Modified Molecular Arrays

Abstract

The invention relates to the preparation of a hydrogel surface useful in the formation and manipulation of arrays of molecules, particularly polynucleotides and to the chemical modification of these and other arrays. In particular, the invention relates to a method of preparing a hydrogel immobilised to a solid support comprising polymerising on the support a mixture of a first comonomer which is acrylamide, methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone and a second comonomer which is a functionalised acrylamide or acrylate.

1 . A method of preparing a hydrogel immobilised to a solid support comprising polymerising on said support a mixture of:
 (i) a first comonomer which is acrylamide, methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone; and   (ii) a second comonomer which is a functionalised acrylamide or acrylate of formula
   H 2 C═C(H)—C(═O)-A-B—C  (I);
 
   
       or a methacrylate or methacrylamide of formula (II):
   or H 2 C═C(CH 3 )—C(═O)-A-B—C—  (II)
 
 
       (wherein:
 A is NR or O, wherein R is hydrogen or an optionally substituted saturated hydrocarbyl group comprising 1 to 5 carbon atoms; 
 —B— is an optionally substituted alkylene biradical of formula —(CH 2 ) n — wherein n is an integer from 1 to 50; and wherein n=2 or more, one or more optionally substituted ethylene biradicals —CH 2 CH 2 — of said alkylene biradical may be independently replaced by ethenylene and ethynylene moieties; and wherein n=1 or more, one or more methylene biradicals —CH 2 — may be replaced independently with an optionally substituted mono- or polycyclic hydrocarbon biradical comprising from 4 to 50 carbon atoms, or a corresponding heteromonocyclic or heteropolycyclic biradical wherein at least 1 CH 2  or CH 2  is substituted by an oxygen sulfur or nitrogen atom or an NH group; and 
 C is a group for reaction with a compound to bind said compound covalently to said hydrogel) to form a polymerised product, 
 characterised in that said method is conducted on, and immobilises the polymerised product to, said support which is not covalently surface-modified. 
 
     
     
         2 . The method as claimed in  claim 1  wherein said support is a silica-based support. 
     
     
         3 . The method as claimed in  claim 2  wherein said silica-based support is fused silica. 
     
     
         4 . The method as claimed in  claim 3  wherein said silica fused silica is SPECTRASlL™. 
     
     
         5 . The method as claimed in  claim 1  wherein said support is a non silica-based support. 
     
     
         6 . A method as claimed in  claim 1  wherein said first comonomer is acrylamide. 
     
     
         7 . A method as claimed in  claim 1  wherein said second comonomer is an acrylamide of formula (I). 
     
     
         8 . A method as claimed in  claim 6  wherein said acrylamide of formula (I) has A=NH. 
     
     
         9 . A method as claimed in  claim 1  wherein —B— is a C 2 -C 10  alkylene biradical. 
     
     
         10 . The method as claimed in  claim 8  wherein —B— is —(CH 2 ) 5 —. 
     
     
         11 . The method as claimed in  claim 1  wherein C is hydroxyl, thiol, amine, acid, ester or haloacetamido. 
     
     
         12 . The method as claimed in  claim 11  wherein said haloacetamido is bromoacetamido. 
     
     
         13 . The method as claimed in  claim 1  wherein said acrylamide of formula (I) is N-(5-bromoacetamidylpentyl)acrylamide (BRAPA). 
     
     
         14 . The method as claimed in  claim 1  wherein said second comonomer is present in an amount of ≧1 mol % relative to the total molar quantity of comonomers. 
     
     
         15 . The method as claimed in  claim 14  wherein said second comonomer is present in an amount of ≧2 mol % relative to the total molar quantity of total comonomers. 
     
     
         16 . The method as claimed in  claim 1  wherein no polyunsaturated crosslinking agent is present during said polymerising. 
     
     
         17 . A solid-supported hydrogel obtainable according to the method of  claim 1 . 
     
     
         18 . The solid-supported hydrogel of  claim 17  wherein the thickness of the hydrogel is less than 100 nm. 
     
     
         19 . A method of preparing a solid supported hydrogel-based molecular array, said method comprising reacting one or more molecules of interest with reactive sites present in a solid-supported hydrogel as defined in  claim 17 . 
     
     
         20 . The method of  claim 19  wherein said molecules of interest are biomolecules. 
     
     
         21 . The method of  claim 19  wherein said molecules of interest are polynucleotides or proteins. 
     
     
         22 . The method of  claim 21  wherein said molecules of interest are polynucleotides. 
     
     
         23 . The method of  claim 22  wherein at least a portion of each polynucleotide is single-stranded. 
     
     
         24 . The method of  claim 22  wherein said polynucleotides comprise from 1 to 20 spacer nucleotides. 
     
     
         25 . The method of  claim 24  wherein said polynucleotides comprise from 1 to 10 spacer nucleotides. 
     
     
         26 . The method of  claim 25  wherein said polynucleotides comprise 10 spacer nucleotides. 
     
     
         27 . The method of  claim 24  wherein said spacer nucleotides each contain the base thymine (T). 
     
     
         28 . The method of  claim 22  wherein said polynucleotides are hairpin polynucleotides. 
     
     
         29 . The method of  claim 19  wherein said molecules of interest contain a sulfur-containing nucleophile. 
     
