at position 14 (61 P( 14)) with 7MP at position 6 (7MP(6)). The introduction of 7MP at position 6 showed a superadditive effect in the context of other modifications such as 6IP(14), 3DS(14), or 2DO(14), all surpassing the ILA formation of the 6-S-dG(14) variant. The increase in ILA formation with 6IP(14), 3DS(14), and 2DO(14) results from 7T-7T-stacking with rGO due to the extended heterocycles.

[0922] The role of the substituent in a position equivalent to 06 in nucleobase dG(14), e.g. in 2-amino-purin and 6-S-dG(14), was identified in our work and led to formulating a structure-activity relationship. An exchange to sulfur as present in 6-S-dG(14) lowers the basicity and strongly decreases Mg2+affinity, which led to an increase in activity. Further modifications of the 06 group, such as in MTG(14) and 6-O-methyl-dG(14), lead to a sharp decrease in ILA formation, which is also corroborated by experimental results (Fig. 21 A). The main reason for this seems to be the size of the methyl group preventing Mg2+from reaching a favorable position for stabilizing the ILA formation. The introduction of a phenyl group instead of the methyl group in P0P(14) leads to an increased 7T-7T-interaction with other nucleobases, improving ILA formation. Despite a potential stereochemical hindrance of the phenyl group, an increase in ILA formation was observed due to similar effects as in 6IP(14), as the phenyl ring interacts with rGO, stabilizing the ILA. This leads to 7±2.1% of the frames reaching a distance below 4 A between rGO and P0P(14), where none was Unser Zeichen: B407-0001W01 67observed for MTG(14) and 6-O-methyl-dG(14). The removal of the 06 position in 2-amino-purine(14) led to an increase in ILA formation but eliminated activity in vitro. This modified DNAzyme shows high Mg2+binding in the catalytic core, especially at T8 and A9, which indicates that higher Mg2+concentrations are necessary to reach full activity. It is claimed herein that an electronegative group, ideally with free electron pairs, is strongly preferred to form the ILA in the context of unmodified DNAzymes.

[0923] In line with the 'chemical sibling' status of Sulfur and Selene due to their chemical similarity, an increased ILA formation was also observed for an exchange of 06 to Selene in 6-Se-dG(14) (Fig. 55). Another structure-activity relationship for fostering the ILA was identified regarding the interaction of rGO with G(6) in terms of 7T-7T-stacking. For DNAzymes with low ILA, also frequent 7T-7T-stacking during the ILA is found (Fig. 20). For DNAzymes with high ILA, 7T-7T-stacking is anti-correlated with the likelihood of ILA formation. This finding led to the use of 7MP(6) which reverses the dipole moment compared to G(6), thus, resulting in an increase in 7T-7T-stacking and, consequently, in ILA formation (Fig. 20). Thus, we claim a super-additive effect for the ILA, if 7T-7T-stacking is reduced via modifications at position 6 when using nucleobases with a high ILA formation at position 14 such as 6IP(14), 3DS(14), 2DO(14), and 2DS(14). See Fig. 20 for the combination of 7MP(6) with one of these modifications in either the A(5) or the C(5) variant (increase of the ILA to 37 ±3.6% or 51 ±2.8% in parallel to a decrease in 7T-7T-stacking between 7MP(6) and rGO); See also Fig. 16.

[0924] The inventors made use of the role of nucleobase dipole moment for the first time in the context of DNAzyme design. Ideally, novel non-natural nucleobases should show beneficial interactions with the RNA, mainly rGO, to direct the cleavage site into a beneficial position. This is given if the dipole moments of rGO and G(6) are collinear.

[0925] Dipole moments of the nucleobases shown in Fig. 22 were computed in the following manner: Using either Orca or Gaussian 16 with the B3-LYP* density functional theory method together with the 6-31G* basis set, the nucleobase structures were minimized using a convergence to within 10-9 Eh as a criterion for successful minimization, and the dipole moments were computed. See Validation for a validation of the computed data against experimental data (Fig. 22).

[0926] Applying this principle led to the invention of non-natural nucleobases with high ILA: The highest ILA formation was observed for nucleobases, in which the direction of the dipole moment of the extended heterocycle is parallel to the remaining nucleobase, pointing away from the attacking amine group with a value of 4-5 D when calculated in water (3-4 D in vacuum). This proved the most beneficial for the ILA of 6IP(14), 3DS(14), 2DO(14), and IOX(14). Furthermore, 2DS(14) and 3DO(14) Unser Zeichen: B407-0001W01 68showed an increase in ILA. In general, heterocycles with more nitrogens compared to 6IP(14) showed a decrease in ILA formation. This is most likely due to an increase in the dipole moment to 6-7 D for 2TA(14) and 3TA(14). On the other hand, a decrease in DM to 0.5 D with 6TP(14) leads to a sharp decrease in ILA formation. As such, a general structure can be formulated to be used for modifications of the DNAzyme, with a heterocycle bound to a position equivalent to C6 in the guanine at position 14 of the DNAzyme. This heterocycle needs to contain a negatively polarized heteroatom (e.g., oxygen, sulphur, selenium, phosphor) at position 2 as well as a less negatively polarized heteroatom at position 4 and / or 5 (e.g., nitrogen, sulphur), with a dipole moment of 5 D.

[0927] Validation of MD data

[0928] When determining the activity of the DNAzyme, other factors besides the ILA formation can play important results, one for example is the change of the entire structure from an inactive state before Mg2+occupies the metal binding sites described previously to an active structure, which can potentially form the ILA, another is the binding and release of the RNA, which would require a different setup of MD simulations to investigate. As such the ILA formation is merely an indicator for determining more active DNAzymes as we assume that an increase in ILA formation also increases the activity due to more opportunities in cleaving the target RNA but there is no quantifiable correlation between ILA and activity. As a reference both the (C(5);6-SdG(14))-variant and the C(5) variant were used. The reported kobs of 4.16x10-3 s’1and 7.32x10-4 s’1, respectively, show a 6-fold increase of activity, which aligns well with the observed ILA formations of 1011.6% for the (C(5);6-SdG(14))-variant and 2.110.2% for the C(5) variant (5-fold increase), which shows the accuracy of our predictions with previous results. Additional experimental results show a kobs of 0.011210.0006 s’1for the (A5);6-SdG14)-variant and 0.005710.0002 s’1for the A(5)-variant, respectively (Fig. 21), which does not align as well but still showcases that our simulations can predict improvements in activity by determining the ILA formation.

[0929] The loss of activity and ILA formation in MTG(14) and BTG(14) happens due to detrimental interactions between the C6-group and the magnesium, decreasing the possibility of Mg2+binding in a position required to form an ILA. As previously explained, 2-amino-purin(14) showcases a far higher cofactor dependency than other DNAzymes, rendering it inactive while being able to form the ILA more easily if reaching an active structure, where all MBS are occupied to initiate the ILA. Nebularine(14) carries no primary amine group but instead a secondary amine at the C6 position, which not only removes the 06 group but also leads to a less accessible ILA, observed both in Unser Zeichen: B407-0001W01 69experiments and MD simulations. Etheno-dA(14) contains no functional group to initiate the cleavage of the RNA, rendering the DNAzyme inactive. In MD simulations the distance between the (inactive) nitrogen and 02' of rGO was investigated, which suggests that they could initiate an ILA if protonated.

[0930] As a counterexample, although the DNAzyme with the PYR(14) modification yielded an increase of ILA to 4.6 ± 0.9% (respectively 7 ± 1.2%), no activity was found in the experiment (Fig. 21).

[0931] In silico methods

[0932] The structure of the C(5)-variant in precatalytic complex with its target RNA in its ILA state was used as start of the simulation. Modified nucleobases were introduced to position G(14) and / or G(6) at the catalytic loop, replacing the respective guanines. Initial structures were obtained by taking the parametrization of the guanosine residue of the DNA OL15 forcefield and then replacing or extending the guanine atoms with gaff2 atoms to describe unnatural heterocycles. RESP charges for each modified nucleotide were calculated via a private account on the RESP ESP charge Derive Server using Gaussian C.16.01 and PyRED. As these nucleotides have not been described in the literature so far, new parameters had to be generated before running simulations. Initial parameters were obtained via parmchk2 and further refined by using mdgx. To sample the dihedral angle of the bond between Cl of the ribose and the respective connecting atom (usually nitrogen), 360 conformations of a nucleotide were generated, each one with a 1° rotation compared to the previous one. Additional restraints were used to prevent intramolecular hydrogen bonds. QM minimization was performed on the obtained structures using Orca at the MP2 / cc-pVDZ level of theory and a tight self-consistent field (10-9 Eh) as an energy convergence criterion. Afterward, energies were extracted, and the dihedral parameters were generated by mdgx. A correlation coefficient of at least 0.85 between the parameters generated by mdgx and the QM-derived energies was considered sufficient to describe the parameters.

[0933] The thus obtained modified DNAzymes were checked and cleaned with pdb4amber. Afterward, Packmol-Memgen was used to solvate the DNAzyme and add hexahydrated Mg2+. All-atom MD simulations were performed, using the gaff2, DNA OL15, and RNA OL3 forcefields, in which the DNA and RNA were surrounded by a shell of TIP3P waters of at least 30 A thickness, 20 mM hexahydrated Mg2+ions as well as counter ions for neutralization, and KCI at a concentration of 150 mM. At the start of the minimization, 17,500 steps of steepest descent and conjugate gradient minimization with a changing force constant to all solute atoms were performed. During the first 2,500, 10,000, Unser Zeichen: B407-0001W01 70and 5,000 steps, positional harmonic restraints with force constants of 25 kcal mol-1A“2, 5 kcal mol-1A“2and zero, respectively, were applied. After that, 50 ps of NVT (constant number of particles, volume, and temperature) MD simulations were conducted to heat the system to 100 K, followed by 300 ps of NPT (constant number of particles, pressure and temperature) MD simulations to adjust the density of the simulation box to a pressure of 1 atm and to heat the system to 300 K. As the last step in thermalization, NVT-MD simulations were performed while gradually reducing the restraint forces on the solute atoms over six steps of 50 ns each from 5 kcal mol1A-2to zero. The gradual reduction of restraints allows Mg2+to bind to energetically favorable sites at the structures and to compensate for high local negative charges of the backbone, which otherwise could lead to a dissociation of the structure. Finally, ten independent production runs of NVT-MD simulations with 1 ps length each were performed. For this, the starting temperatures of the MD simulations at the beginning of the thermalization were varied by a fraction of a Kelvin. After the MD simulations have finished, all results are analyzed via Python as previously described (Fig. 24).

