WO2007107775A2

WO2007107775A2 - Preparation of oligonucleotides with photoswitchable properties

Info

Publication number: WO2007107775A2
Application number: PCT/GB2007/001056
Authority: WO
Inventors: Joseph S. Vyle; James Robert Tucker
Original assignee: Queens University of Belfast
Current assignee: Queens University of Belfast
Priority date: 2006-03-23
Filing date: 2007-03-23
Publication date: 2007-09-27
Anticipated expiration: 2008-09-23
Also published as: WO2007107775A8; WO2007107775A3; GB0605787D0

Abstract

Linkers which provide for the attachment of photoswitchable compounds to nucleic acid are provided. These linkers can be used to provide photo-modulatable compounds wherein the chemical and biological properties of polynucleic acid incorporating these photo-modulatable compounds can be modulated by light. Further, methods for modulation of hybridisation of polynucleic acid sequences and binding of factors, for example polymerases to polynucleic acid sequences incorporating said photo-modulatable compounds are provided.

Description

Preparation of oligonucleotides with pbotoswitchable properties

The present invention relates to linkers which provide for the attachment of photoswitchable • compounds to nucleic acid to provide photo- modulatable compounds wherein the chemical and biological properties of polynucleic acid incorporating these photo-modulatable compounds, for example the hybridisation of polynucleic acid sequences, including duplex formation or triplex, can be modulated by light. Further, the invention relates to methods for modulation of hybridisation of polynucleic acid sequences and binding of factors, for example polymerases to polynucleic acid sequences incorporating said photo-modulatable compounds.

BACKGROUND

Reversible optical control of DNA recognition has been reported using backbone-substituted azobenzene (AB) -derivatives attached via a non-nucleotidic spacer.

In particular, azobenzenes tethered on D-threoninol to allow incorporation of the azobenzenes into DNA have been disclosed (H. Asunama, T. Ito, T.Yoshida, X. Liang, M. Komiyama, 1999 Agnew. Chem. Int. Ed. Engl., 38, 2393-2395). E —> Z photoisomerization of azobenzen.es occurs with high quantum yields at 330 - 370 run, is not particularly environment sensitive, is fatigue resistant and leads to large conformational and polarity changes. Modest discrimination in activities between the so-called irradiated (mainly Z) and more active dark-adapted (mainly JS) states of proteins modifed by non-specific attachment of azobenzenes to surface lysines was reported in 1991 (I. Wiliner, S. Rubin, A. Riklin, J. Am. Chem. Soc. 1991, 113, 3321-3325) and recent reports have demonstrated that localization of azobenzenes at strategic residues of transmembrane proteins or DNA- binding oligopeptides in which the S-isomer is inactive enables highly effective activity- modulation using light (M. Banghart, K. Borges, E. Isacoff, D. Trauner, R. H. Kramer, Nat. Neurosci . 2004, 7, 1381-1386; M. Volgraf, P Goroztiza, R. Numano, R. H. Kramer, E. Y. Isacoff, D. Trauner, Nat. Chem: Biol. 2006, 2, 47-52 and L. Guerrero, O. S. Smart, G. A. Woolley, R. K. Allemann, J. Am. Chem. Soc. 2005, 127, 15624-15629).

A single report of photoswitchable 8-17 deoxy^'ribozyme-mediated RΝA cleavage has been made (Y. Liu, D. Sen, J. MoI. Biol. 2004, 341, 887-892). In this demonstration using 200-fold excess of the deoxyribozyme over substrate, the incorporation of two azobenzenes units for significant irradiated / dark-adapted discrimination and only highly attenuated activities were reported. In addition, there has been reports of photo- regulation of RNA polymerase reaction by use of modified DNA carrying an azobenzene (H. Asanuma, D. Tamaru, T. Yoshida, A. Yamazawa, T. Mitsui, T. Okuni, M. Kimoto, I. Hirao, S. Yokoyama, M. Komiyama, 2001, Nucleic Acids Res Suppl . (l):55-β).

SUMMARY OF INVENTION

It would be advantageous if photoswitenable properties of nucleic acids (e.g., RNA cleavage by nucleic acid catalysts) with improved properties and a greater range of targets could be achieved.

According to a first aspect of the present invention there is provided a linker for linking a photoswitenable compound (PS) to a base, nucleoside or a nucleotide, the linker comprising:

wherein X independently represents

(III) (IV) and a is 0 or 1; Z independently represents

<**>< ^(V» I (R₇)

or

wherein nl, n2 , n3 , n4, n5, nβ, and n7 are independently selected from each other and each of nl, n2 , n.3 , n4, n5, nβ, and n7 is 0 or an integer selected from the range 1 to 12;

(R₇) represents H or CH₃;

Y independently represents

and b is independently selected from 0 or 1 or 2.

In particular embodiments, the linker may be asymmetric .

In one embodiment of a linker of the present invention X is represented by formula (I) , Z is independently selected from the structures as represented by one of formula (V) , (VI) , (VII) , or (VIII) and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

In an another embodiment of a linker of the present invention X is represented by formula (II) , Z is independently selected from the structures as represented by one of formula (V) , (VI) , (VII) , or (VIII) and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

In an another embodiment of a linker of the present invention X is represented by formula (III) , Z is independently selected from the structures as represented by one of formula (V) , (VI) , (VII) , or (VIII) and Y is independently selected from the structures as represented by one of formula ( IX) , (X) or (XI ) .

In an another embodiment of a linker of the present invention X is represented by formula (IV) , Z is independently selected from the structures as represented by one of formula (V) , (VI) , (VII) , or (VIII) and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

In a particular embodiment of a linker of the present invention X is represented by one of the structures as represented by formula (I), (II), (III) , or (IV) , Z is independently selected from the structure as represented by the formula (V) , and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

In other particular embodiments of a linker of the present invention X is represented by one of the structures as represented by formula (I) , (II) , (III), or (IV), Z is independently selected from the structure as represented by the formula (VI) , and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

In other particular embodiments of a linker of the present invention X is represented by one of the structures as represented by formula (I) , (II) , (III), or (IV), Z is independently selected from the structure as represented by the formula (VII) , and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

In other particular embodiments of a linker of the present invention X is represented by one of the structures as represented by formula (I) , (II), (III) , or (IV) , Z is independently selected from the structure as represented by the formula (VIII) , and Y is independently selected from the structures as represented by one of formula (IX), (X) or (XI).

In alternative embodiments of a linker of the present invention X is represented by one of the formula (I), (II), (III), or (IV), Z is independently selected from the structures as represented by the formula (V), (VI), (VII), or (VIII), and Y is a structure as represented by (IX).

In further alternative embodiments of a linker of the present invention X is represented by one of the formula (I), (II), (III), or (IV), Z is independently selected from the structures as represented by the formula (V) , (Vl) , (VII) , or (VIII) , and Y is a structure as represented by (X) .

In further alternative embodiments of a linker of the present invention X is represented by one of the formula (I), (II), (III), or (IV), Z is independently selected from the structures as represented by the formula (V), (VI), (VII), or (VIII) , and Y is a structure as represented by (XI) . In a preferred embodiment a linker is provided wherein Z is represented by formula (V) and (R7) is H i.e. -NH- and Y is represented by formula (IX) i.e. -C(O)-.

In embodiments of the linker wherein X is not present (i.e. a = 0) linkage of the phσtoswitchable compound to the nucleoside or nucleotide may be via nucleoside or nucleotide -NH- (C-linker) -PS or nucleoside or nucleotide - (C-linker) -NH-PS or nucleoside or nucleotide - (C-linker) -PS.

Optionally the aliphatic groups or glycol groups of the linker may suitably contain one or more amine, amide, nitrile, halogen, ether, alcohol, thiol, acid (such as carboxylic, sulphonic or phosphonic acid) , ester, aldehyde, ketone, phosphine or phosphine oxide groups.

Advantageously linkers of the present invention can provide for a photoswitchable compound to be provided to a base, a nucleoside or a nucleotide wherein the base, nucleoside or nucleotide can be part of a polynucleic acid. Suitably a base is selected from adenine, guanine, cytosine, uracil, thymine or an analogue thereof. Suitably a nucleoside can be selected from adenosine, guanosine, cytidine, uridine, thymidine or an analogue thereof. Suitably a nucleotide comprises a nucleoside selected from adenosine, guanosine, cytidine, uridine, thymidine or an analogue thereof and one or more phosphate groups, in particular a mono, di or tri phosphate group.

The linkers of the invention advantageously provide for the attachment of photoswitchable compounds, for example azobenzene moieties, to readily accessible nucleoside derivatives.

Linkers of the invention interposed between a nucleoside or a nucleotide and a photoswitchable compound advantageously provide photo-modulatable compounds.

These photo-modulatable compounds have the potential to maintain essential base contacts and biological activity of nucleic acids. This has been demonstrated using photomodulated deoxyribozyme- catalyzed RNA cleavage under multiple turnover conditions.