     
         30 . The method of  claim 29  wherein said sulfur-containing nucleophile is a moiety of the formula (III): 
       
         
           
           
               
               
           
         
       
       (wherein ˜ denotes the bond or linker connecting the sulfur-based nucleophile to the remainder of the polynucleotide; X represents an oxygen atom, a sulfur atom or a group NR, in which R is hydrogen or an optionally substituted C 1-10  alkyl; Y represents an oxygen or a sulfur atom; and Z represents an oxygen atom, a sulfur atom or an optionally substituted C 1-10  alkyl group). 
     
     
         31 . The method of  claim 30  wherein X is oxygen or sulfur. 
     
     
         32 . The method of  claim 30  wherein Y is oxygen. 
     
     
         33 . The method of  claim 30  wherein Z is an oxygen or sulfur atom or a methyl group. 
     
     
         34 . The method of  claim 30  wherein said moiety is thiophosphate. 
     
     
         35 . The method of  claim 29  wherein said sulfur-containing nucleophile is connected to the molecule of interest by a linker group and wherein said molecule of interest is a polynucleotide. 
     
     
         36 . A method of preparing a solid supported hydrogel-based molecular array which is a clustered array of molecules of interest, the method comprising:
 (i) reacting polynucleotide molecules with reactive sites present in a solid-supported hydrogel according to the method of  claim 22 , wherein said polynucleotide molecules are first and second oligonucleotide primers capable of hybridising to a template to be amplified;   (ii) contacting the first oligonucleotide primers attached to the solid-supported hydrogel in step (i) with one or more templates to be amplified under conditions which permit hybridisation of the templates to the oligonucleotide primers, each template comprising at the 3′ end a sequence capable of hybridising to the first oligonucleotide primer and at the 5′ end a sequence the complement of which is capable of hybridising to a second oligonucleotide primer; and   (iii) performing one or more nucleic acid amplification reactions using the first and second oligonucleotide primers and the template(s), thereby generating a clustered array of molecules of interest.   
     
     
         37 . A method of modifying a molecular array, which molecular array comprises a plurality of molecules of interest immobilised to a surface of a support, said method comprising the step of applying to the array polyelectrolyte or neutral polymers. 
     
     
         38 . The method of  claim 37  wherein said molecules of interest are biomolecules. 
     
     
         39 . The method of  claim 37  wherein the support is comprised of a member selected from the group comprising silica-based substrates, hydrogels and polyelectrolyte multilayers. 
     
     
         40 . The method of  claim 39  wherein the molecules of interest are attached directly or through a linking moiety to a silica-based support. 
     
     
         41 . The method of  claim 39  wherein the hydrogel is a polyacrylamide hydrogel. 
     
     
         42 . The method of  claim 39  wherein the polyelectrolyte multilayer comprises one or more layers of each of polyallylamine and polyacrylic acid wherein the surface to which the biomolecules are attached comprises polyacrylic acid. 
     
     
         43 . The method of  claim 39  wherein the hydrogel is obtainable by a method comprising polymerising on a solid support a mixture of:
 (i) a first comonomer which is acrylamide, methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone; and 
 (ii) a second comonomer which is a functionalised acrylamide or acrylate of formula (I):
   H 2 C═C(H)—C(═O)-A-B—C  (I);
 
 
 
       or a methacrylate or methacrylamide of formula (II):
   or H 2 C═C(CH 3 )—C(═O)-A-B—C—  (II)
 
 
       (wherein:
 A is NR or O, wherein R is hydrogen or an optionally substituted saturated hydrocarbyl group comprising 1 to 5 carbon atoms; 
 —B— is an optionally substituted alkylene biradical of formula —(CH 2 ) n — wherein n is an integer from 1 to 50; and wherein n=2 or more, one or more optionally substituted ethylene biradicals —CH 2 CH 2 — of said alkylene biradical may be independently replaced by ethenylene and ethynylene moieties; and wherein n=1 or more, one or more methylene biradicals —CH 2 — may be replaced independently with an optionally substituted mono- or polycyclic hydrocarbon biradical comprising from 4 to 50 carbon atoms, or a corresponding heteromonocyclic or heteropolycyclic biradical wherein at least 1 CH 2  or CH 2  is substituted by an oxygen sulfur or nitrogen atom or an 
 NH group; and 
 C is a group for reaction with a compound to bind said compound covalently to said hydrogel) to form a polymerised product wherein said polymerising is conducted on, and immobilises the polymerised product to, said solid support. 
 
     
     
         44 . The method of  claim 43  wherein the said solid support is not covalently surface-modified. 
     
     
         45 . The method of  claim 37  wherein the polyelectrolyte applied is polyacrylic acid. 
     
     
         46 . The method of  claim 37  wherein polyallylamine is applied to the array followed by polyacrylic acid. 
     
     
         47 . The method of  claim 37  wherein the neutral polymer is polyethylene glycol. 
     
     
         48 . The method of  claim 37  wherein the method comprises modifying a microarray or a single molecule array. 
     
     
         49 . The method of  claim 48  wherein the method comprises modifying a single molecule array. 
     
     
         50 . The method of  claim 48  wherein the method comprises modifying a clustered microarray. 
     
     
         51 . A molecular array obtainable according to the method of  claim 19 . 
     
     
         52 . The molecular array of  claim 51  which is a single molecule array. 
     
     
         53 . The molecular array of  claim 51  which is a clustered microarray. 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . (canceled)