[0934] Detection of DNAzyme and RNase in vitro cleavage activity - Fluorescence Resonance Energy Transfer (FRET) assay

[0935] To investigate DNAzyme activity in real time, FRET assays were performed with minimal RNA targets. The RNA targets were labeled with a fluorescein at the 5'-end and a black hole quencher at the 3'-end. By adding divalent metal ions (MgCL), DNAzyme-mediated RNA cleavage was initiated, leading to an increase in fluorescence signal. The reaction conditions during the cleavage reaction 50 mM TRIS pH 7.4, 140 mM KCI, 10 mM NaCI, 0.1 mM EDTA, 1 mM MgCI2, and 0.1 pM RNA (FRET-labeled and either 0.4 pM or 0.01 pM DNAzyme for single-turn or multiple-turnover kinetic assays, respectively. Control measurements included FRET-labeled RNAs without DNAzyme as well as the inactive A5G variant with the FRET-labeled-RNA.

[0936] Full-length mRNA fragmentation assay

[0937] For the RNA fragmentation assay, 400 ng of purified target RNA was mixed with 1 pl (10 pM) of the corresponding DNAzyme. The reaction was started with MgCL (1 mM total) and incubated at 37 °C for 90 min in a reaction buffer composed of 50 mM TRIS pH 7.4, 140 mM KCI, 10 mM NaCI and 0.1 M EDTA. The entire reaction volume was loaded onto a 1% agarose gel to separate the RNA bands.

[0938] Unser Zeichen: B407-0001W01 71RNase activity was measured using 400 ng of purified target RNA mixed with 1 unit of RNase Hl. The reaction was started with 1 pl (10 pM) of corresponding DNAzyme and incubated at 37 °C for 20 min in RNase Hl reaction buffer. Parallel handling of an increased number of samples increased the effective incubation time, leading to higher total cleavage yields in some instances. The entire reaction volume was loaded onto a 1% agarose gel to separate the RNA bands.

[0939] Detection of DNAzyme activity in cellular assays:

[0940] Transient eYFP expression in HEK293T cells

[0941] 62500 HEK293T cells were plated in 48-well plates. After 24 h, the cells were transfected using the JetPrime kit. DNAzyme (20 - 200 nM) and eYFP-plasmid (0.5 nM) were transfected simultaneously. As a control, the cells were transfected with water. eYFP expression was monitored after additional 24h using cell imaging and an automated processing of the respective fluorescence values for each captured individual cell. The number of fluorescent cells, the total fluorescence and the median fluorescence of the cell population were used for evaluation.

[0942] For analysis by qPCR, total RNA was extracted from a previous cell culture. The RNA was diluted in a standardized manner and used in a one-step SYBR Green kit with primers specifically designed for the target RNA. Subsequently, the obtained data were normalized to the positive control.

[0943] Cell lines for infection assays

[0944] A549 cells (human bronchial carcinoma, CCL-185) and MC-57G (mouse fibrosarcoma cells, CRL-2295) were purchased from ATCC and grown in DMEM (Pan Biotech) with 10% FBS and penicillin streptomycin-L-glutamine.

[0945] LCMV assay

[0946] MC-57G cells were added to 24-well plates containing diluted viral samples; after 3 h, a medium with 1% methylcellulose (MC) was applied. Forty-eight hours later, the plates were fixed with 4% formalin (Sigma Aldrich), permeabilized with 1% Triton X (Sigma Aldrich) in HBSS medium (Sigma Aldrich), and stained with anti LCMV-Nucleoprotein (Clone VL-4, generated in-house) Ab and peroxidase anti-rat secondary Ab (Jackson ImmunoResearch).

[0947] Unser Zeichen: B407-0001W01 72DNAzymes antiviral assay

[0948] In a 24 well plate A549 cells were seeded in 500ul DMEM at a concentration of 2X 105. The next day cells were infected with LCMV at MOI 0.1 and at the time of infection relevant DNAzyme 0.8uM was added, media was changed after 6hrs. The supernatant was collected 24 hrs post infection for the determination of virus titer.

[0949] Enhanced Selectivity via Auxiliary Mismatches

[0950] As shown in Figs. 11, 13, and 33, the inventors identified a 'selectivity filter' in the DNAzyme arms where a single mismatch between the DNAzyme and its RNA target drastically reduces catalytic activity. This filter typically spans positions -6 to +3. To determine if selectivity could be extended to positions outside this filter, further experiments were conducted. Fig. 14 shows the results of multiple turnover activity experiments (reported as t90 values) for DNAzymes targeting the PrP839 RNA sequence. As shown in Fig. 14A, a DNAzyme with a single mismatch at position +7 (DzPrP839 T(+7)C, SEQ ID NO. 141) shows no significant loss of activity against the wild-type (wt) target RNA (T-PrP839, SEQ ID NO. 172). However, as shown in Fig. 14B, when an additional 'auxiliary' mismatch is introduced at position +3 (DzPrP839 T(+3)A, SEQ ID NO. 136), high selectivity regarding mutations at position +7 (outside of the selectivity filter) is achieved. The DzPrP839 T(+3)A (SEQ ID NO. 136) containing the auxiliary mismatch at position +3 has only minor effects on the activity against the wt target (T-PrP839, SEQ ID NO. 172) as compared to a full-matching target containing a compensating mutation (T-PrP839 A(+3)U, SEQ ID NO. 174). However, it has a strongly decreased activity against an RNA target, which, compared to the wt target, contains a single nucleotide mutation at position +7 (T-PrP839 A(+7)U, SEQ ID NO. 175). This demonstrates that an auxiliary mismatch, which itself has little effect on activity, can be used to confer high single-nucleotide selectivity to positions outside the primary selectivity filter.

[0951] Increased Precision by Reducing RNase Hl Recruitment

[0952] In a cellular environment, the antisense effect of a DNAzyme, mediated by RNase Hl recruitment to the DNA: RNA duplex formed by the binding arms, can lead to less-specific cleavage. To improve precision, modifications designed to disrupt RNase Hl binding were tested (Figs. 15, 31, 32). In this Unser Zeichen: B407-0001W01 73regard, Fig. 15B shows the results of an RNase Hl-mediated cleavage assay using various modified DNAzymes. The data demonstrates that incorporating blocks of RNA nucleotides into the DNAzyme arms (e.g., SEQ ID NO. 169) can significantly reduce RNase Hl recruitment and subsequent cleavage compared to an unmodified DNAzyme (Dz839, SEQ ID NO. 121). Fig. 15C confirms that these same modifications only moderately reduce the DNAzyme's own catalytic activity. Similar results were also obtained with the constructs tested in Fig. 31 and Fig. 32. This strategy therefore allows for the uncoupling of the specific DNAzyme-mediated cleavage from the less specific RNase Hl-mediated antisense effect, leading to a substantial increase in overall precision in environments containing RNases, such as found in most cellular applications.

[0953] Extended Cellular Lifetimes in Antiviral Assays

[0954] The therapeutic potential of modified DNAzymes was assessed in an antiviral assay. A549 cells were infected with Lymphocytic Choriomeningitis Virus (LCMV), and various DNAzymes targeting the viral genome were introduced. As shown in Figs. 3 and 4, DNAzymes with core modifications such as 6-S-dG(14) (e.g., DzLCMV-1497, SEQ ID NO. 113) demonstrated significant reduction in viral titer 24 hours post-infection compared to controls. To assess cellular lifetime, the effect of terminal modifications was investigated. Fig. 5 shows that an unmodified DNAzyme (DzLCMV-1497) loses its antiviral effect by 48 hours. In contrast, a DNAzyme with 2'0Me modifications at its 5' and 3' ends (DzLCMV-1497[5'-2'OMe; 3'-2'OMe], SEQ ID NO. 229) maintains significant antiviral activity for up to 72 hours. This demonstrates that such modifications confer an increased cellular lifetime, prolonging the therapeutic effect of the DNAzyme. In contrast, terminal LNA modifications did not show a similar extension of activity (Fig. 6).

[0955] The inventors further demonstrate that using a pattern comprising one or two 2'OMe-modified nucleotides that are flanked by one LNA-modified nucleotide, i.e. on the 5' arm the pattern: "5'end-(LNA)(2'OMe)(2'OMe)" and on the 3' arm the pattern: "(2'0Me)(2'0Me)(LNA)-3'end", in which at least one LNA-modified nucleotide does not form a regular base-pair with the target RNA (overhang), also provides effective means to increase in vivo effects of DNAzymes, as shown in antiviral in vivo experiments (Figs. 44 and 45, SEQ ID No 297). This pattern can also be combined with the herein reported RNase evasion pattern (Fig. 52, SEQ ID NOs: 325-334), combining multiple benefits into one DNAzyme and confirming the modular nature of the approach.