The photo-modulatable compounds provided may be used to control DNA and / or RNA function using an external stimulus (light) that can be easily and quantitatively provided. The photo-modulatable compounds may be used, for example, in the development of regulatory biocatalysts, spatiotemporal control of gene expression, array- based computation, information storage, biosignal amplifiers or the like.

According to a second aspect of the invention there is provided a nucleic acid complex comprising a nucleic acid and a linker of the first aspect of the invention.

The term nucleic acid comprises DNA or RNA nucleosides or nucleotides. A nucleic acid complex comprising a nucleic acid and a linker of the first aspect of the invention can be produced recombinantly, synthetically, or by any means available to those in the art, including cloning using standard techniques. Polynucleic acids including such a nucleic acid complex may be single stranded or double stranded.

Suitably a linker of the present invention can be attached to the base portion of a nucleoside or nucleotide. In particular embodiments, a linker is attached to a 5-proparglyamino function of a nucleoside.

In other embodiments, a linker is attached to the sugar portion of a nucleoside or nucleotide. In particular embodiments, a linker can be attached to a 2' amino function of a nucleoside.

Advantageously a linker of the first aspect of the invention allows a photoswitchable compound to be conjoined to a nucleotide or nucleoside rather than replace the nucleotide or nucleoside. This may be of benefit as the polynucleic acid comprising such a nucleotide or nucleoside including a photo- switchable compound may retain properties of a nucleic acid, for example base pairing. In particular embodiments of a nucleic acid complex wherein a linker of the first aspect is linked to a modified phosphate diester the nucleic acid complex may comprise at least one of:

or

wherein Rl is O- or is any 5 ' nucleic acid fragment either DNA or RNA; Base is selected from adenosine, cytosine, guanine, thymine or an analogue thereof; R2 is selected from H (DNA), OMe (2' -O-methyl RNA) or OH (RNA)₇ R3 is 0- or is any 3' nucleic acid fragment either DNA or RNA; and R4 is a linker according to a first aspect of the present invention.

In embodiments of nucleic acid complex, the nucleic acid can comprise a linker of the first aspect linked to a 2 ' amino function of a nucleoside.

In such embodiments, a photoswitchable compound linked to a linker at this position will interact with the minor groove of a duplex.

In particular embodiments the nucleic acid complex may comprise at least one nucleotide A, B, C, D, E or F

wherein

Rl is any protecting group typical for 5'; R2 is any protecting group for phosphate or phosphate triesters; R3 is any alkyl group for phosphoramidite; R4 is any protecting group for N⁴ of cytosine;

R5 is a linker according to a first aspect of the present invention.

Suitably Rl can be dimethoxytrityl, pixyl, benzhydryloxy-bis (trimethylsilyloxy) silyl or levulinyl. Suitably R2 can be 2-cyanoethyl, methyl or 3-chlorophenyl . Suitably (R3)2 can be iso-propyl (twice) , methyl (twice) , morpholinyl or pyrrolidinyl. Suitably R4 can be benzoyl, N,N- dimethylformamyl or acetyl. In embodiments of nucleic acid complex the nucleic acid can further comprise a photoswitchable compound linked to a 5-proparglyamino function of a nucleoside via a linker of the first aspect of the invention. In such embodiments the photoswitch interacts in the major groove. In particular embodiments the nucleic acid complex may comprise at least one nucleotide G, H, I, J, K or L

H

J wherein

Rl is any protecting group typical for 5 ' ; R2 is any protecting group for phosphate or phosphate triesters; R3 is any alkyl group for phosphoramidite; R4 is any protecting group for W⁴ of cytosine; R5 is selected from H (DNA), OMe (2 ' -0-methyl RNA), or an 0-protecting group (RNA) ; R6 is a linker according to the first aspect of the present invention. ^• Suitably Rl can be dimethoxytrityl, pixyl, benzhydryloxy-bis (trimethylsilyloxy) silyl or levulinyl. Suitably R2 can be 2-cyanoethyl, methyl or 3-chlorophenyl . Suitably (R3)2 can be iso-propyl (twice) , methyl (twice) , morpholinyl or pyrrolidinyl. Suitably R4 can be benzoyl, N, N- dimethylformamyl or acetyl. Suitably R5 can be 0- tert-butyldimethylsilyl, 2-fluorophenyl) -4- methoxypiperidin-4-yl, bis (2-acetoxyethoxy) methyl or triisopropylsilyloxymethyl.

In particular embodiments the nucleic acid complex can comprise at least one of:

or

wherein

Rl is H or any protecting group for 3 '

R2 is H or any protecting group for Af⁴ of cytosine

R3 is a linker of the first aspect of the invention.

Suitably Rl can be hydrogen or acetyl. Suitably R2 can be hydrogen or acetyl.

In particular embodiments the nucleic acid complex

wherein Rl is H or any protecting group typical for 3 ' ; R2 is H or any protecting group for .Λ7⁴ of cytosine; R3 is selected from H (DNA), OMe (2 ' -0-methyl RNA), or OH (RNA) ; R4 is a linker according to the first aspect of the invention.

Suitably Rl can be hydrogen or acetyl. Suitably R2 can be hydrogen or acetyl.

According to a third aspect of the present invention there is provided a linker of the first aspect of the present invention coupled to a photoswitchable compound.

According to a fourth aspect of the invention there is provided a photo-modulatable compound comprising a nucleic acid complex of the second aspect of the invention and a photoswitchable compound.

Any suitable photoswitchable compound can be attached to the linker of the first aspect of the invention.

Any suitable photoswitchable compound can be attached to the linker of a nucleic acid complex of the second aspect of the invention. Suitably a photoswitchable compound is at least one compound selected from a group consisting of: azobenzenes, asonaphthalenes, azopyridines, azoimidazoles, diaryl alkene, spiropyran, spiroxazine, or azoarenes of formula

wherein AR is independently selected from the group consisting of

wherein each R^a may be independently selected from a Ci to C₄₀ linear or branched alkyl or a C₃ to C₈ cycloalkyl group, wherein said alkyl or cycloalkyl group may be substituted by one to three groups selected from: Ci to C₆ alkoxy, Ci to C₆ (di)alkylamino, C₆ to Cio aryl, Ci to C₃o aralkyl and Ci to C₃₀ alkaryl; NO₂; CO₂R; C(O)NH₂; F; each R^b, R^c, R^d, R^e, R^f, R^g and R^h can be the same or different and are each independently selected from H or any of the defined R^a groups.

Suitably a photoswitchable compound can be selected from the group comprising spiropyrans or spirooxazines of formula

wherein each R^a may be independently selected from a Ci to C₄₀ linear or branched alkyl or a C₃ to Cs cycloalkyl group, wherein said alkyl or cycloalkyl group may be substituted by one to three groups selected from: Ci to C₆ alkoxy, Ci to C₆ (di)alkylamino, C₆ to Ci₀ aryl, Ci to C₃₀ aralkyl and Ci to C₃₀ alkaryl; NO₂; CO₂R; C(O)NH₂; F; each R^b, R^c, R^d, R^e, R^f, R^g, R^h, R¹, R^j, R^k and R¹ can be the same or different and are each independently selected from H or any of the defined R^a groups .

Suitably a photoswitchable compound can be

Suitably a photoswitchable compound can be selected from the group comprising diaryl alkene of formula

wherein each R^a may be independently selected from a Ci to C₄₀ linear or branched alkyl or a C3 to Cs cycloalkyl group, wherein said alkyl or cycloalkyl group may be substituted by one to three groups selected from: Ci to C₆ alkoxy, Ci to Cs (di) alkylamino, Cg to Cio aryl, Ci to C3₀ aralkyl and C_x to C₃₀ alkaryl; NO₂; CO₂R; C(O)NH₂; F; each R^b, R^c, R^d, R^e, R^f, R^g, R^h and R¹ can be the same or different and are each independently selected from H or any of the defined R^a groups .

Preferred photoswitchable compounds are those which are stable to heat, for example zneta-azobenzenes and azopyridines .

As will be appreciated to those of skill in the art, the photoswitchable compounds may be attached to a base, a nucleoside or a nucleotide at an ortho, meta or para configuration, as for example shown in figure 1 in relation to the azobenzene compound

Preferably a 2 ' -deoxy-2 ' -aminouridine is linked to the azobenzene via an amide.

According to a fifth aspect of the present invention there is provided a polynucleic acid comprising a photo-modulatable compound of the fourth aspect of the present invention.

In one embodiment said polynucleic acid comprises DNA. In another embodiment said polynucleic acid comprises RNA.

In a polynucleic acid comprising a particular embodiment of a photo-modulatable compound of the fourth aspect of the invention wherein the phσtoswitchable compound is phenylazobenzene, irradiation of the compound at 350-360 run converts the E-azobenzene to a Z-azobenzene.

