WO2009093188A2 - Sequence specific double-stranded dna binding compounds - Google Patents

Sequence specific double-stranded dna binding compounds Download PDF

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WO2009093188A2
WO2009093188A2 PCT/IB2009/050235 IB2009050235W WO2009093188A2 WO 2009093188 A2 WO2009093188 A2 WO 2009093188A2 IB 2009050235 W IB2009050235 W IB 2009050235W WO 2009093188 A2 WO2009093188 A2 WO 2009093188A2
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residue
stranded dna
general structure
double
formula
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WO2009093188A3 (en
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Anwar Rayan
Mizied Falah
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Antigen Biotechnologies Ltd
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Priority to US12/841,506 priority Critical patent/US20100284959A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base

Definitions

  • the present invention relates to novel compounds which bind dsDNA in a sequence specific manner, to manufacturing method therefor and to methods of using the same.
  • TBMs triplex-forming molecules
  • oligomers that bind to the oligopurines strand via the major groove of oligopyrimidine-oligopurine regions in double stranded DNA.
  • oligomers comprise either nucleotides or nucleopeptides.
  • Designed zinc finger proteins are engineered to display naturally occurring zinc finger motifs as molecular building blocks in a polypeptide chain.
  • sequence specific molecules targeted at the gene of interest would enable to specifically alter gene expression and potentially will entail a wide range of applications in life since research and in therapeutics. For example, such technology could provide a new strategy to knock-out specific genes for therapeutic purposes or function/mechanism study and might be applied in the development of new diagnostic techniques.
  • Specific binders of 16-18 bp in length can be sufficient for a substantially unique binding to a defined sequence in a genome thus affecting an expression of a particular gene therein.
  • Searching after sequence specific molecules targeting the DNA has been the centre of interest of many research groups in the past two decades. Stability to nucleases, sufficient membrane penetration, sequence specificity to a gene of interest and long residence time on the specific target are all crucial issues needed to be discussed when evaluating DNA binding compounds.
  • a major challenge is to provide for molecules that recognize specific sequences on the dsDNA while being long enough, up to twenty base pairs or even more.
  • compound that will fulfil most of the requirements discussed above regarding demands from a binding compound like the ability to cross the membrane and reach the target sequence in the cell nuclei and will be stable to nucleases, shall be beneficial for research and therapy.
  • novel compounds that interact with the dsDNA via the major groove thereof and are capable of complementarily and highly specific binding thererto via the Hoogsteen face of the dsDNA helix.
  • Most of the previously described triplex forming oligonucleotides interact with only one strand while the novel compounds of the present invention are bp-complementary and interact with both strands while able to recognize all four base pairs of the dsDNA, i.e. A-T, T-A, C-G and G-C.
  • novel compounds of the present invention are formed as oligomer structures, in which at least two different types of residues, targeting either A-T/T-A bp or C-G/G-C bp, are incorporated on a backbone.
  • 1A and 1 B are the results of the NMR testing exhibiting structural consistency of synthesized AH-1 1 compound
  • Figs 2A-D are plots of absorbance as a function of temperature, illustrating the T M of complimentary versus non-complimentary dsDNA with and without the addition of synthesized AH-1 1 compound.
  • the residue targeting the A-T or T-A bp hereinafter R1 , has the general structure of Formula 1 ;
  • Xi and X 2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
  • Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
  • R1 residue has the general structure of Formula 2;
  • Xi and X 2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
  • Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
  • the residue targeting the G-C or C-G bp hereinafter R2, has the general structure of Formula 3; wherein:
  • X 1 and X 2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
  • E is oxygen or sulphur
  • W is a methyl, an ethyl, an acetyl, a glycine, a beta-alanine or an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to
  • Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
  • R2 residue has the general structure of Formula 4;
  • Xi and X 2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; and
  • W is a methyl, an ethyl, an acetyl, a glycine, a beta-alanine or an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to
  • Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
  • residue R2 has the general structure of Formula 5;
  • Xi and X 2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
  • Z is a nitrogen or CH
  • Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
  • residue R2 has the general structure of Formula 6;
  • Xi and X 2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; and
  • Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
  • residue R2 has the general structure of Formula 7;
  • X 1 and X 2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; and
  • Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
  • X are each independently an oxygen or sulphur
  • R1 or R2 are each independently one of the compounds the formulae of which are presented supra, preferably linked to the polyamide backbone of Formula 8 via the atom at the position represented by Y.
  • the residues R1 or R1 incorporated onto a backbone form the oligomer structures, which targets dsDNA in a sequence specific manner.
  • the interaction of the R1 residues with A-T or T-A bp is formed via hydrogen bonds, as outlined in Scheme 1 infra; and the interaction of the R2 residues with C-G or G-C bp is also formed via hydrogen bonds, as outlined in Scheme 2 infra;
  • X1 , X2 or R can be a chemical group that reaches the backbone of the dsDNA and interacts in salt bridge thereof with the phosphoric group therein.
  • X1 , X2 or R are each independently a hydrogen, a glycine or related derivatives thereof, a beta- alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor.
  • R is a beta-alanine then preferably the binding group is selective to CG bp, as shown on left side of Scheme 2; whereas if X is a beta-alanine then preferably the binding group is selective to GC bp, as shown on right side of Scheme 2.
  • AH-1 1 The compound, hereinafter referred to as AH-1 1 , having the general structure of Formula 9;
  • T M melting points
  • T M of A-T dsDNA versus C-G dsDNA is known in the art as a common indicator for specificity and selectivity of dsDNA binding.
  • the effect was tested by UV spectra at the wavelength 260 nm and measured over the temperature range of 30-98 °C. Stoppered 1 cm path length quartz cells were used.
  • Fig. 2A shows the plot of absorbance as a function of temperature, illustrating the T M of 25 bp in length A-T ds-primers, without the addition of AH-1 1 , of 53 °C.
  • FIG. 2B shows the plot of absorbance as a function of temperature, illustrating the T M of 25 bp in length A-T ds-primers, with the addition of AH-1 1 , of 84 °C.
  • Fig. 2C shows the plot of absorbance as a function of temperature, illustrating the T M of 25 bp in length G-C ds-primers, without the addition of AH-1 1 , of 84 0 C.
  • Fig. 2D shows the plot of absorbance as a function of temperature, illustrating the T M of 25 bp in length G-C ds-primers, with the addition of AH-1 1 , of 84 °C.