[0956] Unser Zeichen: B407-0001WO1 74Identification of RNase Hl Evasion Patterns

[0957] To overcome the non-specific RNase Hl-mediated cleavage observed when DNAzymes form heteroduplexes with target RNA, a systematic screening methodology was employed. Reactions were conducted on full-length target RNA at 37°C for 20 minutes, utilizing 400 ng of RNA per measurement, with the reaction terminated by the addition of 0.125 M EDTA. To further increase band resolution and allow identification of specific RNase interaction regions, gel-base cleavage assays were also carried out with minimal RNA-substrate using established Urea-PAGE methodologies. Initial control assays established that RNase Hl alone exerted no digestion effect, whereas RNase Hl in the presence of standard ASOs, active DNAzymes (e.g., Dz839), or catalytically inactive DNAzymes (e.g., Dz839 A5G mutant) led to significant, non-specific RNA cleavage. To identify structural evasion mechanisms, particularly for targeting single-point mutations in heterozygote disease models, DNAzymes carrying systematically placed mismatches were evaluated (Figs. 31, 32 and 41-43). The systematic substitution of specific nucleotides with RNA or 2'0Me modifications revealed that arm substitutions focusing on the 5' and 3' ends were insufficient to prevent nuclease cleavage on full-length RNA. However, targeted modifications to the inner 6 nucleotides of the binding arms positions flanking the catalytic core on both sides demonstrated promising evasion capabilities. Further progressive screening culminated in the identification of optimal 3' arm and 5' arm configurations, leading to the development of the REP-1 and REP-2 motifs. These coordinated patterns take the asymmetry of the pre-catalytic DNA: RNA complex into consideration and successfully disrupt the minimum 5-base-pair RNA: DNA contiguous helical structure required for RNase Hl binding, conferring near-complete RNase evasion while maintaining near-complete DNAzyme catalytic efficacy. One skilled in the art will understand that the identified RNAse evasion pattern (REP-1 and REP-2) reflect DNAzyme constructs that comprise an, often beneficial, minimal total amount of incorporated modified nucleotides and that increasing the number of modified nucleotides, in particular between area I and II, as well as between area III and IV (REP-1 and REP-2) and between the 5'end and area I in REP-2, is also possible and in line with the herein presented data (Figs. 41-43).

[0958] Efficacy of the RNase Evasion Patterns

[0959] The REP-1 and REP-2 architectures rely on precisely coordinated suitable modifications such as 2'-O-methyl (2'0Me) modifications. For the 5' arm, the pattern coordinates from the catalytic loop outward towards the 5' end. For the 3' arm, it coordinates from the 3' end inward towards the loop. Unser Zeichen: B407-0001W01 75When applied to targets including the mRNAs of Prion Protein (PrP) via Dz839, Enhanced Yellow Fluorescent Protein (EYFP) via Dzl84, and KRAS via Dzl89, the modifications dramatically reduced RNase Hl induced cleavage. Assays confirmed that inactive DNAzyme variants containing these evasion patterns (SEQ ID NOs: 284, 286, 288, 290, 293, 295) demonstrated near-zero background RNase Hl cleavage compared to wild-type controls. Conversely, the catalytically active pattern-modified DNAzymes (SEQ ID NOs: 285, 287, 289, 291, 294, 296) retained high DNAzyme-mediated catalytic RNA cleavage, confirming that the structural modifications inhibit nuclease recruitment without hindering the DNAzym's catalytic mechanism.

[0960] In Vivo Limitation of Chronic LCMV Infection and T-Cell Modulation

[0961] The biological efficacy of the DNAzymes was assessed in a complex chronic infection model. C57BL / 6J mice were infected with 2xlOA6 pfu of LCMV clone 13, and received 20 pg of DNAzyme via spleen-targeted liposomes on Days 6 and 8 post-infection. Phenotypic analysis on Day 10 postinfection demonstrated a highly significant reduction in blood viral titers (p < 0.0001) alongside a massive physiological upregulation in IFN-y and TNF-a cytokine production by CD8+ T cells in the blood (p < 0.0001). Furthermore, the specific percentage of Short-lived effector cells (SLECs; IL7R-, KLRG1+) within the virus-specific tet-gp33+ T cell populations was increased. By Day 14 postinfection, systemic viral loads across blood and major organs remained significantly decreased. Importantly, DNAzyme treatment was shown to rescue T cells from immunological exhaustion by limiting viral replication, evidenced by the downregulation of surface exhaustion markers TIM-3 and PD-1 (p < 0.0001) on virus-specific T cells. This immunological functional rescue coincided with a broad reduction in systemic clinical pathology, determined via significantly lowered ALT and AST serum enzyme activities.

[0962] Assessment of anti-cancer efficacy by measurement of VEGF and TGF- 1 secretion via ELISA B16-F10-OVA or MC38-OVA tumor cells were injected subcutaneously into C57BL / 6 mice to establish tumors. Once tumors became measurable (day 8), mice were randomized into treatment groups. Mice received intratumoral injections of DNAzymes targeting VEGF or TGF-P encapsulated in lipid nanoparticles (LNPs), modified DNAzyme-LNP, or empty LNP controls on days 8, 12, and 14. Tumor size was monitored on days 8, 10, 12, 14, and 16. Mice were sacrificed on day 16, and tumors were excised and weighed for endpoint analysis.

[0963] Unser Zeichen: B407-0001WO1 76Assessment of anti-cancer efficacy by measurement of in vivo tumor growth

[0964] B16-F10-OVA or MC38-OVA tumor cells were injected subcutaneously into C57BL / 6 mice to establish tumors. Once tumors became measurable (day 8), mice were randomized into treatment groups. Mice received intratumoral injections of DNAzymes targeting VEGF or TGF-P encapsulated in lipid nanoparticles (LNPs), modified DNAzyme-LNP, or empty LNP controls on days 8, 12, and 14. Tumor size was monitored on days 8, 10, 12, 14, and 16. Mice were sacrificed on day 16, and tumors were excised and weighed for endpoint analysis. All animal experiments were performed under authorization of the Landesamt fur Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV) in accordance with German animal protection laws.

[0965] FIGURE CAPTIONS

[0966] Fig. 1 discloses a schematic representation of an RNA-cleaving DNAzyme (capital letters) in complex with its RNA substrates (lower case letters). N-n preferentially but not necessarily is a Watson-Crick base pair. The shown DNAzyme core region represents the so-called 10-23 DNAzyme. The flanking binding arms can be adjusted to recognize a given RNA sequence (target). The DNAzymes catalyse RNA cleavage between a specific dinucleotide pair (cleavage site) in a reaction that is dependent on divalent metal ions.

[0967] Fig. 2 discloses various nucleotide modifications as claimed herein. The modified nucleotides are given in the chain form, as found as an internal modification of the finished nucleic acid. The IUPAC names given below the structures describe the modification exemplary as the free 3'-Nucleotide-monophosphate, which corresponds to the partially hydrolyzed form of the closed chain structure. Fig. 3 shows that DNAzyme's antiviral activity in cells - DNAzymes were able to reduce virus titer in cells. A -D A549 cells infected with LCMV at MOI 0.1 and DNAzyme 0.8 uM was added at the time of infection and washed away after 6 hours; media collected 24hrs post infection for virus titer determination (n=4). Error bars show SEM. ****p < 0.0001.

[0968] Fig. 4 shows the DNAzymes antiviral activity in cells- DNAzymes were able to reduce virus titer in cells A — C A549 cells infected with LCMV at MOI 0.1 and DNAzyme 0.8 pM was added at the time of infection and washed away after 6 hours; media collected 24hrs post infection for virus titer determination (n=4 Error bars show SEM. ****p < 0.0001.

[0969] Unser Zeichen: B407-0001W01 77Fig. 5 shows the DNAzymes antiviral activity in cells- DNAzymes were able to reduce virus titer in cells A -I A549 cells infected with LCMV at MOI 0.1 and DNAzyme 0.8 pM was added at the time of infection and washed away after 6 hours; media collected 24 hrs, 48 hrs and 72 hrs, respectively, post infection for virus titer determination (n=4). Error bars show SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0970] Fig. 6 shows the DNAzymes antiviral activity in cells- DNAzymes were able to reduce virus titer in cells A — C A549 cells infected with LCMV at MOI 0.1 and DNAzyme 0.8 pM was added at the time of infection and washed away after 6 hours; media collected 24 hrs (A), 48 hrs (B) and 72 hrs (C) post infection, respectively for virus titer determination (n=4).

[0971] Fig. 7 shows the DNAzymes antiviral activity in cells - DNAzymes were able to reduce virus titer in cells A -B A549 cells infected with LCMV at MOI 0.1 and DNAzyme 0.8 pM was added at the time of infection and washed away after 6 hours; media collected 24 hrs post infection for virus titer determination (n=3). It shows extended data, and an activity test of pyrazol modification at G(14) (inactivated).

[0972] Fig 8 shows the DNAzymes antiviral activity in cells- DNAzymes were able to reduce virus titer in cells A -B A549 cells infected with LCMV at MOI 0.01 (A) and MOI 0.1 (B) and DNAzyme 0.8 pM was added at the time of infection and washed away after 6 hours; media collected 24 hrs post infection for virus titer determination (n=3). It shows X6 extended data, it shows conformation that A5G is inactive and a good control. It confirms that results are independent on initial MOI value (between 0.1 and 0.01).

[0973] Fig. 9. Validation of readout parameter and cellular response to DNAzyme treatment targeting eYFPgag mRNA. Two different DNAzyme variants were tested, i.e. Dz839 (comprising an adenine at position 5 (Dz A(5)) and its A5G mutation (Dz G(5)). HEK293 cells were co-transfected with indicated concentrations of respective DNAzymes and constant amount of eYFPggg-plasmid. Neg. = negative control without plasmid; Pos. = positive control without DNAzyme. Number of eYFP positive cells were determined via fluorescence imaging (A) or copy number of mRNA was determined via qPCR (B). The data show clear dose response behaviour, narrowing down the required DNAzyme concentration range. Since the Dz G(5) variant is catalytically inactive, it serves as valuable control to assess the antisense effect. According to the data the active DNAzyme Dz A(5) shows significantly increased effects, indicative of cellular activity surpassing the antisense effect.

[0974] Fig. 10. In vitro assessment of DNAzyme performance. Plot of relative cleavage rate ratios (kobs / kobs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard Unser Zeichen: B407-0001W01 78assay conditions (FRET reaction buffer). Dz-ref corresponds to SEQ. ID No. 1. Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. Data was recorded using DzPrP839 comprising the indicated modifications in the catalytic core.

[0975] Fig. 11 Experimental data showing the effect of single point mismatches in each position of the Dz arm on the cleavage activity (kobs). The relative cleavage activity (kobs / kobs, Dz-ref) is shown, with Dz-ref being the regular Dz sequence that is fully complementary to the RNA target as shown in Fig.

[0976] 1. Data was recorded on DNAzymes targeting the indicated different RNAs, showing individual variations but overall similar pattern, allowing to define the nucleotide positions that determine specificity, i.e. the selectivity filter. Respective sequence variations of used DNAzymes are provided in Table 1.