In a polynucleic acid comprising a particular embodiment of a photo-modulatable compound of the fourth aspect of the invention wherein the photoswitchable compound is spiropyran, irradiation of the compound at 350-360 nm converts the spiropyran to a merocyanine zwitterion or cation depending upon the pH.

The £-azobenzene and spiropyran isomers promote nucleic acid duplex stability whilst Z-azobenzene and merocyanine zwitterion isomers decrease nucleic acid duplex stability.

In particular embodiments a polynucleic acid comprising a photo-modulatable compound of the fourth aspect of the present invention may further comprise a fluorescent-quencher probe.

In said embodiments a fluorescent-quencher probe may be used to measure distance and geometry within nucleic acids, and/ or to monitor hybridisation of target sequences .

According to a sixth aspect of the present invention there is provided a method of modulating hybridisation of at least a first polynucleic acid with at least a second polynucleic acid comprising the steps: - providing the at least first polynucleic acid with at least one photo-modulatable compound of the fourth aspect of the invention; - providing the at least second polynucleic acid capable of hybridising to the at least first polynucleic acid in conditions suitable to allow hybridisation of the at least second polynucleic acid to the at least first polynucleic acid; and - providing a wavelength of light capable of inducing photoisomerization of the photo- modulatable compound in said at least first polynucleic acid, wherein photoisomerization of the photo-modulatable compound modulates hybridisation of the at least first and second polynucleic acids to each other.

According to a seventh aspect of the present invention there is provided a method of modulating the activity of a ribozyme comprising the steps of: - providing a ribozyme wherein at least one nucleotide of the catalytic core of the ribozyme is replaced by at least one photo- modulatable compound of the fourth aspect of the invention; and - providing a wavelength of light capable of inducing photoisomerization of the photo- modulatable compound, wherein the activity of the ribozyme is modulated.

Suitably the ribozyme is a deoxyribozyme.

According to an eighth aspect of the present invention there is provided a method of modulating the binding of a polymerase to a polynucleic acid comprising the steps of: - providing a polynucleic acid with at least one a photo-modulatable compound of the fourth aspect of the invention; and - providing a wavelength of light capable of inducing photoisomerization of the photo- modulatable compound, wherein the binding of the polymerase to the polynucleic acid is modulated.

In one embodiment the polymerase is a RNA polymerase.

Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

Embodiments of the present invention will now be described by way of example only and with reference to the accompanying drawings in which:

Figure 1 illustrates a structure of azobenzene- appended to uridylate analogues;

Figure 2 illustrates 10-23 deoxyribozymes as prepared (FAM = 6-fluoresceinyl-) , cleavage site indicated by arrow.

Figure 3 illustrates photo-isomerization of azobenzene unit (irrDR and d-aDR refer to irradiated and dark-adapted 10-23 deoxyribozymes respectively) ;

Figure 4 illustrates reversibility of RNA (20 μM) cleavage by 10-23 deoxyribozymes DRo (1 μM) , DRm (1 μM) and DRp (2 μM) under multiple turnover conditions. Key: O d-aDRo; • irrDRo; Δ d-aDRm; A irrDRm; D d-aDRp; ■ irrDRp;

Figure 5 is an illustration of a photo- modulatable compound of the fourth aspect of the invention comprising a linker of the first aspect of the invention interposed between a photoswitchable compound (PS) and a nucleotide, nucleoside or base;

Figure 6 is an illustration of a nucleic acid complex of the second aspect of the invention comprising a linker of the first aspect of the invention linked to base, nucleotide or nucleoside;

Figure 7 is an illustration of a linker of the first aspect of the invention linked to a photoswitchable compound (PS) ;

Figure 8 is an illustration of photoswitchable compounds (PS) which may be bound to a linker . of the invention;

Figure 9 illustrates photoswitching of azobenzenes (E<→Z) or spiropyran (SP) «→ merocyanine (MC) ;

Figure 10 illustrates a methodology for preparing photoswitchable 2'amino- 2 ' deoxyuridine derivatives, noting that the photoswitch compound (PS) as illustrated in (2a-d) , (3a-d) and (4a-d) can be replaced by any of (4a) , (4b) , (4c) or (4d) as shown;

Figure 11 illustrates photoswitching of d(ACC4dGGTA) . a) HPLC chromatagrams of oligomer following purification and irradiation b) degradation pathway during irradiation; and

Figure 12 illustrates Arrhenius plots derived from first order rate constants of the thermal isomerisation of Z-ABs within Jrr-d(ACC4a- cGGTA) under isothermal conditions at 55⁰C - 8O⁰C.

Synthesis of phosphoramidites

Dichloromethane (DCM) was dried under reflux over calcium hydride, distilled and stored over activated 3A molecular sieves under argon. Triethylamine and rz-butylamine were dried under reflux over calcium hydride and distilled immediately prior to use; N, N- diisopropylethylamine (DIPEA) was similarly purified but stored over activated 3A molecular sieves for upto 6 weeks. DMF was obtained as anhydrous solvent over molecular sieves from Fluka. Dry MeOH was obtained by distillation from magnesium turnings and stored over activated 3A molecular sieves. Tetrahydrofuran (THF) was distilled from sodium/benzophenone, stored over activated 3A molecular sieves and used within 7 days. Acid-free ethyl acetate was prepared by prewashing with saturated Na₂CO₃. CDCl₃ was passed through activated basic AI₂O₃ immediately prior to use and NMR' s obtained within 1 hour (³¹P, ¹³C) or 12 hours (¹H) of the CDCI₃ solution being prepared. All other reagents were purchased from commercial suppliers and used without further purification. Silica gel (particle size 30-60 μm) for flash column chromatography was obtained from BDH and dried at 300°C immediately prior to use.

UV-Visible data were obtained with a VARIAN CARY 100 Spectrometer. Nuclear Magnetic Resonance (NMR) spectra were run on Bruker AC-250 and AMX-400 spectrometers .

5 '-O- (4,4 '-dimethoxytrityl ) -2 ' - (N- ( o- phenylazo)benzamido) -2 '-deoxyuridine-3 '-O- (2- cyanoethyl-iV,.iV-diisopropyl-amino) -phosphoramidite .

To a stirred solution of o-phenylazobenzoic acid-N- hydroxysuccinimidyl ester (260 mg, 0.80 mmol) in anhydrous DMF (5 ml) at room temperature was added Λ-butylamine (10 μl, 0.10 mmol) and the reaction mixture maintained under these conditions for a further 10 min. This solution was transferred under argon to a flask containing 5 '-O- (4, 4'- dimethoxytrityl) -2 '-amino-2 '-deoxyuridine (250 mg, 0.46 mmol) and iV,IV-diisopropylethylamine (DIPEA: 80 μl, 0.47 mmol) added. The reaction was stirred under ambient conditions for a further 6 hours, a further aliquot of DIPEA (100 μl, 0.59 mmol) was added and the reaction stored at 3⁰C overnight. Excess activated ester was quenched by addition of n- butylamine (190 μl, 1.92 mmol) and stirring at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate (250 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 150 ml) and saturated NaCl (2 x 150 ml) . The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant red gum was purified by silica gel column chromatography, eluting with a gradient of 0 - 2% (v/v) methanol in DCM containing 1% (v/v) triethylamine to give the title compound as an orange solid (250 mg, 72 %) .

To a stirred solution of 5'-0-(4,4'- dimethoxytrityl) -2 '- (N- (o-phenylazo)benzamido) -2' - deoxyuridine (180 mg, 0.24 mmol) and DIPEA (90 μl, 0.98 mmol) in anhydrous DCM (4 ml) and THF (1 ml) at room temperature was added 2-cyanoethyl--V,-V- diisopropylaminochlorophosphoramidite (86 μl, 0.53 mmol) dropwise over 3 min. The reaction was maintained under these conditions for a further 30 min and then quenched following addition of anhydrous methanol (60 μl, 1.48 mmol) and DIPEA (60 μl, 0.65 mmol) and stirring for a further 10 min. The reaction mixture was diluted with acid-free ethyl acetate (50 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 50 ml), saturated NaCl (1 x 50 ml) . The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant solid was purified by silica gel column chromatography, eluting with 2% (v/v) solutions of triethylamine in 3:1 DCM: 40-60 petroleum ether; pure DCM and 3:1 DCM : ethyl acetate. The title compound was isolated as a brown foam (194 mg, 0.20 mmol, 85 %) .

5 ' -O- (4, 4 '-dimethoxytrityl) -2 '- (N- (m- phenylazo) benzamido) -2 '-deoxyuridine-3 '-O- (2- cyanoethyl-JV,JJ-diisopropylamino) -phosphoramidite .