Abstract

Novel compounds that interact with the dsDNA via the major groove thereof and are capable of complementarily and highly specific binding thererto via the Hoogsteen face of the dsDNA helix are disclosed.

Description

SEQUENCE SPECIFIC DOUBLE-STRANDED DNA BINDING COMPOUNDS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of priority to US Provisional Patent Application Serial Number 61/022,833, filed January 23, 2008, entitled "DNA Major Groove Selective Binders and Uses Thereof"; the aforementioned application is hereby incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to novel compounds which bind dsDNA in a sequence specific manner, to manufacturing method therefor and to methods of using the same.
BACKGROUND ART
Transcription of a gene gives rise to many copies of mRNA, which is translated into a large number of proteins. Thus, an inhibition of the transcription via targeting a dsDNA presents several advantages over inhibition at any other level. Blocking protein translation from mRNA will not prevent the corresponding gene from being transcribed. In contrast, interfering with gene transcription by targeting dsDNA is expected to attenuate the expression of a given gene more efficiently and for a longer time. Hitherto, however, there are only few compounds known in the art that directed to interact with DNA, i.e. Nitrogen mustards and Decarbazine that react covalently with DNA, often cross-linking the strands or Bleomycin, which causes DNA breakage. Due to the lack of sequence specificity, most of these compounds are highly toxic and used for chemotherapy as anticancer drugs. Groove binding by selective molecules is almost exclusively limited to the minor groove while selective recognition of the major groove has remained elusive. The design of artificial sequence specific molecules, which bind DNA specifically and stably, can provide a means of interfering with gene expression more safely and efficiently.
Targeting of the DNA as a means of alltering gene expression is a very attractive strategy. This approach was first contemplated about 20 years ago with the description of triplex-forming molecules (TFMs) that can bind double stranded DNA. The molecules that are able to stably bind dsDNA with sequence specificity can be classified into three groups:
1 - Triplex-forming molecules that bind to the oligopurines strand via the major groove of oligopyrimidine-oligopurine regions in double stranded DNA. Such oligomers comprise either nucleotides or nucleopeptides.
2- Small molecules comprising hairpin polyamides that bind short DNA sequences, of up to seven base pairs (bp), with high affinity and sequence specificity. The recognition depends on side-by-side amino acid pairings in the minor groove.
3- Designed zinc finger proteins are engineered to display naturally occurring zinc finger motifs as molecular building blocks in a polypeptide chain. The polyfinger peptide units specifically recognize DNA triplets XNN (with X=G or T, and N=G, T, C, A) and have been proved to be efficient for the binding of up to 18 bp (six triplets).
The use of sequence specific molecules targeted at the gene of interest would enable to specifically alter gene expression and potentially will entail a wide range of applications in life since research and in therapeutics. For example, such technology could provide a new strategy to knock-out specific genes for therapeutic purposes or function/mechanism study and might be applied in the development of new diagnostic techniques. Specific binders of 16-18 bp in length, can be sufficient for a substantially unique binding to a defined sequence in a genome thus affecting an expression of a particular gene therein. Searching after sequence specific molecules targeting the DNA has been the centre of interest of many research groups in the past two decades. Stability to nucleases, sufficient membrane penetration, sequence specificity to a gene of interest and long residence time on the specific target are all crucial issues needed to be discussed when evaluating DNA binding compounds.
It is to be emphasized that two of the main limitations in the triplex strategy is the need to extend the range of the recognition sequences and the design of bases that would recognize all four base pairs of DNA, i.e. A-T, T-A, C-G and G-C, when binding to the major groove. Heretofore, all of the developed molecules targeting DNA will probably target many sequences in the human genome since 16-18 base pairs is minimal to afford the recognition of a unique target.