[0977] Fig. 12 DNAzyme-mediated cleavage but not RNase Hl-mediated cleavage has single-nucleotide selectivity. (A) DNAzyme-mediated cleavage of full-length mRNA transcripts. Cleavage yield was determined via EMSA. Dz839 was used. The data show that a single-nucleotide mutation, such as present in DzT(-3)A, can strongly reduce cleavage activity of the substrate comprising a single-nucleotide mismatch (RNA_wt), while full activity is recovered for the matching target (RNA_A(-3)U). Note, that the sequence similarity between RNA_wt and RNA_A(-3)U is 99.9%, highlighting the extraordinary substrate selectivity of the DNAzyme-mediated reaction. (B) RNase Hl-mediated cleavage of full-length mRNA transcripts. The data show that the presence of RNase Hl cause RNA cleavage in the presence of complementary DNA sequences. The effects are comparable between the ASO control (comprising a gapmer design, known to favor RNAse Hl recruitment) and all tested DNAzyme variants. The inactive Dz_A(5)G variant confirms that RNase and not DNAzyme-mediated cleavage is the predominant contribution to cleavage. The data show that RNase-mediated cleavage has little or no selectivity towards the single-nucleotide mismatch present in the DzT(-3)A variants.

[0978] Fig. 13. Dz: RNA schematic highlighting the regions of the selectivity filter and the auxiliary selectivity filter.

[0979] Fig. 14. Auxiliary mismatches strongly expand the DNAzyme's accessible high-selectivity region (as shown in Fig. 13). Results of multiple turnover activity experiments (t_90 values; i.e. the time required to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNA over Dz) are shown for indicated combinations of Dz and RNA target. While a single nucleotide mismatch at position +7 (outside of the selectivity filter) does not reduce activity (A), the nucleotide at position +7 can be selectivity targeted with an auxiliary mismatch at position +3 leading to a double Unser Zeichen: B407-0001W01 79mismatch at positions +3 and +7 (B). Note that the auxiliary mismatch has no or little effect on the cleavage activity towards the wt RNA.

[0980] Fig. 15. Increasing DNAzyme precision by reducing RNase Hl mediated cleavage. (A) RNase Hl mediated cleavage induced by presence of antisense oligonucleotide or DNAzyme. (B) RNase Hl mediated cleavage induced by presence of different DNAzymes. The data shows that DNAzymes with modifications in the arm regions that disturb the double helix conformation can gradually reduce RNase Hl recruitment. (C) DNAzyme-mediated cleavage of variants used in (B), showing weak or only moderately reduced cleavage capabilities of variants with reduced RNase Hl recruitment.

[0981] Fig. 16. Priority ranking according to in silico data. The likelihood for in-line attack (ILA) formation in the DNAzyme carrying the respective modification(s), the number of replicas showing at least five state changes over the simulation time, and the likelihood for in-line attack formation using only verified replicas are shown. Modifications replace the nucleotide present at the respective position and are either included at position 14 (14) and / or position 6 (6) by replacing the respective nucleotide, and simulated in DNAzyme variants comprising the indicated nucleotide at position 5. Fig. 17. Angle of attack distribution of all frames below 4 A between 02' of rGOand N2 of a DNAzyme carrying a POP modification at position 14 (SEQ ID No. 78). All angles above 120° (dashed line) are considered attack angles, able to introduce the ILA. A total of 7084 out of 100,000 frames reached a distance <4 A. Only Replicas 0, 1, 4, and 6 were able to close the distance, of which only Replicas 1 and 6 initiate an ILA.

[0982] Fig. 18. Preferred sites for modifications have been marked.

[0983] Fig. 19. Nucleotide numbering in the catalytic core.

[0984] Fig. 20. Relative time of 7T-7T-stacking during ILA versus relative time in the in-line attack formation. Modifications are arranged from lowest to highest ILA. Above 10% ILA as indicated by the dashed line, less relative time of 7T-7T-stacking for the modifications is found. Notably, 7MG (SEQ ID No. 64) together with 6IP (SEQ ID No. 66) shows almost no 7T-7T-stacking but increases ILA formation to 36%. Fig. 21. Validation of ILA formation against experimental activity data. The correlation of experimental data (left axis) with the likelihood of in-line attack formation (right axis) is shown. " Recalculated in-line attack" considers only those trajectories in which at least 5 state changes are observed. The highest activity and ILA formation were previously observed with a thioguanine modification at position 14 (6-S-dG(14)), while an ethenoadenine modification reduces the activity Unser Zeichen: B407-0001WO1 80to zero as predicted. The simulations align well with the experimental data except for PYR(14) and 2-amino-purine(14).

[0985] Fig. 22. Calculated dipole moment in Debye. All calculations were performed with the B3LYP functional and the 6-31G** basis set in ORCA and Gaussian 16. Overall, both the magnitude and the direction of the dipole moments align well with their previously reported experimental values. The gas phase calculations of ORCA resulted in p = 0.027 and R2 = 0.993, while Gaussian reached p = 0.003 and R2 = 0.985, showcasing high reproducibility.

[0986] Fig. 23. Directions of experimental dipole moments (top row) compared to directions of calculated dipole moments (bottom row). From left to right: Imidazole, 1,3,4-thiadiazole (same direction as 1,3,4-oxadiazole), and isoxazole. The directions align well in each case.

[0987] Fig. 24. Workflow to generate and analyze non-natural nucleobases. All nucleobases were designed and simulated using the AMBER software package.

[0988] Fig. 25. List of used DNAzyme sequences.

[0989] Fig. 26. Target RNA sequences.

[0990] Fig. 27. In vitro assessment of DNAzyme activity. (A) Plot of relative cleavage rate ratios (kObs / kObs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard assay conditions (FRET reaction buffer). Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. Data was recorded using DNAzyme variants comprising binding arm SEQ ID Nos 215 and 216 and a core region with the shown SEQ-ID Nos. (B) Results of multiple turnover activity experiments using the same constructs as in (A). Relative tgo values; i.e. the fold change in time (as compared to SEQ ID NO. 1) to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNA over Dz. Higher values indicate faster substrate turnover. The data show that modifications associated to SEQ ID NO. 24 and 69 effectively inactivate the DNAzyme.

[0991] Fig. 28. In vitro assessment of DNAzyme activity. (A) Plot of relative cleavage rate ratios (kObs / kObs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard assay conditions (FRET reaction buffer). Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. Data was recorded using DNAzyme variants comprising binding arm SEQ ID NOs 215 and 216 and a core region with the shown SEQ-ID NOs. Data was recorded with higher sampling rates as compared to data shown in Fig. 10, allowing to detect faster rates with higher accuracy. (B) Results of multiple turnover activity experiments using the same Unser Zeichen: B407-0001WQ1 81constructs as in (A). Relative tgo values; i.e. the fold change in time (as compared to SEQ ID NO. 1) to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNA over Dz. Higher values indicate faster substrate turnover. The data show that modifications associated to SEQ ID NO. 54 can increase catalytic activity.

[0992] Fig. 29. In vitro assessment of DNAzyme activity. (A) Plot of relative cleavage rate ratios (kObs / kObs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard assay conditions (FRET reaction buffer). Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. DNAzyme variants comprising binding arm SEQ ID Nos 221 and 222 and a core region with the shown SEQ-ID NOs. Data was recorded with same sampling rates as data in Fig 28. (B) Results of multiple turnover activity experiments using the same constructs as in (A). Relative tgo values; i.e. the fold change in time (as compared to SEQ ID NO. 1) to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNA over Dz. Higher values indicate faster substrate turnover. The data support findings reported in Fig. 28, showing that modifications associated to SEQ ID NO. 54 can increase catalytic activity, and confirm transfer of this effect to a different target RNA.

[0993] Fig. 30. In vitro assessment of DNAzyme activity. (A) Plot of relative cleavage rate ratios (kObs / kObs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard assay conditions (FRET reaction buffer). Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. Data was recorded using DNAzyme variants comprising the indicated SEQ ID NOs, which contain methyl phosphonate modifications at specific positions in the arms and / or the catalytic core. By reducing the net charge of the DNAzyme backbone the respective methyl phosphonate modifications will reduce Mg2+interactions at the selected positions, with the aim to reduce the unwanted Mg2+dependency. (B) Results of multiple turnover activity experiments using the same constructs as in (A). Relative tgo values; i.e. the fold change in time (as compared to SEQ ID NO. 1) to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNA over Dz. Higher values indicate faster substrate turnover. The data reveal variants with increased activity, including SEQ ID NOs: 182, 183, 184, associated with enhanced cleavage rates and / or improved catalytic turnover.

[0994] Fig. 31. Increasing DNAzyme effective selectivity (precision) by reducing RNase Hl mediated cleavage. (A) Co-incubation of indicated RNA, DNA and / or DNA-RNA hybrids with RNase Hl. Minimal RNA targets were used. (B) Cleavage activity of different DNAzyme variants in the presence or the absence of RNase Hl. Same buffer conditions, Mg2+level, and incubation times for DNAzyme mediated and RNase Hl mediated cleavage reaction were used. The data show that DNAzymes with Unser Zeichen: B407-0001WQ1 82modifications in the arm regions that disturb the double helix conformation can gradually reduce RNase Hl recruitment (including SEQ ID NOs: 167, 168, 169, 170, 186, 187). Variants that reduce RNase Hl recruitment and maintain (partly) their own catalytic capabilities are particularly promising and include (SEQ ID NOs: 167, 170).

[0995] Fig. 32. Increasing DNAzyme effective selectivity (precision) by reducing RNase Hl mediated cleavage. (A, B) Cleavage of full-length RNA by indicated DNAzyme variants in the absence of RNase Hl. Reaction was stopped after 90 min. (C, D) Cleavage of full-length RNA in the presence of indicated DNAzyme variants and RNase Hl. Reaction was stopped after 20 min. Variants that reduce RNase recruitment include SEQ ID NOs: 194, 188, 189, 190, 191, 192, 195, 196, 197, 198. Variants that maintain (partly) their own catalytic capabilities are particularly promising and include (SEQ ID NOs: 188, 189, 190, 191, 192, 193, 195, 196, 197, 198).