To a stirred solution of m-phenylazobenzoic acid-iV- hydroxysuccinimidyl ester (260 mg, 0.80 mmol) in anhydrous DMF (5 ml) at room temperature was added n-butylamine (10 μl, 0.10 mmol) and the reaction mixture maintained under these conditions for a further 10 min. The reaction mixture was transferred under argon to a flask containing 5'-O- (4, 4'- dimethoxytrityl) -2'-aminσ-2'-deoxyuridine (250 mg, 0.46 mmol) and -V,iV-diisopropylethylamine (DIPEA: 80 μl, 0.47 mmol) added. The reaction was stirred under ambient conditions for a further 6 hours, a further aliquot of DIPEA (100 μl, 0.59 mmol) was added and the reaction stored at 3°C overnight. Excess activated ester was quenched by addition of n- butylamine (190 μl, 1.92 mmol) and stirring at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate (250 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 150 ml) and saturated NaCl (2 x 150 ml) . The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant red gum was purified by silica gel column chromatography, eluting with a gradient of 0 - 2% (v/v) methanol in DCM containing 1% (v/v) triethylamine to give the title compound as an orange solid (250 mg, 72 %) .

To a stirred solution of 5'-O- (4,4'- dimethoxytrityl) -2'- (-V-(in-phenylazo)benzamido) -2'- deoxyuridine (134 mg, 0.18 mmol) and DIPEA (67 μl, 0.73 mmol) in anhydrous DCM (3 ml) and THF (0.5 ml) at room temperature was added 2-cyanoethyl-I\J,2V- diisopropylaminochlorophosphoramidite (57 μl, 0.36 mmol) dropwise over 3 min. The reaction was maintained under these conditions for a further 30 min and then quenched following addition of anhydrous methanol (90 μl, 2.22 mmol) and DIPEA (90 μl 0.98 mmol) and stirring for a further 10 min. The reaction mixture was diluted with acid-free ethyl acetate (50 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 50 ml), saturated NaCl (1 x 50 ml) . The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant solid was purified by silica gel column chromatography, eluting with 2% (v/v) solutions of triethylamine in 3:1 DCM: 40-60 petroleum ether; pure DCM and 3:1 DCM : ethyl acetate. The title compound was isolated as an orange foam (160 mg, 0.17 mmol, 93 %) . 5 '-O- ( 4 , 4 '-dimethoxytrityl ) -2 ' - (N- (p- phenylazo) benzamido) -2 ' -deoxyuridine-J? ' -O- { 2- cyano ethyl -AT, AT-diisopropylamino ) -phosphoramidite .

To a stirred solution of p-phenylazobenzoic acid-IV- hydroxysuccinimidyl ester {260 mg, 0.80 mmol) in anhydrous DMF (5 ml) at room temperature was added zz-butylamine (10 μl, 0.10 mmol) and the reaction mixture maintained under these conditions for a further 10 min. The reaction mixture was transferred under argon to a flask containing 5'-0-{4,4^r- dimethoxytrityl) -2 '-aminσ-2 ' -deoxyuridine (250 mg, 0.46 mmol) and JVViV-diisopropylethylamine (DIPEA: 80 μl, 0.47 mmol) added. The reaction was stirred under ambient conditions for a further 6 hours, a further aliquot of DIPEA (100 μl, 0.59 mmol) was added and the reaction stored at 3°C overnight. Excess activated ester was quenched by addition of n- butylamine (190 μl, 1.92 mmol) and stirring at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate (250 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 150 ml) and saturated NaCl (2 x 150 ml) . The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant red gum was purified by silica gel column chromatography, eluting with a gradient of 0 - 2% (v/v) methanol in DCM containing 1% (v/v) triethylamine to give the title compound as an orange solid (250 mg, 72 %) .

To a stirred solution of 5 '-O- {4,4'- dimethoxytrityl) -2 '-(N- (p-phenylazo)benzamido) -2 ' - deoxyuridine (250 mg, 0.33 mmol) and DIPEA (125 μl, 1.36 mmol) in anhydrous DCM (5 ml) and THF (2 ml) at room temperature was added 2-cyanoethyl-ΛT_/-V- diisopropylaminochlorophosphoramidite (106 μl, 0.66 mmol) dropwise over 3 min. The reaction was maintained under these conditions for a further 30 min and then quenched following addition of anhydrous methanol (60 μl, 1.48 mmol) and DIPEA (60 μl 0.65 mmol) and stirring for a further 10 min. The reaction mixture was diluted with acid-free ethyl acetate (250 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 250 ml) , saturated NaCl (1 x 150 ml) . The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant solid was purified by silica gel column chromatography, eluting with 2% (v/v) solutions of triethylamine in 3:1 DCM: 40-60 petroleum ether; pure DCM and 3:1 DCM : ethyl acetate. The title compound was isolated as an orange foam (308 mg, 0.32 mmol, 97 %) .

5 ' -O- (4, 4 ' -Dimethoxytrityl) -2 ' -N- (3- [3 ' , 3 ' -dimethyl- 6-nitrospiropyro[2H-l-benzopyran-2_# 2 ' -indoline] -1- yl] -prσpanamidyl) -2 ' -deoxyuridine To a stirred solution of N-hydroxysuccinimidyl 3- [3 ' , 3 ' -dimethyl-6-nitrospiropyro [2H-l-benzopyran- 2, 2 '-indoline] -1-yl propanoate (P. Zhang, J. B. Meng, X. L. Li, T. Matsuura, and Y. M. Wang, Journal of Heterocyclic Chemistry, 2002, 39, 179) (381 mg, 0.80 mmol) in anhydrous DMF (5 ml) under argon at room temperature was added π-butylamine (10 μl, 0.10 mmol) and the reaction mixture maintained under these conditions for a further 10 min. The reaction mixture was transferred under argon to a flask containing 5 '-O- (4, 4 '-dimethoxytrityl) -2 '-amino-2 '- deoxyuridine (250 mg, 0.46 mmol) and to the stirred suspension was added N,iV-diisopropylethylamine (DIPEA: 80 μl, 0.47 mmol) following which addition a clear violet solution resulted. The reaction was stirred under ambient conditions for a further 4 hours, a further aliquot of DIPEA (190 μl, 1.12 mmol) was added and the reaction stored at 3⁰C overnight. Excess activated ester was quenched by addition of n-butylamine (190 μl, 1.92 mmol) and stirring at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate (250 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 150 ml) and saturated NaCl (150 ml) . The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant violet oil was purified by silica gel column chromatography, packed with DCM containing 2% (v/v) triethylamine and eluting with a gradient of 0 - 2% (v/v) methanol in DCM to give the title compound as an impure violet foam which was used directly in the subsequent phosphorylation (370 mg, <0 .41 minol , <90 % ) . ^" .

5 ' -O- (4, 4 ' -Dimethoxytrityl) -2 ' -N- (3- [3 ' , 3 ' -dimethyl- β-nitrospiropyro[2Jif-l-benzopyran-2,2 ' -indoline] -1- yl] -propanamidyl) -2 ' -deoxyuridine-3 ' -O- (2- cyanoethyl-AT,-V-diisopropylamino)phosphoramidite To a stirred red-purple solution of 5 '-O- (4,4'- Dimethoxytrityl ) -2 ' -N- (3- [3 , 3-dimethyl-6 ' - nitrospiropyro [2 'ff-chromene-2 , 2 '- (2,3- dihydro-liJ- indole) ] -1-yl] -propanamidyl) -2 ' -deoxyuridine (370 mg, <0.41 mmol) and DIPEA (154 μl, 1.69 mmol) in anhydrous DCM (6 ml) and THF (1 ml) at room temperature under argon was added J^-cyanoethyl-iV,^- diisopropylaminochloro-phosphoramidite (132 μl, 0.83 mmol) dropwise over 3 min during which time the solution decoloured. The reaction was maintained under these conditions for a further 30 min and then quenched following addition of anhydrous methanol (80 μl, 1.97 mmol) and DIPEA (80 μl, 0.88 mmol) and stirring for a further 10 min. The reaction mixture was diluted with acid-free ethyl acetate (50 ml) and washed successively with aqueous saturated NaHCO₃ (2 x 50 ml), saturated NaCl (1 x 50 ml). The organics were dried over anhydrous sodium sulfate, filtered and solvent removed in vacuo. The resultant solid was purified by silica gel column chromatography, eluting with 2% (v/v) solutions of triethylamine in 3:1 DCM: 40-60 petroleum ether; pure DCM and 3:1 DCM : ethyl acetate. Linking of a photoswitchable compound to phosphate

An example of a method of linking a photoswitchable compound to a phosphate of an oligonucleotide is described. An amino-modified oligonucleotide (typically 1 μmol) was dissolved in sodium carbonate/bicarbonate buffer at pH 9 (500μl) and DMSO (500μl) . Solid NHS ester (typically 5 mg) was added to give a saturated solution following sonication and vortex mixing. The reaction mixture was incubated at 37°C overnight. The oligo- azobenzene conjugate was separated from salts by size exclusion on a NAP-IO column. It was then further purified by RP HPLC.