A major challenge is to provide for molecules that recognize specific sequences on the dsDNA while being long enough, up to twenty base pairs or even more. In addition, compound that will fulfil most of the requirements discussed above regarding demands from a binding compound, like the ability to cross the membrane and reach the target sequence in the cell nuclei and will be stable to nucleases, shall be beneficial for research and therapy.
SUMMARY OF THE INVENTION
There are provided in accordance with some embodiments of the present invention novel compounds that interact with the dsDNA via the major groove thereof and are capable of complementarily and highly specific binding thererto via the Hoogsteen face of the dsDNA helix. Most of the previously described triplex forming oligonucleotides interact with only one strand while the novel compounds of the present invention are bp-complementary and interact with both strands while able to recognize all four base pairs of the dsDNA, i.e. A-T, T-A, C-G and G-C.
In accordance with some preferred embodiments the novel compounds of the present invention are formed as oligomer structures, in which at least two different types of residues, targeting either A-T/T-A bp or C-G/G-C bp, are incorporated on a backbone.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended drawings in which:
1A and 1 B are the results of the NMR testing exhibiting structural consistency of synthesized AH-1 1 compound;
Figs 2A-D are plots of absorbance as a function of temperature, illustrating the TM of complimentary versus non-complimentary dsDNA with and without the addition of synthesized AH-1 1 compound.
DISCLOSURE OF THE INVENTION
Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.
In one preferred embodiment of the present invention the residue targeting the A-T or T-A bp, hereinafter R1 , has the general structure of Formula 1 ;
Figure imgf000006_0001
wherein:
Xi and X2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
In another preferred embodiment of the present invention the R1 residue has the general structure of Formula 2;
Figure imgf000006_0002
wherein:
Xi and X2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
In one preferred embodiment of the present invention the residue targeting the G-C or C-G bp, hereinafter R2, has the general structure of Formula 3;
Figure imgf000007_0001
wherein:
X1 and X2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
E is oxygen or sulphur;
W is a methyl, an ethyl, an acetyl, a glycine, a beta-alanine or an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to
10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; wherein W preferably compromise a terminal amine;
Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
In another preferred embodiment of the present invention the R2 residue has the general structure of Formula 4;
Figure imgf000007_0002
wherein:
Xi and X2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; and
W is a methyl, an ethyl, an acetyl, a glycine, a beta-alanine or an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to
10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; wherein W preferably compromise a terminal amine;
Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
In yet another preferred embodiment of the present invention the residue R2 has the general structure of Formula 5;
Figure imgf000008_0001
wherein:
Xi and X2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor;
Z is a nitrogen or CH
Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
In still another preferred embodiment of the present invention the residue R2 has the general structure of Formula 6;
Figure imgf000008_0002
wherein:
Xi and X2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; and
Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
In yet still other preferred embodiment of the present invention the residue R2 has the general structure of Formula 7;
Figure imgf000008_0003
wherein:
X1 and X2 are each independently a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; and
Y is a hydrogen, a carbon chain linker or preferably -CH2-COOH.
The above-identified residues R1 and/or R2 can be incorporated on a polyamide backbone moiety having the general structure of Formula 8;
Figure imgf000009_0001
wherein:
X are each independently an oxygen or sulphur; and
R1 or R2 are each independently one of the compounds the formulae of which are presented supra, preferably linked to the polyamide backbone of Formula 8 via the atom at the position represented by Y.