[0996] Fig. 33. Experimental data showing the effect of single point mismatches in each position of the Dz arm on the cleavage activity (kobs). The relative cleavage activity (kobs / kobs, Dz-ref) is shown, with Dz-ref being the regular Dz sequence that is fully complementary to the RNA target as shown in Fig.

[0997] 1. Data was recorded on DNAzymes targeting the indicated different RNAs. Data for PrP binding arms (SEQ-ID NOs: 215 and 216 and SEQ ID NOs: 123 - 144) and KRAS biding arms (SEQ ID NOs: 225 and 226 and SEQ ID NOs: 145 - 163) are identical to Fig. 11 and are compared to effects on EYFP binding arms (SEQ ID NOs: 1 1 and 228 and SEQ ID NOs: 199 - 214). Note that the EYFP targeted region is one nucleotide shorter on each arm as compared to the other two targets. The mean values of the respective sequence positions are shown as dashed line. Although outliers are detected in different systems, the selectivity filter encompasses all positions where a single mismatch in the binding arm sequence results in an activity decrease to less than 40% of the reference activity in the majority of tested systems. Therefore, mismatches within the selectivity filter represent the most likely positions to strongly affect activity and consequently determine the selectivity of the DNAzyme. The developed knowledge of the selectivity filter hence allows to identify the positions in the substrate binding sequence that have the highest probability to be sensitive to single point mutations in the targeted RNA and to identify positions that, without additional modifications, are not selective towards single point mutations.

[0998] Fig. 34. List of used DNAzyme sequences with various modifications used in the experiments shown in Figs. 30, 31, 32, and 33.

[0999] Fig. 35. Table listing the SEQ IDs for the binding arms of DNAzymes targeting various genes (PrP, LCMV, KRAS, EYFP).

[1000] Unser Zeichen: B407-0001W01 83Fig. 36. Table listing the SEQ IDs for DNAzyme variants with 2'0Me and LNA end-modifications and PYR core modifications used in the antiviral assays of Figs. 5-7.

[1001] Fig. 37. Table correlating the names of modified DNAzymes used in Figs. 16, 20, and 21 with their corresponding core sequences and SEQ IDs.

[1002] Fig. 38. Table linking the data presented in each figure to the relevant claims, SEQ IDs, and technical topic, providing a map of the application's disclosure.

[1003] Fig. 39. Comparison of RNase Hl induced cleavage and DNAzyme-mediated cleavage. (A-C) RNase Hl induced cleavage using catalytically inactive DNAzymes (A5G mutation) targeting different RNAs (A) PrP839, (B) EYFP184, (C) KRAS189. For each RNA target the unmodified binding arms are compared to two RNase Hl evasion pattern (see SEQ ID NOs for details). (D-F) DNAzyme mediated cleavage with wild-type DNAzyme core sequences (SEQ ID NO. 1) targeting the different RNAs (D) PrP839, (E) EYFP184, (F) KRAS189. For each RNA target the unmodified binding arms are compared to the two RNase Hl evasion pattern used in (A-C) (see SEQ ID NOs for details). (G-l) FRET-based determination of kObs for indicated variants. Data in (D-l) was recorded in absence of RNase Hl. The data demonstrate that for all three different RNA targets the two RNase Hl evasion pattern effectively suppress RNase Hl mediated RNA cleavage, while having no or only minor negative effects on the DNA-mediated cleavage activity. Thus, the data reveal that the herein introduced RNase evasion pattern represent a general beneficial strategy to effectively increase precision of the DNAzyme technology.

[1004] Fig. 40 represents an optical schematic outlining the arrangements of RNase evasion pattern 1 and 2, i.e. REP-1 (A) and REP-2 (B). DNAzyme representation and numbering refers to representation in Fig. 1 with 'Loop' referring to the catalytic core region. Underscored nucleotide positions indicate positions for chemical modifications capable to hinder RNase binding. Herein, in particular 2'0Me modifications were used at the indicated positions to disrupt DNA-RNA duplex arrangement and thus RNase interaction. One skilled in the art will understand that other modifications with similar effects are also available, which is additional supported by our data using RNA substitutions or 2'MOE modifications. The sequence positions comprising these chemical modifications are indicated by roman numbers ( l-IV). The inventors have found an asymmetry in the binding arms in the pre-catalytic Dz: RNA complex, which is associated with different RNase evasion pattern on the 5' and 3' substrate binding arms. In this regard the 5' arm in REP-1 comprises modifications at positions -8,-7 (area I) and -4,-3 (area II). For REP-2 the 5' arm comprises modifications at positions -6,-5 (area I) and -2 (area II). Shortening and elongation of the 5' arm will keep theses numbering Unser Zeichen: B407-0001WQ1 84constant for REP-1 and REP-2. The 3' arm is identical in REP-1 and REP-2 and comprises modifications at the last nucleotide at the 3' end (area IV), e.g. position +9 for an 3' arm length of 9 nucleotides or position +8 for an 3'arm length of 8 nucleotides. In addition, the 3' arm comprises a modification in area III which is preceding area IV by four nucleotides, e.g. position +5 for an 3' arm length of 9 and position +4 for an 3' arm length of 8. REP-1 and REP-2 have been identified to represent the variants with the minimally required number of modifications to realize an efficient RNase evasion. One skilled in the art will understand that increasing the number of modifications, in particular between area I and II and area lll-IV and between the 5'end and area I in REP-2, is also possible and in line with the herein presented data (Figs. 41-43). The molecular structures of the precatalytic Dz839: RNA (shown at the bottom of A and B) support the asymmetry of the 5' and 3' binding arm accessibility as well as the minimal pattern required to interfere with RNase binding. In this regard a central feature is the absence of accessible DNA: RNA duplex regions comprising four or more unmodified consecutive DNA nucleotides. In the shown representation of the DNAzyme: RNA complex nucleotides in the respective areas l-IV are highlighted in surface representation.

[1005] Fig. 41 displays RNA gel electrophoresis images for characterization of RNase evasion pattern. Using systematic RNA substations or 2'MOE modifications at different position in the substrate binding arms, the data identify different pattern that promote RNase Hl evasion and narrow down minimal requirements that enable the RNase Hl evasion.

[1006] Fig. 42 displays RNA gel electrophoresis images for characterization of RNase evasion pattern. Using systematic RNA substations at different position in the substrate binding arms, the data identify different pattern that promote RNase Hl evasion and narrow down minimal requirements that enable the RNase Hl evasion.

[1007] Fig. 43 displays RNA gel electrophoresis images for characterization of RNase evasion pattern. (A) demonstrating that RNA substitutions as well as 2'0Me and 2'MOE modifications have comparable effects on RNase Hl evasion when placed at identical position in the substrate binding arms. In addition, a minimal evasion pattern for the 3' arm is identified. (B) Identification of the two modification pattern in the 5' arm leading to REP-1 and REP-2 when combined with the 3' pattern identified in (A). (C) Comparison of DNA-mediated activity and RNase Hl-mediated cleavage for Dz839 variants comprising wild-type, inactive A5G, inactive A5G REP-1, active REP-1, inactive A5G REP-2, and active REP-2 variants. The data demonstrate effective RNase Hl evasion for REP-1 and REP-2. In addition, it is shown that DNAzymes comprising either REP-1 or REP-2 maintain a high level of catalytic activity.

[1008] Unser Zeichen: B407-0001W01 85Fig. 44. Antiviral DNAzymes limit chronic LCMV infection in vivo. C57BL / 6J mice were injected with LCMV clone 13. On day 6 and 8 p.i., mice were divided into groups and injected with empty lipid nanoparticles (LNPs), and LNPs loaded with wild-type DNAzyme and stabilized DNAzyme. Analyses were performed on day 10 p.i. (A) LCMV titers were determined in blood. Data presented as mean±SEM, n=6. (B) IFN-y and TNF-a production by CD8+T cells in blood after re-stimulation with LCMV-specific peptides (gp33) was determined. Data presented as percentage of CD8+T cells, mean±SEM, n=6. (C) Tet-gp33+T cells were determined in the blood. Data presented as percentage of CD8+T cells. Short-lived effector cells (SLECs, IL7R“, KLRG1+) within tet-gp33+T cell subset were determined in blood. Data presented as percentage of tet-gp33+T cells. Mean±SEM, n=6.

[1009] Fig. 45. Antiviral DNAzymes modulate T cell function during chronic LCMV infection. C57BL / 6J mice were injected with LCMV clone 13. On day 6 and 8 p.i., mice were divided into groups and injected with empty lipid nanoparticles (LNPs), and LNPs loaded with wild-type DNAzyme and stabilized DNAzyme. Analyses were performed on day 14 p.i. (A) Tet-gp33+T cells were determined in blood and spleen. Data presented as percentage of CD8+T cells. Short-lived effector cells (SLECs, IL7R“, KLRG1+) and memory precursor effector cells (MPECs, I L7 R+, KLRG1 ) within tet+T cell subsets were determined. Data presented as percentage of tet+T cells. Mean±SEM, n=6. (B) Surface expression of TIM-3 and PD-1 was determined on tet-gp33+T cells. Data presented as MFI, mean±SEM, N=6. (C) IFN-y and TNF-a production by CD8+T cells in blood and spleen after re-stimulation with LCMV-specific peptides (gp33) was determined. Data presented as percentage of CD8+T cells, mean±SEM, n=6.

[1010] Fig. 46. Antiviral DNAzymes decrease viral load and pathology during chronic LCMV infection. C57BL / 6J mice were injected with LCMV clone 13. On day 6 and 8 p.i., mice were divided into groups and injected with empty lipid nanoparticles (LNPs), and LNPs loaded with wild-type DNAzyme and stabilized DNAzyme. Analyses were performed on day 14 p.i. (A) LCMV titers were determined in blood and organs. Data presented as mean±SEM, n=6. (B) ALT and AST activity in the serum was evaluated. Data presented as mean±SEM, n=6 mice per condition.

[1011] Fig. 47. (A-D) Effects of DNAzyme transfection on cytokine secretion in B16-F10 melanoma cells. VEGF secretion at 24 h (A) and 48 h (B), and TGF-pi secretion at 24 h (C) and 48 h (D) were measured in culture supernatants collected from B16-F10 cells following transfection with the indicated DNAzymes. (E-F) same as (A-D) but using MC38 cells. Cytokine concentrations were quantified using ELISA (n=3). Error bars show SEM.