Key p = -O-PO₂-O- Synthesis of oligonucleotides Oligosynthesis was carried out on Beckman Oligo IOOOM and Expedite 8909 DNA/RNA synthesizers on a 1 .μmol scale using standard DNA and RNA phosphoramidites from Link Technologies . Standard nucleobase protecting groups and 2'-0-TBDMS- protection were used. Benzylthiotetrazole (0.2 M) was used as activating agent. Oxidation was performed using 8:1:1 THF:pyridine .-H₂O containing either 50 mM or 20 mM I₂ for, respectively, Beckman- or Expedite-run syntheses. Unmodified DNA and RNA were synthesized DMT-on and deprotected and purified according to standard procedures. The coupling time was extended (5-6 min) for 2 '-azobenzene-modified am'idites. Deprotection of azobenzene-modified oligonucleotides was performed with 1.5 ml of an anhydrous mixture of NH₃ZMeOH (dry MeOH saturated with gaseous ammonia at -1O⁰C) overnight at room temperature. The decanted supernatant was evaporated and the crude oligonucleotides purified by RP-HPLC (column: Hichrom KR100-5C18; buffer A: 0.1 M TEAAc, pH 6.5, 5% (v/v) MeCN; buffer B: 0.1 M TEAAc, pH 6.5, 65% (v/v) MeCN) monitoring absorbance in the range 200 - 400 (or 600) nm with a diode array detector. After evaporation of the buffer the oligonucleotides were desalted using standard SepPak protocols (Waters) and concentrations determined using UV absorbance at 260 nm. MALDI-TOF analysis of DNA-azobenzene conjugates with reference figure 1

Photoswitching of DNA-azobenzene conjugates

For the irradiation a Medium Pressure Hg Arc-lamp (100 W, Engelhard Hanovia of Canada Ltd.) was utilized. A band-pass filter was used for irradiation at 366 run (4.13 W) and a cutoff filter was used for irradiation >400 nm (435 nm, 3.36 W) in combination with a water filter (1 cm) to prevent warming of the samples during prolonged irradiation.

As the high concentrations of deoxyribozyme and labeled substrate used prevented full kinetic analysis, all constructs were assayed at a fixed set of standard conditions (20 μM substrate, 2 μM deoxyribozyme) , and from the measured initial rates, apparent first order rate constants of the Z- and E- isomers were determined.

UV-spectroscopy to determine the thermal stability

Following irradiation to the Z-form at 366 run for 10 min, UV spectra of the ortho, meta and para modified oligonucleotides (5 μM in buffer: 50 mM pH 7.5 tris, 50 mM NaCl, 25 mM MgCl₂) were measured every 30 min for 6 hours. This procedure was carried out at the following temperatures; 25, 37, 45, 55 and 65 ⁰C. Before the final UV measurement samples were irradiated to the U-form at >400 nm for 2 min. The changing absorbances at 325 nm were converted into concentrations of the E-form of the photoisomerised oligonucleotides . The initial rates of these concentration changes were calculated and used in Arrhenius plots. Analysis of these data provided the activation energies.

Activity assays for 10-23 deoxyribozymes in the PAGE format

For the measurements of RNA-cleavage activities under multiple turnover conditions the deoxyribozyme construct (final concentration 2 μM or lμM) was added to a buffered solution containing 50 mM Tris, pH 7.5, 40 mM NaCl and 25 mM MgCl₂ (all final concentrations) . Preirradiation of these mixtures was performed at 366 nm for 10 min and at 435 nm for 2 min in clear Eppendorf™ tubes . The reaction was initiated by addition of the deoxyribozyme solutions to the substrate RNA (20 μM) , yielding a total volume of 50 or 100 μl . Incubation was performed in the absence of light at 26⁰C, except for irrDRp, which was incubated under continuous irradiation at 366 nm (temperature under these conditions was determined to be 26°C) . After appropriate time intervals aliquots (10 μl) were withdrawn, mixed with a stop solution (6 M urea, 50 mM EDTA) and frozen at -20°C. The samples were loaded on a 15% (w/v) (3% cross-linked) denaturing polyacrylamide sequencing gel and run at 900 V for 2 hr. The gel was then removed from the plates and transferred onto a gel documentation system (Versadoc, Biorad) and analyzed. Quantitation of the bands was performed with the accompanying software (QuantityOne) such that the intensity of the product bands was set in relation to the sum of the intensities of the product and substrate bands which resulted in relative cleavage values.

Activity assays for 10-23 deoxyribozymes using RP- HPLC analysis

For the measurements of RNA-cleavage activities under multiple turnover conditions the deoxyribozyme construct (final concentration 2 μM) was added to a buffered solution containing 50 mM Tris, pH 7.5, 40 mM NaCl and 25 mM MgCl₂ (all final concentrations) . Preirradiation of these mixtures was performed at 366 run for 10 min and at 435 run for 2 iαin in clear Eppendorf™ tubes . The reaction was initiated by addition of the deoxyribozyme solutions to the substrate RNA (20 μM) , yielding a total volume of 50 μl. Incubation was performed in the absence of light at 26°C, except for irrDRp, which was incubated under continuous irradiation at 366 nm (temperature under these conditions was determined to be 26⁰C) . After 1.5 hr an aliquot (45 μl) was removed and quenched with 10 μl 250 mM EDTA. For the injection 40 μl 0.1 M TEAAc were added and the samples analyzed on RP-HPLC (column: Hichrom KR100-5C18, buffer A: 0.1 M TEAAc, pH 6.5; buffer B: 0.1 M TEAAc, pH 6.5, 65% (v/v) MeCN; UV detection at 260 nm; gradient indicated by turquoise line) . Quantitation was performed by determining peak areas for the two product peaks and set in relation to the sum of the peak areas of the product and substrate peaks which resulted in relative cleavage values .

Chemical synthesis and analysis of nucleoside conformation (wherein references apply to figure 10).

Methodology for the preparation of photoswitchable 2 '-aminσ-2 '-deoxyuridine derivatives (3a-d; as illustrated in figure 10) was developed intially using phenyl para-azobenzoic acid in trial reactions. Direct condensation of this acid with 5 '-dimethoxytrityl-2 '-amino-2 '-deoxyuridine (1) mediated by EEDQ or IIDQ using literature protocols (D. P. C. McGee, A. Vaughnsettle, C. Vargeese, and Y. S. Zhai, Journal Of Organic Chemistry, 1996, 61, 781; B. Belleau and G. MaIek, Journal of the American Chemical Society, 1968, 90, 1651 and Y. Kiso, Y. Kai, and H. Yajima, Chemical and Pharmaceutical Bulletins, 1973, 21, 2507) yielded high (upto 30%) levels of side-products (in particular, from esterification of the 3'-hydroxyl) which were difficult to resolve from the desired product except following saponification.

Alternatively, reaction of 1 with the N- hydroxysuccinimidyl activated ester (2a) was found to overcome these side-reactions under conditions in which residual DCC and anhydrides were quenched prior to addition of the aminonucleoside. Quenching of these contaminants was achieved by treatment of 2a with sub-stoichiometric quantities of n- butylamine prior to addition of the aminonucleoside. This methodology was therefore adopted for the preparation of the photoswitchable nucleoside analogues 3b - 3d.

The large polarity difference between E-3a. and Z-3a enabled facile resolution of these isomers using silica gel chromatography. However, attempted phosphitylation of pure Z-3a was not successful. Both E-ZB. and Z-3a exhibited DNA-like C2'-endo ring puckers characterised by strong coupling between Hl' and H2 ' (J = 6.5 - 7.0 Hz) and weak H3'-H4' interactions (<lHz) . The C2'-endo conformation of the furanoside of both azobenzene isomers was maintained following detritylation of E-3a, and subsequent irradiation of the unprotected nucleoside at 300-350 run; this gave a mixture of E-3a and Z-3a (ca. 4/5) and revealed no significant change in the ring conformation. These observations contrast with recent descriptions of RNA-like C3'-endo sugar conformations observed for related deoxynucleotide 2 '-amine and 2 '-amide analogues⁴³ and also for 2'-O- para-phenylazobenzyl uridine.³⁷

Methodology for the phosphitylation of E-/Z-3a-c and their subsequent application to the solid-phase synthesis of oligodeoxyribonucleotides incoporating the corresponding nucleotide analogues (4a-c) has been described in S. Keiper and J. S. VyIe, Angewandte Chemie-International Edition, 2006, 45, 3306. Synthesis of the previously unreported spiropyran-nucleoside derivative 3d was performed by adapting related protocols developed in the laboratory of Meng (P. Zhang, J. B. Meng, T. Matsuura, and Y. M. Wang, Chinese Chemical Letters, 2002, 13, 299 and D. P. C. McGee, A. Vaughnsettle, C. Vargeese, and Y. S. Zhai, Journal Of Organic Chemistry, 1996, 61, 781) . Oligodeoxyribonucleotides incorporating the corresponding nucleotide analogue (4d) were synthesised using an RNA coupling protocol for the coupling of the modified phosphoramidite.

