Patent Application
20170073673
This invention relates to methods for purifying the products of nucleic acid conjugation reactions from unreacted reactants, in particular, in the production of purified nucleic acid-conjugates for nucleic acid encoded chemical libraries.
Nucleic acid encoded chemical libraries are collections of chemical moieties covalently linked to identifier oligonucleotides encoding the identity of the chemical moieties. The members of nucleic acid encoded chemical libraries display pharmacophores made up of one or more chemical moieties (also called “building blocks”). These chemical libraries can be used to identify pharmacophores which are candidate binding agents or have improved characteristics, for example improved binding. DNA-encoded chemical libraries (DEL) are a particular type of nucleic acid encoded chemical libraries for which the nucleic acids is deoxyribonucleic acid (DNA).
Diverse populations of pharmacophores are produced by different combinations of chemical moieties. Each library member is tagged with a nucleic acid strand comprising nucleotide sequences that encode the chemical moieties that constitute the pharmacophore that is displayed by the member. This allows rapid identification of selected library members during screening.
DNA-encoded chemical library (DEL) technology allows the synthesis and screening of pharmacophores libraries of unprecedented size. DEL represents an advance in medicinal chemistry, which bridges the fields of combinatorial chemistry and affinity selection techniques (e.g., phage display, mRNA display) and promises to revolutionise the drug discovery field and to reshape the way pharmaceutically relevant compounds are traditionally discovered. Recent advances in ultrahigh-throughput nucleic acid sequencing (e.g., Illumina sequencing, SOLiD technology, etc.) indicate that it should be possible to sequence even billions of sequence tags per sequencing run. With suitable synthetic and encoding procedures, it should be possible to construct, perform selections, and decode nucleic acid-encoded libraries comprising multiple building blocks and containing millions to billions of chemical compounds [1].
DNA encoded chemical libraries may display pharmacophores that are formed of chemically linked chemical moieties that are all attached to a single strand of nucleic acid (“single pharmacophore libraries”) or pharmacophores that are formed of chemical moieties that are attached to two different strands of nucleic acid hybridised together, one or more chemical moieties being attached to each strand (“dual pharmacophore libraries”).
DNA-encoded chemical libraries were proposed first by Sydney Brenner and Richard Lerner in 1992 ([2]; U.S. Pat. No. 5,573,905; WO93/20242). These authors postulated the alternating stepwise synthesis of a polymer (e.g. a peptide) and an oligonucleotide sequence (serving as a coding sequence) on a common linker (e.g. a bead) in split and pool cycles. After affinity capture on a target protein, the population of identifier oligonucleotides of the selected library members would be amplified by PCR and, in theory, used for enrichment of the bound molecules by serial hybridisation steps to a subset of the library. In principle, the affinity-capture procedure could be repeated, possibly resulting in a further enrichment of the active library members. Finally, the structures of the chemical entities would be decoded by cloning and sequencing the PCR products. It was postulated that encoding procedures could be implemented by a variety of methods, including chemical synthesis, DNA polymerization or ligation of DNA fragments [2].
The feasibility of the orthogonal, solid-phase synthesis of peptides and oligonucleotides was demonstrated by attaching a test peptide (the pentapeptide leucine enkephalin) and an encoding identifier oligonucleotide onto controlled-pore glass beads [3]. The peptide bound to a specific antibody and the corresponding DNA coding tag was amplified by PCR. The technology was used to construct a collection of ˜106 heptapeptide sequences and their corresponding identifier oligonucleotide tags on beads. The library was incubated with a fluorescently labelled anti-leucine enkephalin antibody, and binders were selected successfully by fluorescent-assisted cell sorting [4].
In their original paper, Brenner and Lerner suggested that the alternate synthesis of chemical compounds and oligonucleotides could also be implemented in the absence of beads. The use of enzyme catalysed ligation of coding DNA fragments is now established in the field (US 2006-0246450; WO 02/103008; WO2004/013070; WO2004/074429; WO2007/062664 and WO2005/058479).
Various methods of generating DNA-encoded chemical libraries have been described in the art (see for example [1, 2, 5-9]; WO2009/077173; WO2003/076943). Standard strategies for encoding DNA-encoded chemical libraries based on two sets of building blocks involve the stepwise ligation of double-stranded DNA-fragments (containing a code for the unambiguous identification of each building block) after each addition of a chemical moiety in the library ([2], US 2006-0246450; WO 02/103008; WO2004/013070; WO2004/074429; WO2007/062664 and WO 2005/058479). Other methods of encoding include polymerase extension of single stranded DNAs with overlapping sequences, enzymatic ligation, and chemical ligation. In other words, the insertion of a chemical building block in a nascent chemical structure is associated with the ligation of a DNA-fragment that serves as code for that building block. The final code of the complete molecule is provided by the sum of the codes, corresponding to the individual building blocks.