[1012] Unser Zeichen: B407-0001W01 86Fig. 48. DNAzyme-LNP treatment suppresses tumor growth in a B16-OVA melanoma model (A-D) as well as a MC38-OVA tumor model (E-H). Tumor growth curves (A, C) and endpoint tumor weights (B, D) in C57BL / 6 mice bearing B16-OVA tumors treated with DNAzyme-LNP formulations targeting VGEF (A, B) or TGF-P (C, D). Either Wild type DNAzyme (SEQ ID NO. 298 / 300, for VGEF / TGF, respectively) or modified DNAzymes (SEQ ID NO. 299 / 301 for VGEF / TGF, respectively) were used. Empty LNPs served as control. (E-H) Same as (A-D) but using a MC38-OVA tumor model. Treatments were administered on days 8, 12, and 14, tumor volumes were monitored on days 8-16, and tumors were excised and weighed on day 16. Error bars show SEM.

[1013] Fig. 49. In vitro assessment of DNAzyme performance. Plot of relative cleavage rate ratios (kobs / kobs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard assay conditions (FRET reaction buffer). Dz-ref corresponds to SEQ ID NO. 1. Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. Data was recorded using DzPrP839 (Binding arms (5' / 3'): SEQ ID NO: 215 / 216) comprising the indicated modifications (given SEQ ID NO.) in the catalytic core.

[1014] Fig. 50. In vitro assessment of DNAzyme activity. (A) Plot of relative cleavage rate ratios (kObs / kObs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard assay conditions (FRET reaction buffer). Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. DNAzyme variants comprising binding arm SEQ ID NOs 215 and 302 and a core region with the shown SEQ-ID NOs. The used RNA target comprises a UGUU cleavage site sequence (SEQ ID NO. 303) that differs from the CGUU cleavage site of the regular targeted RNA sequence (SEQ ID NO. 172). This difference has been reported before to strongly affect other modifications in the catalytic core, such as a 2'-MOE-dG modification at position G14. (B) Results of multiple turnover activity experiments using the same constructs as in (A). Relative tgo values; i.e. the fold change in time (as compared to SEQ ID NO. 1) to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNA over Dz. Higher values indicate faster substrate turnover. The data support findings reported in Fig. 28, showing that modifications associated to SEQ ID NO. 54 can increase catalytic activity, and confirm transfer of this effect to a different cleavage site pattern of targeted RNA. This cleavage site tolerance is an advantage over other activity increasing modifications reported in the literature before.

[1015] Fig. 51. In vitro assessment of DNAzyme activity. (A, B) Plot of relative cleavage rate ratios (kObs / kobs, Dz-ref) determined via FRET-based activity assays of indicated DNAzyme variants at standard assay conditions (FRET reaction buffer). Values above or below 1 identify variants with increased or decreased catalytic activity, respectively. (A) Data for DNAzyme variants with shown SEQ ID NOs Unser Zeichen: B407-0001WQ1 87(for SEQ ID NOs: 1, 304, 305, 101 comprising binding arm SEQ ID NOs 215 and 216). The used RNA target comprises a CGUU cleavage site sequence (SEQ ID NO. 172). (B) Data for DNAzyme variants with shown SEQ-ID NOs (for SEQ ID NOs: 1, 304, 101 comprising binding arm SEQ ID NOs 215 and 302). The used RNA target comprises a UGUU cleavage site sequence (SEQ ID NO. 303). The data demonstrate the activity increasing benefits of the used multiple chemical modifications at position 14 that surpass the wild-type DNAzyme (SEQ ID NO. 1) as well as a previously reported 2'OME modification at position 14 (SEQ ID NO. 306 / 307, for a CGUU and UGUU cleavage site, respectively). The data reveal strongly increased cleavage rates with kObs values surpassing 2 min1, representing a more than 10-fold increase in catalytic activity. In addition and in contrast to the single 2'OME modification at G14 (SEQ ID NOs 306 and 307), all shown variants with multiple chemical modifications at position G14 (SEQ ID NOs. 101, 304, 305) lead to an increase over the unmodified variant (SEQ ID NO. 1) independent of the cleavage site nucleotide pattern, rendering these variants more tolerant for a broad range of targets. (C-E) Representation of nucleotide modifications as claimed herein. The modified nucleotides are given in the chain form, as found as an internal modification of the finished nucleic acid.

[1016] Fig. 52. demonstrates compatibility of RNase evasion pattern and established modification pattern to increased cellular life time. (A-C) RNase Hl induced cleavage using catalytically inactive DNAzymes (A5G mutation) targeting different RNAs (A) PrP839, (B) KRAS189, (C) LCMV2337. For each RNA target the unmodified binding arms are compared to two variants combining an RNase Hl evasion pattern (REP-1) with stabilizing modification pattern at the 5' and 3'ends (see SEQ ID NOs for details). For the LCMV2337 system two additional mismatches / overhang pattern at the 5'end were considered (see SEQ ID NOs for details). In general, stabilizing and RNase evasion pattern can be combined via addition of an unpaired LNA-modified nucleotide at the 3' (overhang) following the 2'0Me modification of the REP-1 and REP-2 pattern. Similarly, on the 5' -end the REP-1 pattern can be combined with a terminal LNA-modified nucleotide in a suitable way, which includes overhang of an unpaired LNA-nucleotide, a LNA-modified nucleotide matching the target RNA or LNA-substition of the 2'0Me modification of the first nucleotide in the REP-1 pattern. One skilled in the art will understand that a large variety of similar possibilities of combining stabilizing and RNase evasion pattern will realize the same features as shown here, and that the lengths and composition of the binding arms should also be adjusted to enable optimal substrate turnover rates. The data demonstrate effective Rnase evasion also for the DNAzyme comprising the combined set of modifications.

[1017] Unser Zeichen: B407-0001W01 88Fig. 53. assesses the effects of charge-reducing modifications at the DNAzyme 'interface' region. Data from In vitro FRET assays using DNAzyme variants comprising the indicated SEQ ID NOs, which contain methyl phosphonate modifications at specific positions in the 'interface' region, are shown. By reducing the net charge of the DNAzyme backbone the respective methyl phosphonate modifications will reduce Mg2+interactions at the selected positions, with the aim to reduce the unwanted Mg2+dependency. (A) Results of multiple turnover activity experiments. Relative tgo values; i.e. the fold change in time (as compared to the respective unmodified variant SEQ ID NO.

[1018] 121) to reach 90% of the maximal RNA cleavage amount using 10-fold access of RNAover Dz. Higher values indicate faster substrate turnover. The data reveal variants with beneficial catalytic features, including SEQ ID NOs: 341, 342, 343, associated with improved catalytic turnover. (B) Assessment of Mg2+dependency of indicated variants using the same assay as in (A) but at varying concentrations of Mg2+, revealing particularly beneficial effects at the lowest tested Mg2+concentrations (i.e. 0.3 mM).

[1019] Fig. 54. List of used DNAzyme sequences with various modifications used in the experiments shown in Figs. 39-53.

[1020] Fig. 55. provides additional data obtained in FRET-based activity assays or MD simulations. The FRET data support the non-trivial cross talk between modifications at positions 5 and 14 in the catalytic loop. In this regard, the FRET data reveal a clear decrease in activity for the 2'0Me modification at position G14 when used in combination with a C(5) modification, while the same 2'0Me modification leads to a strong increase in activity in the A(5) variant. Furthermore, MD simulations reveal that both a 6-S-dG and a 6-Se-dG modification at position 14 lead to a strong increase in formation of the in-line-attack conformation, supporting that the chemical similarity of Selene and Sulphur also translates to its beneficial role in increasing activity of the DNAzyme.

[1021] Fig. 56. Table linking the data presented in figures 39-54 to the relevant claims, SEQ IDs, and technical topic, providing a map of the application's disclosure.

[1022] Unser Zeichen: B407-0001WQ1 89

Claims

HEINRICH HEINE UNIVERSITY CH Kilger Anwaltspartnerschaft mbB Dusseldorf Fasanenstraße 29 Our Ref.: B407-0001W01 10719 BerlinCLAIMS1. Nucleic acid encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence SEQ ID NO. 1, wherein the number in parenthesis designates the position number, 5'-G (1), G (2), C (3), T (4), A (5), G (6), C (7), T (8), A (9), C (10), A (11), A (12), C (13), G (14), A (15)-3' (SEQ ID NO. 1), and wherein said core region nucleic acid sequence comprises one or more of the following modifications,i. a modification that increases activity,ii. a modification that reduces or abolishes activity,ill. a modification that influences target association and / or dissociation resulting in a changed, reduced or improved catalytic turnover,iv. a modification that increases target RNA selectivity,v. a modification that reduces target RNA selectivity,vi. a modification that reduces the antisense effect via reduced RNase recruitment to increase the DNAzyme precision in (cellular) applications, vii. a modification that promotes the antisense effect via enhanced RNase recruitment to increase the overall effect of DNAzyme treatment in (cellular) applicationsviii. a modification that increases affinity to, or reduces the need for, metal ions, including but not limited to Mg2+,ix. a modification that increases cellular life-time,x. a modification that decreases innate immune response,xi. a modification that increases innate immune response.