The synthesis of the oligoribonucleotides incorporating 4a-c was performed trityl-on using 2'- tBDMS-protected monomers. Deprotection was thus effected following treatment with anhydrous saturated methanolic ammonia overnight at ambient temperatures followed by a 33% (v/v) solution of triethylamine trishydrofluoride in 1-methyl-2- pyrrolidinone: triethylamine (2:1) at 65 ⁰C. Significant loss of the photoswitch from oligomers (both DNA and RNA) was observed upon initial treatment with either aqueous ammonia at room temperature or methylamine solutions under heating. The deprotected oligomers were then purified by reversed-phase HPLC, detritylated and desalted.

Example 1 - with reference to figures 1 to 4

Decocyribozymes were prepared in which a single nucleotide (T₈) within the catalytic core was replaced by a 2 ' -deoxyuridylate analogue. Ortho-, meta- or para-phenylazobenzoyl moieties were appended to this residue via a 2 ' -amido linkage. This site was chosen as deletion of Ts or substitution by 2 ' -O-methyluridine has previously been shown to only minimally perturb deoxyribozyme activities.

The deoxyribozyme constructs were engineered with short binding arms to promote product-release under multiple turnover conditions.

Modified nucleoside precursors were prepared by reaction of the protected 2 ' -aminonucleoside with the iV-hydroxysuccinimidyl ester of the appropriate azobenzene, for example, but not limited to,

and subsequently phosphitylated under standard conditions. These were incorporated into model 8- mers (ACClGGTA) and also 10-23 deoxyribozyme sequences using automated solid-phase synthesis with extended reaction times for introduction of the modified residue. Coupling yields as monitored by trityl release were greater than 98%. Following deprotection under anhydrous conditions, the oligonucleotides were purified by reversed-phase HPLC and their identities confirmed by MALDI-TOF analysis.

The photoisomerization of oligodeoxynucleotide- appended azobenzenes under irradiation at 366 run was investigated by UV/Vis spectroscopy of the model 8- mers . When the photoswitch.es were in the thermally stable S-configuration, the spectra showed local maxima at 330 (lo) , 321 (Im) , 329 (Ip) run typical for azobenzes π-π* transitions.

Irradiation at 366 run led to loss of these absorption maxima with photostationary states being achieved within eight minutes. These results were reproduced with azobenzene-appended deoxyribozymes and the E ~» Z conversion yields determined immediately by HPLC. Peak quantification at 260 run was used to determine the level of E- and 2-isomers either directly for DRm and DRp or by inference using the model 8-mer sequence containing Io. Following irradiation, E -» Z conversion yields of 86% for irrDRo, 75% for irrDRm and 61% for irrDRp were thus determined. At 26⁰C the ortho- and meta- photoswitches within both the model sequences and also deoxyribozymes were found to be stable towards Z —> E thermal back-isσmerization. In all cases Z —> E photoisomerization was complete following irradiation at 435 nm for two minutes.

Both azoberizene-modified and unmodified (wild type; DRwt) , deoxyribozymes catalyzed the site-specific cleavage of a 13-mer oligoribonucleotide substrate labelled at its 3 ' -terminus with a fluoresceinyl compound (FAM) to yield a' labelled 6-mer and a 2 ',3 '-cyclic phosphate terminated 7-mer. The cleavage reactions were thus resolved, visualized and quantified in a PAGE assay. Deoxyribozyme solutions were exposed to light at 366 nm for ten minutes or at 435 nm for two minutes and reactions initiated by addition of substrate RNA and incubated at 26⁰C in the absence of light. To assess the activity of the more thermally labile irrDRp, continuous irradiation at 366 nm was performed during the assay.

Under multiple turnover conditions, irradiated azobenzene-deoxyribozymes maintained essentially wild-type activities; thus irrDRo showed 100%, irrDRm 84% and irrDRp 50% activity. In contrast, RNA cleavage rates by dark-adapted (d-a) deoxyribozymes were considerably attenuated. k_irr/k_d-._a ratios in this assay were determined to be 9 for DRo and 8 for DRm and DRp) . Photocontrol of RNA-cleavage by DR- azobenzene conjugates was also demonstrated using an unlabeled RNA substrate and reversed-phase HPLC analysis. The effects observed in these assays compare well with the results from the PAGE analyses. Thus, the relative cleavage activities of irrDRo and irrDRm are both the same as for the unmodified deoxyribozyme DRwt, and irrDRp shows 44% of the wild-type activity. Dark-adapted deoxyribozymes give significantly less conversion than the irradiated constructs; ki_rr/kd-a discrimination factors of six for DRo and five for DRm and DRp constructs were observed.

The effect of photoswitching upon catalysis by the azobenzene-conjugated deoxyribozymes was also demonstrated to be reversible under multiple turnover conditions with either 5 mol% (DRo or DRm) or 10 mol% (DRp) deoxyribozyme. Following initial irradiation of deoxyribozyme solutions at 366 run, the reactions were initiated by addition of substrate and RNA-cleavage activities comparable with those previously described were observed. After 120 minutes the reactions were irradiated at 435 nm for two minutes to afford the dark-adapted deoxyribozymes which gave rise to significantly retarded reaction rates although these were higher than previously observed using purified E-DRs. This observation might be accounted for by perturbation of the Z —^■> E photoisomerization process in the presence of substrate RNA or kinetic folding traps, which do not respond to this isomerization. However, initial cleavage rates were recovered following irradiation at 366 nm. Preliminary NMR investigations indicate that both Ξ and Z isomers of Ip reside in the C2 '-endo furanoside pucker typical of 2 ' -amido deoxyribonucleoside analogues and their unmodified congeners and so the active conformation of the catalytic core containing the photoswitch may be modulated in some other fashion.

Example 2

Photoswitching efficiencies and thermal stabilities of irradiated states Spiropyran derivatives of indolene exhibit both thermochromism and photochromism arising from the heat- or UV-induced isomerisation to the corresponding highly coloured merocyanine- (MC) isomers. This was also observed for CPG-supported oligonucleotides which upon irradiation at 366 nrα for 130 s gave a deep purple colouration. However, in contrast to the stability observed for the irradiated AB-modified oligonucleotides, irradiation of SP-modified DNA at 36βnm over extended periods of time (as illustrated in figure 11) led to both the expected photoisomerisation product (R_t 17.6 min) but also significant decomposition of the photoswitchable moiety (Rt 14.2 min.

The decomposition product was isolated by HPLC (R_t 14.2 min) and characterised by MALDI mass spectrometry. The degradation process was thereby attributed to a reteroaldol reaction of the merocyanine to the corresponding methyleneindole- derivative. Such reactivity has previously been described for the preparation of substituted salicaldehydes from the corresponding spiropyrans under heating. Thermal degradation of the spiropyran modification via this route was also observed in aqueous solution.

The photochemical and thermal isomerisations of model octadeoxynucleotides bearing photoswitchable moieties at an internal site - d(ACC4a-dGGTA) - were investigated by UV / Vis spectroscopy and reversed- phase HPLC.

Irradiated photostationary states of the azobenzene- appended oligomers (containing 4a-c) were achieved following exposure to near-UV radiation from a medium pressure Hg Arc-lamp at 366 nm. At this wavelength, excitation of the π - π* transition induced E —> Z photoisomerisation of the azobenzene which was monitored by decrease in absorption at 325 nm. Irr states were thus induced within six minutes and the E/Z ratio determined after 10 minutes following analysis by RP-HPLC; oligomers bearing the more hydrophobic S-AB 's were resolved from those incoporating the corresponding Z-isomer

Dark-adapted photostationary states were induced much more rapidly following excitation of the n - π* transition at 436 nm. Thus, within two minutes full restoration of the original E-n - π* absorbances was observed and this was found to be independent of the nature of the AB.

The mole fractions of Z-AB-appended 8-mers (χ_z) in both photostationary states (Table 1) were quantified at 26⁰C and 40⁰C either directly by monitoring the HPLC output at the isosbestic points of the modified oligomers or indirectly from the apparent χ_z values at 260nm. High E —> Z photoswitching yields (>90%) are typical of the Irr- states of azobenzenes belonging to the "azobenzene type" due to large separation of the π - π* transitions in the different isomers (λma_x ~325nm for E-AB's and ~260nm for Z-AB' s) . However, attenuated yields of Z-para-AB in particular were observed following irradiation at 366nm at 26⁰C arising from the large positive structural volume change associated with the light-induced helix-coil transition at this temperature. This is illustrated in Table 1.

Table 1 Mole fractions (χ_z ) of oligomers containing Z-4a, Z-4b or Z-4c (wherein Z-4a is the para, zO4b is the meta and z-4c the ortho azobenzene photo- modulatable compounds as illustrated in figure 10) in irradiated and dark-adapted photostationary states at 26°C and 40⁰C.