The development of methods for the purification of nucleic acid conjugates is useful in the preparation of nucleic acid chemical libraries with a high degree of purity.
A first aspect of the invention provides a method of producing a purified population of nucleic acid conjugate products comprising;
The reaction members comprise unreacted reactants and reaction products produced by a reaction between reactants. Generally the reaction members will comprise nucleic acid conjugate products, which are desired, and unreacted nucleic acid or nucleic acid conjugate reactants, which it is desirable to isolate from the products.
The reaction members may be provided by reacting a population of nucleic acid or nucleic acid conjugate reactants having a first reactive group with a population of chemical moieties that react with the first reactive group to produce reaction members comprising nucleic acid conjugate products that contain the chemical moiety and unreacted nucleic acid or nucleic acid conjugate reactants.
The reaction occurs between corresponding reactive groups on each of the reactants and results in the formation of a bond, generally a covalent bond, between the reactants. The reaction between the reactants produces a population of reaction members.
The reaction between the reactants may not go to completion (i.e. the reaction may be incomplete). Thus, the reaction members comprise both the desired reaction products and unreacted reactants.
Optionally, the methods described herein may further comprise:
Steps (a) to (c) may be repeated one or more times to produce a purified population of nucleic acid conjugate products comprising two or more additional chemical moieties.
Preferably, the nucleic acid is DNA.
A purified population contains an increased proportion of desired reaction products compared to unreacted reactants, relative to the unpurified population. For example, a purified population of reaction products may comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleic acid conjugate products compared to unreacted reactants.
Before purification as described herein the reaction members comprise both nucleic acid conjugate products and nucleic acid or nucleic acid conjugate reactants. The nucleic acid or nucleic acid conjugate reactants in the reaction members are unreacted reactants. In order to increase the purity of the nucleic acid conjugate products, it is desirable to remove the unreacted reactants.
A nucleic acid is a molecule formed from a plurality of nucleotide monomers. The nucleic acid may be single stranded or double stranded. The nucleic acid may be a single stranded nucleic acid, for example a single stranded DNA strand or a DNA conjugate comprising single stranded DNA. The nucleic acid may be a double stranded nucleic acid, for example a double stranded DNA strand or a DNA conjugate comprising double stranded DNA.
Generally, the nucleic acid is less than 1 kilobase (or kilobase pairs) in length, preferably less than 500 bases or base pairs in length. The nucleic acid may be from 50-500 base pairs in length, 100-400, 100-300, 100-200 base pairs in length or any combination of these ranges. Preferably the nucleic acid is from about 100-200 base pairs in length.
The nucleic acid may comprise DNA, RNA, PNA (peptide nucleic acid) LNA (locked nucleic acid), 2′-fluoro DNA, 2′-methoxy DNA or HNA (hexose nucleic acid) bases. Preferably the nucleic acid is DNA.
A nucleic acid conjugate comprises a nucleic acid molecule linked (conjugated) to one or more chemical moieties.
The nucleic acid conjugate may be a product of a conjugation reaction (nucleic acid conjugate product). Nucleic acid conjugate products are formed when two or more reactants are reacted together. For example, nucleic acid conjugate products may be formed when a nucleic acid is conjugated to a chemical moiety or when a further chemical moiety is conjugated to a nucleic acid conjugate.
The nucleic acid conjugate may be a reactant (nucleic acid conjugate reactant), which is reacted with a further reactant to form a reaction product. For example, a nucleic acid conjugate reactant may be conjugated with one or more chemical moieties to form a nucleic acid conjugate product.
Nucleic acid conjugate reactants may comprise a nucleic acid strand linked to n chemical moieties, where n is 1 to 10, (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 chemical moieties), and the nucleic acid conjugate products may comprise a nucleic acid molecule linked to n+1 chemical moieties.
The nucleic acid conjugate may be a member of a nucleic acid chemical library, including nucleic acid-encoded chemical libraries such as a DNA-encoded chemical library, or may be a nascent chemical library member. The chemical moieties linked to the nucleic acid or nucleic acid conjugate may form a pharmacophore for screening and selection on a target of interest. Generally the chemical moieties in the pharmacophore will have different structures to each other.
DNA chemical libraries and DNA-encoded chemical libraries (DELs) are well-known in the art.
Nucleic acid reactants may comprise nucleic acid strands having a first reactive group.
In some embodiments, where the reactant is a nucleic acid strand, the reactive group may be preferably located at a terminal of the nucleic acid strand. For example, the first reactive group may be located at the 5′ or 3′ terminal of the nucleic acid strand.
The terminal of the nucleic acid strand is not limited to the nucleotide at the very end of the strand (the terminal nucleic acid) but includes a number of nucleotides located adjacent to the terminal nucleotide. For example the terminal of the nucleic acid strand generally comprises the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides located adjacent to the terminal nucleic acid.