2. Nucleic acid according to claim 1, encoding a catalytic core region of a catalytic deoxyribozyme (DNAzyme), wherein said catalytic core region comprises the following sequence SEQ ID NO. 1, wherein the number in parenthesis designates the position number, 5'-G (1), G (2), C (3), T (4), A (5), G (6), C (7), T (8), A (9), C (10), A (11), A (12), C (13), G (14), A (15)-3' (SEQ ID NO. 1), and wherein said core region nucleic acid sequence comprises one or more of the following modifications, at position 5,Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'SEQ ID5' G G C T A G C T A C A A C G A 3' pos. / Modification NO.1SEQ ID5' G G C T N* G C T A C A A C G A 3' A*(5)=etheno-dA NO.2SEQ ID5' G G C T N* G C T A C A A C G A 3' N* (5)=dNebularine NO.3SEQ ID5' G G C T N* G C T A C A A C G A N*(5)=ANT NO.4SEQ ID5' G G C T N* G C T A C A A C G A A*(5)=Me-Ph NO.5at position 6,Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'SEQ ID G G C T A G C T A C A A C G A 3' Pos. / Modification NO.1SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=6-S-dG NO.6SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=6-Se-dG NO.7SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=8-oxo-dG NO.8SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=Iso-dG NO.9Unser Zeichen: B407-0001W01 2SEQI DG*(6)=6-O-methyl- 5' G G C T A N* C T A C A A C G A 3' NO. dG 10 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=7-deaza-dG NO.11 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2'-OMe-dG NO.12 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2'-MOE-dG NO.13 SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=etheno-dA NO.14SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=ANT NO.15SEQ ID G*(6)=2-amino- 5' G G C T A N* C T A C A A C G A 3' NO. purine 16 SEQ JO 5' G G C T A N* C T A C A A C G A 3' G*(6)=2-amino-dA NO.17 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=dNebularine NO.18 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=7MG NO.19 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=IOX NO.20 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=6IP NO.21Unser Zeichen: B407-0001W01 3SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=3DS NO.22 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2DO NO.23 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=PYR NO.24 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=NIP NO.25 SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=FIP NO.26SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=3NA NO.27SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=7OP NO.28SEQO 5' G G C T A N* C T A C A A C G A 3' G*(6)=3DO NO.29SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2DS NO.30 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=B0G NO.31 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=2TA NO.32 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=P0P NO.33Unser Zeichen: B407-0001W01 4SEQID G T A N* C T A C A C G A 3' G*(6)=NTZ NO.34SEQ ID5' G G C T A N* C T A A C A 3' G*(6)=MES NO.35SEQ IDG G T A N* C T A A C G A 3' G*(6)=TTZ NO.36SEQ IDG G C T A N* C T A A C A 3' G*(6)=MER NO.37SEQ ID G T A N* C T A A C G A 3' G*(6)=M2R NO.38SEQ IDG G C T A N* C T A C A C A 3' G*(6)=M2S NO.39SEQ IDG G T N* C T A C A C A 3' G*(6)=BTG NO.40SEQ IDG G C T A N* C T A A C A 3' G*(6)=6TP NO.41SEQ IDG G T A N* C T A A C A 3' G*(6)=PHY NO.42SEQ ID G G C T A N* C T A C A C A 3' G*(6)=3TA NO.43SEQ IDG T N* C T A A C A 3' G*(6)=MTG NO.44SEQ IDG G C T A N* C T A A C A 3' G*(6)=AIP NO.45Unser Zeichen: B407-0001W01 5SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=D0P NO.46 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=AEG NO.47 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=AMG NO.48 SEQI D5' G G C T A N* C T A C A A C G A 3' G*(6)=IOX2 NO.49 SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=Me-Ph NO.50SEQ ID5' G G C T A N* C T A C A A C G A 3' G*(6)=6aG NO.51SEQ ID5' G G C T A N* C T A C A A C G A 3' N*(6)=6-cG NO.52SEQO 5' G G C T A N* C T A C A A C G A 3' N*(6)=7MP NO.53at position 14,Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3' SEQ ID5' G G C T A G C T A C A A C G A 3' Pos. / Modification NO.1 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=6-S-dG NO.54 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=6-Se-dG NO.55SEQ5' G G C T A G C T A C A A C N* A 3' G*(14)=8-oxo-dG IDUnser Zeichen: B407-0001W01 6NO.56 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=Iso-dG NO.57 SEQG*(14)=6-O-methyl- JO 5' G G C T A G C T A C A A C N* NO. dG 58 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=7-deaza-dG NO.59SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=etheno-dA NO.60SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=ANT NO.61SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=2-amino-dA NO.62SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=dNebularine NO.63SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=7MG NO.64SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=IOX NO.65SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=6IP NO.66SEQO 5' G G C T A G C T A C A A C N* A 3' G*(14)=3DS NO.67SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=2DO NO.68SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=PYR NO.69S|EDQ5' G G C T A G C T A C A A C N* A 3' G*(14)=NIP Unser Zeichen: B407-0001W01 7NO.70 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=FIP NO.71 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=3NA NO.72 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=7OP NO.73 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=3DO NO.74 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=2DS NO.75 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=B0G NO.76 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=2TA NO.77 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=P0P NO.78 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=NTZ NO.79 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=MES NO.80 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=TTZ NO.81 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=MER NO.82 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=M2R NO.83 S|EDQ5' G G C T A G C T A C A A C N* A 3' G*(14)=M2SUnser Zeichen: B407-0001W01 8NO.84 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=BTG NO.85 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=6TP NO.86 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=PHY NO.87 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=3TA NO.88 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=MTG NO.89 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=AIP NO.90 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=D0P NO.91 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=AEG NO.92 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=AMG NO.93 SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=IOX2 NO.94 SEQ ID 5' G G C T A G C T A C A A C N* A 3' G*(14)=Me-Ph NO.95 SEQI D5' G G C T A G C T A C A A C N* A 3' G*(14)=6aG NO.96 SEQ ID G*(14)=Me-Ph-6-S- 5' G G C T A G C T A C A A C N* NO. dG 97 SEQ G*(14)=Me-Ph-6-|D5' G G C T A G C T A C A A C N*Se-dG Unser Zeichen: B407-0001W01 9NO.98SEQ ID5' G G C T A G C T A C A A C N* A 3' G*(14)=PSO-6-S-dG NO.99SEQG*(14)=PSO-6-Se- O 5' G G C T A G C T A C A A C N* A 3'NO. dG100SEQI DG*(14)=2'-MOE-6-S- 5' G G C T A G C T A C A A C N* A 3' NO. dG 101 SEQ ID G*(14)=2'-MOE-6- 5' G G C T A G C T A C A A C N* A 3' NO. Se-dG 102 SEQI DG*(14)=Me-Ph-2'- 5' G G C T A G C T A C A A C N* A 3'NO. MOE-6-S-dG 103SEQ ID G*(14)=Me-Ph-2'- 5' G G C T A G C T A C A A C N* A 3'NO. MOE-6-Se-dG 104SEQI D5' G G C T A G C T A C A A C N* A 3' N*(14)=6-cG NO.105SEQ ID5' G G C T A G C T A C A A C N* A 3' N*(14)=7MP NO.1063. Nucleic acid encoding a catalytic core region according to claim 1 or 2, wherein the modification is combined with an internucleotide linkage modification and / or ribose modification.

4. Nucleic acid according to claim 3, wherein the internucleotide linkage modification is methyl phosphonate (Me-Ph) or phosphorothioate (PSO).

5. Nucleic acid according to claims 1 to 4, wherein the one or more modifications is further combined with a backbone modification selected from the group consisting of phosphorothioate, phosphorodithioate, and methyl phosphonate.

6. Nucleic acid according to claim 1, wherein at least one nucleotide at position 5, 6, or 14 within said catalytic core region is replaced by a modified nucleotide selected from the group consisting of:Unser Zeichen: B407-0001W01 10a. 6-S-dG, 6-Se-dG, 8-oxo-dG, iso-dG, 6-O-methyl-dG, 7-deaza-dG, etheno-dA, 2- amino-dA, dNebularine, 7-methylguanosine (7MG), IOX, 6IP, 2'-OMe-dG, 2'-MOE- dG, 2-amino-purine,b. a methyl phosphonate (Me-Ph) or phosphorothioate (PSO) internucleotide linkage, c. or any combination thereof, thereby forming a modified deoxyribozyme capable of cleaving an RNA substrate.

7. Nucleic acid encoding a catalytic deoxyribozyme (DNAzyme) comprising,a. a catalytic core region according to claim 1 to 6,b. and, at least one flanking variable target recognition sequence, wherein the target recognition sequence is between 5 and 30 nucleotides in length, and c. wherein said at least one flanking target recognition sequence can be either 5' or 3' of said catalytic core region.

8. Nucleic acid according to claim 7, encoding a catalytic deoxyribozyme (DNAzyme) comprising two flanking variable target recognition sequences, wherein each of the two target recognition sequences is between 5 and 30 nucleotides in length.

9. Nucleic acid according to claims 7 to 8, encoding a catalytic deoxyribozyme (DNAzyme) comprising one or more of the following modifications or substitutions in one or more positions in the flanking variable target recognition sequence,i) PEG spacer in various lengths or composition,ii) hexaethylene glycol spacer in various lengths or composition,iii) digoxigenin with or without linker,iv) biotin with or without linker,v) fluorophore-spacer conjugates such as J0E-C12 spacer,vi) Propynyl-dC as framework for the addition of suitable steric blocker via click chemistry, vii) Azid-dT on its own and as framework for the addition of suitable steric blocker via click chemistry,viii) Dibenzocyclooctynes (DBCO)-dT on its own or in combination with suitable azid modification at a different position to establish an RNase-inhibiting inter-nucleotide bridge,ix) dSpacer (a basic site),x) RNA,Unser Zeichen: B407-0001W01 11xi) LNA,xii) 2'0Me,xiii) 2'MOE,xiv) FANA,xv) auxiliary mismatches,xvi) overhang of unpaired nucleotides on one or both ends of the DNAzyme arms.

10. Nucleic acid according to claims 7 to 9, wherein the nucleic acid is selected from the group consisting of SEQ. ID NO. 107 to 112 as shown below, wherein the flanking variable regions of the core regions of are modified by 2'-0Me on 3'- and 5'-ends,Pos. 5' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3'SEQ (5'=2'OMe),ID(G*(14)=6-S- 5' G G C T A G C T A C A A C N* A 3' NO. dG), (3'=2'OMe) 107 SEQ (5'=2'OMe), ID (G*(14)=6-Se- 5' G G C T A G C T A C A A C N* A 3'NO. dG), (3'=2'OMe) 108SEQ (5'=2'OMe),(G*(14)=Me-Ph- O 5' G G C T A G C T A C A A C N* A 3'NO. 6-S-dG),109 (3'=2'OMe)SEQ (5'=2'OMe),(G*(14)=Me-Ph-ID5' G G C T A G C T A C A A C N* A 3'NO. 6-Se-dG),110 (3'=2'OMe)SEQ (5'=2'OMe), ID (G*(14)=2'MOE- 5' G G C T A G C T A C A A C N* A 3'NO. 6-S-dG),111 (3'=2'OMe)SEQ (5'=2'OMe),(G*(14)=2'MOE- O 5' G G C T A G C T A C A A C N* A 3'NO. 6-Se-dG),112 (3'=2'OMe)11. Nucleic acid of any one of claims 1 to 10, wherein the modified nucleotide confers enhanced catalytic activity and / or improved stability in vivo relative to the corresponding unmodified 10- 23 deoxyribozyme.Unser Zeichen: B407-0001W01 1212. Nucleic acid of any one of claims 1 to 10, wherein said nucleic acid polymer has a 10- to 100- fold increased cleavage rate (k_obs) toward an RNA substrate under physiologically relevant conditions compared to an unmodified 10-23 deoxyribozyme.