The stabilities of the Jrr-oligonucleotides towards thermal Z —> E isomerisation were also investigated. Thus, the absorbances of Jrr-d(ACC4a-cGGTA) were monitored at 325 nm over six hours at temperatures between 55°C and 80⁰C. First order rate constants were derived for each experiment and Arrhenius and Eyring plots of these data were made as illustrated in Figure 12.

The kinetic parameters derived from the plots for oligonucleotides containing Z-4a and Z-4c (Table 2) are typical for the "azobenzene" class described by Rau. In contrast, thermal isomerisation of the meta isomer (Z-4a) displayed an elevated activation energy (high ΔH*) and fast pre-exponential factor (positive ΔS*) (e.g., E_a = 129 KJmol^"1; A = 1.71 x 10¹⁵ s^"1) . Such values have previously only rarely been described for azobenzenes in which rotation around the MN-C bond is inhibited by the presence of tetra-alkyl substituents ortho-to the azo bridge, by short alkyl tethers within azobenzenophanes , or azobenzene-linked crown ethers embedded within cast PMMA.

Table 2 Kinetic parameters describing the thermal Z - E isomerisation (wherein Z-4a comprises a para azobenzene photoswitchable compound, z-4b comprises a meta azobenzene photoswitchable compound and z-4c comprises an ortho azobenzene photoswitchable compound as illustrated in figure 10) .

Duplex stability T_m-measurements Self-complementary 8-mers Sequence context

To investigate the influence of the position and conformation of the nucleotide analogues la-c (as illustrated in figure 1) on the stability of the duplex as measured by melting temperatures a octanucleotide sequence was designed. The modification of an internal position, as shown in Table 1, led to an overall destabilisation of the duplexes (Table 3), whereas modification near the end was considerably less perturbing (Table 4) .

Table 3 Melting temperatures of oligonucleotides (wherein Z-4a comprises a para azobenzene photoswitchable compound, z-4b comprises a meta azobenzene photoswitchable compound and z-4c comprises an ortho azobenzene photoswitchable compound as illustrated in figure 10) .

_.

Target Probe T_M / oC ΔT_M / OC

DA Irr

5 '-d (TACCAGGT) 5 ^r-d (ACCTGGTA) 31 .1

5'-d(ACC4cGGTA) <20 <20 ND

5^r-d(ACC4bGGTA) <20 <20 ND

5'-d(ACC4aGGTA) 32.0 <15 >17

5'-d(ACCTGG4cA) 29.0 28.2 0, .8

5'-d(ACCTGG4bA) 30.0 27.5 2, .5

5'-d(ACCTGG4aA) 29.0 31.3 -2 .3

5 ^r-r (UACCAGGU) 5 '-d (ACCTGGTA) 35, .0

5'-d(ACC4cGGTA) 27.5 24.5 3. 0

5'-d(ACC4bGGTA) 27.0 23.1 3. 9

5'-d(ACC4aGGTA) 28.5 31.0 -2 .5

5'-d(ACCTGG4cA) 32.5 33.1 -0 .6

5'-d(ACCTGG4bA) 33.0 32.8 0. 2 5'-d(ACCTGG4aA) 35.5 33.2 2.3

5'-d(TCGT4aGAC) 5 ' -d(GTC4aACGA) 31.6 28.0 3.6

5 '-d (TCGTCGAC) 5'-d(GTC4aACGA) 29.2 29.3 -0.1

5 '-d (TCGTTGAC) 5'-d(GTC4aACGA) 22.3 22.0 0.3

5 ' -d (TCGTAGAC) 5 ' -d (GTC4aACGA) <20 <20 ND

5 '-d (TCGTGGAC) 5 ' -d(GTC4aACGA) <20 <20 ND

5'-d(ACC4aGGTA) 30.5 <15 >15.5

5'-d(ACG4aCGTA) 37.2 31.5 5.7

5^f-d(ACC4aGGTA) 32.0 27.1 4.9

5'-d(CGT4aACGA) 34.5 27.7 7.8

5 ' -d ( ACC4a4aGGTA) 31.5 29.1 2.4

5 ' -d (GATATC4aGATATC) SEQ ID NO 1 41.8 39.9 1.9

Table 4

(wherein Z-4a comprises a para azobenzene photoswitchable compound, z-4b comprises a meta azobenzene photoswitchable compound and z-4c comprises an ortho azobenzene photoswitchable compound as illustrated in figure 10) Target Probe T_M / oC ΔT_M / oC

DA Irr

5 ^r-d (TACCAGGT) 5' -r (ACCTGGTA) 32 .5 -

5' -r(ACC4aGGTA) 30 .5 <20 >10.5

5' -r(ACC4bGGTA) 32 .0 <20 >12.0

5' -r(ACC4cGGTA) 25 .0 28 .1 -3.1

5' -r(ACCTGG4aA) 27 .9 28 .5 -0.6

5' -r (ACCTGG4bA) 28 .5 28 .7 -0.2

5^f -r(ACCTGG4cA) <2 0 <20 ND

5 '-r (UACCAGGU) 5' -r (ACCTGGTA) 48 .1 _

5' -r (ACC4aGGTA) 33, .0 31. .7 1.3

5' -r(ACC4bGGTA) 33, .0 34. .1 -0.9

5' -r(ACC4cGGTA) 32. .5 32. .6 -0.1

5^r -r(ACCTGG4aA) 48, .0 45. .3 2.7

5^f -r(ACCTGG4bA) 45. .0 45. ,0 0.0

5' -r(ACCTGG4cA) 44. .5 46. ,5 -2.0

Only minor effects of the conformation of the azobenzene moieties were observed in most cases. Incorporation of Ia leads to a strong destabilisation of the DNA duplex when switched to the Z-conformation.

IA

Key p = -0-PO₂-O-

Surprisingly, in the presence of an RNA counter strand both the irradiated and dark-adapted sequences demonstrated similar melting behaviours.

Upon further investigation, the strong destabilisation of the DNA duplex modified with Ia was found to result from the self-complementary nature of the oligonucleotide sequence. When the experiment was performed in the absence of the counter strand a similar destabilisation was measured (Table 2) . A further set of duplex stablity measurements were then performed designed to probe the generality of this phenomenom. Thus, for the melting behaviour of three related self-complementary octanucleotides with a single internal AB modification in which the heptanucleotide duplex region is flanked by 5'- overhangs, the magnitude of the effect is sequence- dependent .

The lack of difference of duplex stability observed for the double-substituted sequence following photoswitching was rationalised by the low efficiency encountered for this process which, would result in the presence of over 50% of irradiated sequences containing an E-AB (and also the well precendented diminishing efficiency of photoisomerisation of proximal AB' s)

Oligo d (CnGn) sequences are known to adopt A-form DNAand in order to probe the potential for conformational switching, Ia was inserted into a central postion of a 13-mer oligonucleotide flanked by two EcoRV restriction sites. A → B switching was observed upon irreadiation at 366 run.

From these data the inventors conclude that a strong effect (i.e. >6°C) is only found for symmetrical sequences that bear the photoswitch opposite each other in both strands and 1 nt overhangs on either end for the particular modification under investigation. The inventors have also determined that for unmodified counterstrands (as is the case in potential applications) a cytidine residue opposite the modified base gives the most stable duplex.

The above demonstrates light-modulated self- recognition by DNA and describes conformational photoswitching of a DNA duplex.

Various modifications may be made to the invention herein described without departing from the scope thereof .

Claims

1. A linker for linking a photoswitenable compound (PS) to a base, nucleoside or a nucleotide, the linker comprising:

wherein X independently represents

(III) (IV) and a is 0 or 1;

Z independently represents

or

wherein nl, n.2 , n3 , n.4, n.5, nβ, and n7 are independently selected from each other and each of nl , n2 , n3 , n4, n5, nβ, and n7 is 0 or an integer selected from the range 1 to 12;

(R₇) represents H or CH₃;

Y independently represents

(IX) (X) ⁽XI)

and b is 0 or 1 or 2.

2. A linker as claimed in claim 1 wherein X is represented by formula (I) , Z is independently selected from the structures as represented by one of formula (V), (VI), (VII), or (VIII) and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

3. A linker as claimed in claim 1 wherein X is represented by formula (II) , Z is independently selected from the structures as represented by one of formula (V), (VI), (VII), or (VIII) and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

4. A linker as claimed in claim 1 wherein X is represented by formula (III) , Z is independently selected from the structures as represented by one of formula (V), (VI), (VII), or (VIII) and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

5. A linker as claimed in claim 1 wherein X is represented by formula (IV) , Z is independently selected from the structures as represented by one of formula (V), (VI), (VII), or (VIII) and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

6. A linker as claimed in claim 1 wherein X is represented by one of the structures as represented by formula (I), (II), (III), or (IV), Z is independently selected from the structure as represented by the formula (V) , and Y is independently selected from the structures as represented by of formula (IX) , (X) or (XI) .