It should be acknowledged that above-identified polyamide backbone moiety having the general structure of Formula 8 is for exemplification purposes merely and the residues R1 or R2 can be incorporated onto any other suitable backbone moiety having a general polymeric structure.
The residues R1 or R1 incorporated onto a backbone form the oligomer structures, which targets dsDNA in a sequence specific manner. The interaction of the R1 residues with A-T or T-A bp is formed via hydrogen bonds, as outlined in Scheme 1 infra; and the interaction of the R2 residues with C-G or G-C bp is also formed via hydrogen bonds, as outlined in Scheme 2 infra;
Figure imgf000010_0001
Scheme 2 wherein:
X1 , X2 or R can be a chemical group that reaches the backbone of the dsDNA and interacts in salt bridge thereof with the phosphoric group therein. X1 , X2 or R are each independently a hydrogen, a glycine or related derivatives thereof, a beta- alanine or related derivatives thereof, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor. If R is a beta-alanine then preferably the binding group is selective to CG bp, as shown on left side of Scheme 2; whereas if X is a beta-alanine then preferably the binding group is selective to GC bp, as shown on right side of Scheme 2.
An exemplary method of synthesizing R1 residue having the general structure of Formula 1 elaborated supra, which can be conjugated thereafter with a backbone, such as the polyamide backbone having the general structure of Formula 8 elaborated supra, outlined in Scheme 3 infra. ;
Figure imgf000011_0001
Example 1
The compound, hereinafter referred to as AH-1 1 , having the general structure of Formula 9;
Figure imgf000011_0002
was synthesized, from widely available reactants and by techniques known in the art, in accordance with the reaction equation as outlined in Scheme 4 infra.
Figure imgf000011_0003
Acidification
Figure imgf000011_0004
Figure imgf000011_0005
The product was subjected to NMR testing to verify the consistency with the structure of the AH-1 1 compound. Reference is now made to Figs 1A and 1 B, in which the results of the NMR testing are shown, exhibiting consistency with the structure of the AH-1 1 compound. Example 2
After the structural integrity of the AH-1 1 compound was verified, the quality and magnitude of the selective physical interaction of the compound with complimentary dsDNA primers, namely comprising A-T bp, was tested versus the interaction with non-complimentary dsDNA primers, namely comprising G-C bp, was tested by measuring the melting points (TM) of complimentary and non- complimentary primers, with and without the addition of AH-1 1 ; TM as hereinafter referred to is defined as the temperature at which approximately 50% of dsDNA has underwent a denaturation and is found in the form of a single-stranded DNA (ssDNA), not annealed to the complimentary strand thereof. The effect on TM of A-T dsDNA versus C-G dsDNA is known in the art as a common indicator for specificity and selectivity of dsDNA binding. The effect was tested by UV spectra at the wavelength 260 nm and measured over the temperature range of 30-98 °C. Stoppered 1 cm path length quartz cells were used.
It was found that the addition of AH-1 1 , designated to be specific for A- T bp, has increased the TM of 25 bp in length A-T ds-primers, namely the TM has shifted from 53°C to 84 °C, whereas the TM of 25 bp in length CG ds-primers hasn't been affected. Reference is now made to Figs 2A-D, in which the results of the TM tests are shown. Fig. 2A shows the plot of absorbance as a function of temperature, illustrating the TM of 25 bp in length A-T ds-primers, without the addition of AH-1 1 , of 53 °C. Fig. 2B shows the plot of absorbance as a function of temperature, illustrating the TM of 25 bp in length A-T ds-primers, with the addition of AH-1 1 , of 84 °C. Fig. 2C shows the plot of absorbance as a function of temperature, illustrating the TM of 25 bp in length G-C ds-primers, without the addition of AH-1 1 , of 840C. Fig. 2D shows the plot of absorbance as a function of temperature, illustrating the TM of 25 bp in length G-C ds-primers, with the addition of AH-1 1 , of 84 °C.
It will be appreciated that the present invention is not limited by what has been particularly described and shown hereinabove and that numerous modifications, all of which fall within the scope of the present invention, exist. Rather the scope of the invention is defined by the claims which follow:

Claims

1. A chemical residue incorporated onto a backbone moiety, wherein said backbone moiety having essentially a general polymeric structure; or a reactant, an intermediate, a derivative, a salt or a conjugate comprising said residue, used as a monomer unit for the synthesis of at least a fragment of said backbone moiety respectively having said chemical residue incorporated thereon; wherein said residue having the general structure of any selected from the group consisting of:
■ a residue having the general structure of Formula 1 ;
Figure imgf000013_0001
a residue having the general structure of Formula 2;
Figure imgf000013_0002
a residue having the general structure of Formula 3;
Figure imgf000013_0003
a residue having the general structure of Formula 5;
Figure imgf000013_0004
a residue having the general structure of Formula 6;
Figure imgf000014_0001
a residue having the general structure of Formula 7;
Figure imgf000014_0002
wherein X1, X2 and X are each independently represent a hydrogen, a glycine or related derivatives thereof, a beta-alanine or related derivatives thereof, a hydrocarbon chain of 1 to 10 carbon atoms, a hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; wherein E represents a nitrogen, oxygen or sulphur; wherein W represents a methyl, an ethyl, an acetyl, a glycine, a beta-alanine or an aliphatic hydrocarbon chain of 1 to 10 carbon atoms, an aliphatic hydrocarbon chain of 1 to 10 carbon atoms including nitrogen, oxygen, sulphur or phosphor; a terminal amine; wherein Z represents a nitrogen or CH; wherein Y represents a hydrogen, a nitrogen, a carbon chain linker, a carboxyl, a -CH2-COOH group or a covalent bond with an atom of said backbone moiety.
2. A chemical residue or a reactant, an intermediate, a derivative, a salt or a conjugate thereof as in claim 1 , wherein said backbone is a polyamide.
3. A chemical residue or a reactant, an intermediate, a derivative, a salt or a conjugate thereof as in claim 1 , being a chemical group binding a specific base pare of double-stranded DNA.
4. A sequence specific double-stranded DNA binding compound, having a general hetero-oligomeric structure, said compound comprising a plurality of the residues set forth in claim 1.
5. A sequence specific double-stranded DNA binding compound as in claim 4, wherein said residues having the general structure of Formulae 1 or 2 is the residue (R1 ) binding a base pare of double-stranded DNA selected from the group consisting of: an A-T base pare and a T-A base pare.
6. A sequence specific double-stranded DNA binding compound as in claim 4, wherein said residues having the general structure of Formulae 3 or any of 5 to 7 is the residue (R2) binding a base pare of double-stranded DNA selected from the group consisting of: a C-G base pare and a G-C base pare.
7. A sequence specific double-stranded DNA binding compound as in claim 4, interacting with said double-stranded DNA via the major groove thereof.
8. A method of affecting DNA transcription comprising exposing a double-stranded DNA to the sequence specific double-stranded DNA binding compound as set forth in claim 4.
9. A method of altering gene expression comprising administrating to a living organism the sequence specific double-stranded DNA binding compound as set forth in claim 4.
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WO2012011114A2 (en) 2010-07-22 2012-01-26 Genearrest Ltd Sequence specific double-stranded dna/rna binding compounds and uses thereof

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WO2012011114A3 (en) * 2010-07-22 2012-07-26 Genearrest Ltd Sequence specific double-stranded dna/rna binding compounds and uses thereof

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