13. Medicament comprising a nucleic acid according to claims 1 to 12.

14. A pharmaceutical composition comprising a medicament according to claim 13 and a pharmaceutically acceptable carrier.

15. Medicament according to claim 13, or composition according to claim 14, for use in treating a disease or disorder associated with the expression of a target RNA, wherein the deoxyribozyme is designed to cleave said target RNA in vivo and ameliorate the disease.

16. A method of cleaving a target RNA in a sample or in a subject, the method comprising contacting the sample or administering to the subject an effective amount of the nucleic acid of any one of claims 1 to 12 under conditions suitable for cleavage of said target RNA.

17. Nucleic acid encoding a catalytic deoxyribozyme (DNAzyme) comprising:a) a catalytic core region, andb) at least one flanking variable target recognition sequence, wherein the target recognition sequence is between 5 and 30 nucleotides in length; wherein said at least one flanking variable target recognition sequence comprises one or more modifications selected to confer one or more of the following characteristics:(i.) a modification that increases activity,(ii.) a modification that reduces or abolishes activity,(iii.) a modification that influences target association and / or dissociation resulting in a changed, reduced or improved catalytic turnover,(iv.) a modification that increases target RNA selectivity,(v.) a modification that reduces target RNA selectivity,(vi.) a modification that reduces the antisense effect, thus increasing the DNAzyme's precision, via reduced RNase recruitment,(vii.) a modification that promotes the antisense effect, thus reducing the DNAzyme's precision, via enhanced RNase recruitment,(viii.) modification that increases affinity to, or reduces the need for, metal ions,(ix.) a modification that increases cellular life-time,Unser Zeichen: B407-0001W01 13(x.) a modification that decreases innate immune response,(xi.) a modification that increases innate immune response.

18. Nucleic acid according to claim 17, wherein the one or more modifications in the at least one flanking variable target recognition sequence is a modification selected from the group consisting ofi) PEG spacer in various lengths or composition,ii) hexaethylene glycol spacer in various lengths or composition,iii) digoxigenin with or without linker,iv) biotin with or without linker,v) fluorophore-spacer conjugates such as J0E-C12 spacer,vi) Propynyl-dC as framework for the addition of suitable steric blocker via click chemistry, vii) Azid-dT on its own and as framework for the addition of suitable steric blocker via click chemistry,viii) Dibenzocyclooctynes (DBCO)-dT on its own or in combination with suitable azid modification at a different position to establish an RNase-inhibiting inter-nucleotide bridge,ix) dSpacer (a basic site),x) RNA,xi) LNA,xii) 2'0Me,xiii) 2'MOE,xiv) FANA,xv) auxiliary mismatches,xvi) overhang of unpaired nucleotides on one or both ends of the DNAzyme arms.

19. Nucleic acid according to claim 17 or 18, wherein the one or more modifications in the at least one flanking variable target recognition sequence is an internucleotide linkage modification selected from the group consisting of a methyl phosphonate (Me-Ph) and a phosphorothioate (PSO).

20. Nucleic acid according to any one of claims 17 to 19, wherein the one or more modifications are located within the first 5 nucleotides of the at least one flanking variable target recognition sequence, adjacent to the catalytic core region.Unser Zeichen: B407-0001W01 1421. Nucleic acid according to any one of claims 17 to 20, wherein the modification reduces the antisense effect, thus increasing the DNAzyme's precision, via reduced RNase Hl recruitment.

22. Nucleic acid according to claims 19 and 20, wherein the internucleotide linkage modification is a methyl phosphonate (Me-Ph) modification that reduces the negative charge of the backbone.

23. Medicament comprising a nucleic acid according to claims 1 to 12 and 17 to 22.

24. A pharmaceutical composition comprising a medicament according to claim 23 and a pharmaceutically acceptable carrier.

25. Medicament according to claim 23, or composition according to claim 24, for use in treating a disease or disorder associated with the expression of a target RNA, wherein the deoxyribozyme is designed to cleave said target RNA in vivo and ameliorate the disease.

26. A method of cleaving a target RNA in a sample or in a subject, the method comprising contacting the sample or administering to the subject an effective amount of the nucleic acid of any one of claims 1 to 12 and 17 to 22 under conditions suitable for cleavage of said target RNA.

27. A kit for performing the method according to claim 26.

28. A catalytic deoxyribozyme (DNAzyme) according to claim 18, wherein the DNAzyme comprises chemical modifications in the 5' substrate-binding arm and 3' substrate-binding arm systematically arranged to evade RNase Hl recruitment. This is characterized in that the DNAzyme comprises:a. an " REP-1" modification pattern, wherein the modified nucleotides on the 5' arm are paired in two groups (positions -3 and -4, and positions -7 and -8 relative to the 5' -start of the catalytic core), and the 3' arm comprises modifications at the absolute 3'-terminal nucleotide and at the relative position located four nucleotides upstream of said 3'- terminal nucleotide (e.g., position +5 in a 9-nucleotide arm);b. an " REP-2" modification pattern, comprising modifications on the 5' arm at positions -5, -6, and -2, with the 3' arm modified identically to REP-1; orUnser Zeichen: B407-0001W01 15c. a hybrid modification pattern incorporating the architecture of REP-1 or REP-2 in conjunction with supplementary modifications in the substrate binding arms or the catalytic loop.

29. A catalytic deoxyribozyme (DNAzyme) according to claim 28, wherein said chemical modifications are 2'0Me or 2'MOE modifications.

30. The DNAzyme according to claim 28 or 29, wherein the catalytic core region comprises the nucleotide sequence of SEQ ID NO. 1, wherein at least one nucleotide at position 5, 6, or 14 within said catalytic core region that is replaced by a modified nucleotide selected from the group consisting of:a. 6-S-dG, 6-Se-dG, 8-oxo-dG, iso-dG, 6-O-methyl-dG, 7-deaza-dG, etheno-dA, 2- amino-dA, dNebularine, 7-methylguanosine (7MG), IOX, 6IP, 2'-OMe-dG, 2'-MOE- dG, 2-amino-purine,b. a methyl phosphonate (Me-Ph) or phosphorothioate (PSO) internucleotide linkage, c. or any combination thereof, such as a (PSO, 2'OME, 6-S)-dG at position 14, thereby forming a modified deoxyribozyme capable of cleaving an RNA substrate31. The DNAzyme according to any of the claim 28 to 30, wherein the DNAzyme comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 284 to 296 and 325 to 340.

32. An isolated deoxyribozyme comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 145 to SEQ ID NO: 163, or a variant thereof comprising one or more chemical modifications to the nucleobase, ribose moiety, or internucleotide linkage.

33. The isolated deoxyribozyme according to claim 32 for use as a medicament.

34. The isolated deoxyribozyme according to claim 32 for use in a method of treating cancer, preferably wherein the cancer is associated with a KRAS mutation, more preferably a KRAS Q61K mutation.Unser Zeichen: B407-0001WO1 1635. The isolated deoxyribozyme for use according to claim 34, wherein the cancer is selected from the group consisting of non-small cell lung cancer, pancreatic adenocarcinoma, colorectal cancer, leukemia, and melanoma.

36. The DNAzyme according to any of claims 28 to 31, for use as a medicament.

37. The DNAzyme for use according to claim 36, or the medicament or pharmaceutical composition for use according to any of claims 23 to 25, wherein the disease or disorder is a viral infection.

38. The DNAzyme for use according to claim 36, or the medicament or pharmaceutical composition for use according to any of claims 23 to 25, wherein the disease or disorder is a cancer.

39. The DNAzyme for use according to any of claims 36 to 38, wherein the DNAzyme, medicament, or pharmaceutical composition is formulated in targeted lipid nanoparticles (LNPs).

40. The DNAzyme for use according to any of claims 36 to 39, or the medicament or pharmaceutical composition for use according to any of claims 23 to 25, wherein the treatment further comprises improving immune function in vivo, characterized in that the administration of the DNAzyme can either:(a) increase the production of pro-inflammatory cytokines, including IFN-y and TNF-a, by CD8+ T cells;(b) expand Short-lived effector cells (SLECs) and Memory precursor effector cells (MPECs); or (c) downregulate the surface expression of T-cell exhaustion markers, including TIM-3 and PD- 1.

41. The DNAzyme for use according to any of claims 36 to 40, wherein the treatment reduces systemic pathology and organ damage.

42. A DNAzyme according to claim 6 or 30 that combines at least two chemical modifications at position G(14) involving a modification of the nucleobase region selected from the group:6-S-dG or 6-Se-dG,and at least one additional modification involving either an internucleotide linkage modification and / or ribose modification, whereas the internucleotide linkage modification is selected from the group:Unser Zeichen: B407-0001W01 17- PSO or Me-Phand the ribose modification is selected from the group:- 2'MOE or 2'0Me.

43. A DNAzyme according to claim 42, wherein G(14) is replaced by:a. (PSO, 2'0Me, 6-S)-dG;b. (PSO, 2'MOE, 6-S)-dG;c. (2'MOE, 6-S)-dG;d. (2'0Me, 6-S)-dG;e. (PSO, 2'0Me, 6-Se)-dG;f. (PSO, 2'MOE, 6-Se)-dG;g. (2'MOE, 6-Se)-dG; orh. (2'0Me, 6-Se)-dG44. The DNAzyme according to any of claims 42 to 43, for use as a medicament.Unser Zeichen: B407-0001W01 18

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