7. A linker as claimed in claim 1 wherein X is represented by one of the structures as represented by formula (I), (II), (III), or (IV), Z is independently selected from the structure as represented by the formula (VI) , and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

8. A linker as claimed in claim 1 wherein X is represented by one of the structures as represented by formula (I), (II), (III), or (IV), Z is independently selected from the structure as represented by the formula (VII) , and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

9. A linker as claimed in claim 1 wherein X is represented by one of the structures as represented by formula (I), (II), (III), or (IV), Z is independently selected from the structure as represented by the formula (VIII) , and Y is independently selected from the structures as represented by one of formula (IX) , (X) or (XI) .

10. A linker as claimed in claim 1 wherein X is represented by one of the formula (I), (II), (III), or (IV) , Z is independently selected from the structures as represented by the formula (V) , (VI) , (VII) , or (VIII) , and Y is a structure as represented by (IX) .

11. A linker as claimed in claim 1 wherein X is represented by one of the formula (I) , (II) , (III) , or (IV) , Z is independently selected from the structures as represented by the formula (V) , (VI) , (VII), or (VIII), and Y is a structure as represented by (X) .

12. A linker as claimed in claim 1 wherein X is represented by one of the formula (I) , (II) , (III) , or (IV) , Z is independently selected from the structures as represented by the formula (V) , (VI) , (VII) , or (VIII) , and Y is a structure as represented by (XI) .

13. A nucleic acid complex comprising a nucleic acid and a linker as claimed in any one of claims 1 to 12.

14. A nucleic acid complex as claimed in claim 13 wherein the nucleic acid comprises a DNA or RNA nucleoside or nucleotide.

15. A nucleic acid complex as claimed in claim 13 or claim 14 wherein the linker is attached to a base of a nucleoside or nucleotide.

16. A nucleic acid complex as claimed in any claim 13 or claim 14 wherein a linker is attached to the sugar portion of a nucleoside or nucleotide.

17. A nucleic acid complex as claimed in claim 13 or claim 14 wherein the linker is attached to a 5- proparglyamino function of a nucleoside.

18. A nucleic acid complex as claimed in claim 13 or claim 14 wherein the linker is attached to a 2 ' amino function of a nucleoside.

19. A nucleic acid complex as claimed in claim 13 or claim 14 wherein the linker is attached to a modified phosphate diester such that the nucleic acid complex is selected from at least one of:

Ri \ Base

OH or

wherein

Rl is O- or is any 5' nucleic acid fragment either DNA or RNA; Base is selected from adenosine, cytosine, guanine, thymine or an analogue thereof; R2 is selected from H (DNA), OMe (2 '-0-methyl RNA) or OH (RNA) , R3 is 0- or is any 3 ' nucleic acid fragment either DNA or RNA; and R4 is the linker.

20. A nucleic acid complex as claimed in claim 13 or claim 14 wherein said nucleic acid complex is selected from A, B, C, D, E or F

wherein

Rl is any protecting group typical for 5 ' ;

R2 is any protecting group for phosphate or phosphate triesters;

R3 is any alkyl group for phosphoramidite;

R4 is any protecting group for N⁴ of cytosine; and

R5 is the linker.

21. A nucleic acid complex as claimed in claim 13 or claim 14 wherein said nucleic acid complex is selected from G, H, I, J, K or L

H

J K wherein

Rl is any protecting group typical for 5'; R2 is any protecting group for phosphate or phosphate triesters; R3 is any alkyl group for phosphoramidite; R4 is any protecting group for iV⁴ of cytosine; R5 is selected from H (DNA), OMe (2 ' -0-methyl RNA), or an O-protecting group (RNA) ; R6 is the linker.

22. A nucleic acid complex as claimed in claim 13 or claim 14 wherein said nucleic acid complex is selected from:

or

wherein Rl is H or any protecting group for 3' R2 is H or any protecting group for JV⁴ of cytosine R3 is the linker.

23. A nucleic acid complex as claimed in claim 13 or claim 14 wherein said nucleic acid complex is selected from:

Rl is H or any protecting group typical for 3 ' ;

R2 is H or any protecting group for iV⁴ of cytosine;

R3 is selected from H (DNA), OMe (2 ' -O-methyl RNA), or OH (RNA) ;

R4 is the linker.

24. A photo-modulatable compound comprising a nucleic acid complex as claimed in any one of claims 13 to 23 and a photoswitenable compound.

25. A photo-modulatable compound as claimed in claim 24 wherein the photoswitenable compound is at least one compound selected from a group consisting of: azobenzene, asonaphthalene, azopyridine, azoimidazole, diaryl alkene, spiropyran, spiroxazine, or azoarene of formula

wherein AR is independently selected from the group consisting of

wherein each R^a may be independently selected from a Ci to C40 linear or branched alkyl or a C3 to Cs cycloalkyl group, wherein said alkyl or cycloalkyl group may be substituted by one to three groups selected from: Ci to Ce alkoxy, Ci to Ce (di ) alkylamino, C₆ to Ci₀ aryl, Ci to C₃o aralkyl and Ci to C₃₀ alkaryl; NO₂; CO₂R; C(O)NH₂; F; each R^b, R^c, R^d, R^e, R^f, R^g and R^h can be the same or different and are each independently selected from H or any of the defined R^a groups.

26. A photo-modulatable compound as claimed in claim 24 or claim 25 wherein the spiropyran wherein X is CH or the spirooxazine wherein X is N is of the formula

wherein each R^a may be independently selected from a

Ci to C40 linear or branched alkyl or a C₃ to Cs cycloalkyl group, wherein said alkyl or cycloalkyl group may be substituted by one to three groups selected from: Ci to Ce alkoxy, Ci to C₆

(di) alkylamino, C₆ to Cio aryl, Ci to C₃₀ aralkyl and

Ci to C₃₀ alkaryl; NO₂; CO₂R; C(O)NH₂; F; each R^b, R^c, R^d, R^e, R^f, R^g, R^h, R¹, R^j, R^k and R¹ can be the same or different and are each independently selected from H or any of the defined R^a groups .

27. A photo-modulatable compound as claimed in claim 24 or claim 25 wherein the diaryl alkene is of the formula

wherein each R^a may be independently selected from a Ci to C₄₀ linear or branched alkyl or a C₃ to C₈ cycloalkyl group, wherein said alkyl or cycloalkyl group may be substituted by one to three groups selected from: Ci to Ce alkoxy, Ci to C_δ (di)alkylamino, C₆ to Cio'aryl, Ci to C₃0 aralkyl and Ci to C₃₀ alkaryl; NO₂; CO₂R; C(O)NH₂; F; each R^b, R^c, R^d, R^e, R^f, R^g, R^h and R¹ can be the same or different and are each independently selected from H or any of the defined R^a groups.

28. A photo-modulatable compound as claimed in claim 24 or claim 25 wherein the photoswitchable compound is

29. A polynucleic acid comprising a photo- modulatable compound of any one of claims 24 to 28

30. A polynucleic acid as claimed in claim 29 wherein said polynucleic acid comprises DNA.

31. A polynucleic acid as claimed in claim 29 wherein said polynucleic acid comprises RNA.

32. A polynucleic acid as claimed in any one of claims 29 to 31 wherein the photo-modulatable compound comprises the photoswitenable compound phenylazobenzene wherein irradiation of the compound at 350-360 run converts the E-azobenzene to a Z- azobenzene.

33. A polynucleic acid as claimed in any one of claims 29 to 31 wherein the photo-modulatable compound comprises the photoswitchable compound spiropyran wherein irradiation of the compound at 350-360 nm converts the spiropyran to a merocyanine zwitterion or cation depending upon the pH.

34. A linker of any one of claims 1 to 12 further comprising a photoswitchable compound.

35. A method of modulating hybridisation of at least a first polynucleic acid with at least a second polynucleic acid comprising the steps: - providing the at least first polynucleic acid with at least one photo-modulatable compound of any one of claims 24 to 28; - providing the at least second polynucleic acid capable of hybridising to the at least first polynucleic acid in conditions suitable to allow hybridisation of the at least second polynucleic acid to the at least first polynucleic acid; and - providing a wavelength of light capable of inducing photoisomerization of the photo- modulatable compound in said at least first polynucleic acid, wherein photoisomerization of the photo-modulatable compound modulates hybridisation of the at least first and second polynucleic acids to each other.

36. A method of modulating the activity of deoxyribozyme comprising the steps of: - providing a deoxyribozyme wherein at least one nucleotide of the catalytic core of the deoxyribozyme is replaced by at least one photo-modulatable compound of any one of claims 24 to 28; and - providing a wavelength of light capable of inducing photoisomerization of the photo- modulatable compound, wherein the activity of the deoxyribozyme is modulated.

37. A method of modulating the binding of a polymerase to a polynucleic acid comprising the steps of: - providing a polynucleic acid with at least one a photo-modulatable compound of any one of claims 24 to 28; and - providing a wavelength of light capable of inducing photoisomerization of the photo- modulatable compound, wherein the binding of the polymerase to the polynucleic acid is modulated.