In other embodiments, for example where the reactant is a nucleic acid strand which forms a hairpin structure (e.g. hairpin DNA or RNA), the reactive group may be located outside a terminal of the nucleic acid strand, for example at the centre.
Nucleic acid-conjugate reactants may comprise nucleic acid conjugate reactants having a first reactive group.
Where the reaction members comprise nucleic acid conjugate reactants having a first reactive group, the nucleic acid conjugate reactants may comprise a nucleic acid strand conjugated to at least one chemical moiety. The conjugated chemical moiety may comprise the first reactive group.
The reaction members may also comprise chemical moieties having a first reactive group. Unreacted chemical moieties are generally removed from the reaction members by existing purification techniques known in the art such as HPLC, ethanol precipitation, gel electrophoresis, ion exchange chromatography, affinity purification (for example on a C12 resin but without a HPLC). In the case of synthesis on solid phase or DNAs immobilized on (ion exchange) resins, the unreacted small molecules and side products may be removed by washing the solid support.
This is because the chemical moiety is generally small and easily separated from the nucleic acid or nucleic acid conjugate reactants. However, unreacted chemical moieties may also be removed using a suitable capping molecule in the same way that unreacted nucleic acid or nucleic acid conjugate reactants are removed. Unreacted chemical moieties may be removed from the reaction members before or after the purification as described herein.
The unreacted reactants (e.g. unreacted nucleic acid strands or nucleic acid conjugates) may be removed by contacting the reaction members with a capping molecule comprising a capture group. The capping molecule reacts with a first reactive group located on the unreacted reactants. This links the capping molecule to the reactants. The reactants may then be removed from the reaction members using a binding member which binds the capture group. Removing the unreacted reactants from the reaction members increases the purity of the reaction products, which do not bind the capping molecule and therefore do not bind the binding member.
In the reaction products, the first reactive group has already been reacted to form the products. This means that the capping molecule is unable to react with the first reactive group in the reaction products.
A reactive group is a chemical group that is capable of forming a bond, preferably a covalent bond with a further reactive group.
One or more reactive groups are located on the reactants. For example, one or more reactive groups may be located on a nucleic acid strand or nucleic acid conjugate.
A reaction between reactive groups located on different reactants forms a bond which links the reactants together to form a reaction product. The formation of a reaction product means that the reactive groups located on the reactants will not be available to react with the capping molecule. However, unreacted reactants will still comprise a reactive group which is capable of reacting with the capping molecule. Thus, during purification as described herein, the capping molecule can be used to remove unreacted reactants from the reaction members, leaving the reaction products.
Where the nucleic acid strand or nucleic acid conjugate comprises a chemical moiety, the chemical moiety may comprise a first reactive group.
The chemical moiety may be a reactant which is reacted with first reactive group on a nucleic acid or nucleic acid conjugate to produce a nucleic acid conjugate product.
The chemical moiety may comprise one or more further reactive groups which may participate in subsequent reactions. Such further reactive groups may be protected, or may be orthogonal to the first reactive group, as described herein. The further reactive groups may be used to link a further chemical moiety to a nucleic acid conjugate comprising the chemical moiety. Thus, multiple chemical moieties may be added to a nucleic acid conjugate.
The chemical moiety and capping molecule are both capable of reacting with the same functional group on the reactants. Thus, any reactants which have not reacted with the chemical moiety can be removed by reacting the functional group with the capping molecule.
The reactive groups located on the chemical moiety and capping molecule may be the same. Alternatively, the reactive groups may be different, but still capable of reacting with the same reactive group (e.g. because they have the same chemistry).
Suitable reactive groups are well-known in the art. For example, the reactants may comprise a reactive group which is an amine and the chemical moiety and capping molecule may comprise a reactive group which is an activated carboxylic acid-derivative, or vice versa. Here, the reactive group on the chemical moiety and capping molecule is the same.
Alternatively, the reactive group on the chemical moiety and capping molecule may be different. For example, one of the reactive groups may be an alkoxyamine or 1, 2-mercaptoamine and the other may be an amine. The reactive group on the reactants may then be an aldehyde.
Alternatively, one of the reactive groups may be a nucleophilic group, such as thiol or phosphorothioate, and the other may be a primary amine. The reactive group on the reactants may then be a chloroacetyl group.
When a reactant comprises more than one reactive group, any reactive groups which are not required to participate in a particular reaction may be protected with a suitable protecting group. This prevents any reactions from occurring at that reactive group. The protected reactive groups may later be deprotected so that they may participate in a reaction. Protective groups and techniques for protecting and de-protecting reactive groups are well known in the art, (see for example, [10]) as is explained further below.
In addition to protecting the reactive groups with a protective group, a further reactive group may be orthogonal to the first reactive group to prevent them from reacting with the chemical moiety or capping molecule. Orthogonal reactive groups have sufficiently different chemistries so that they do not react with the same further reactive groups.
Because reactants may comprise more than one reactive group, the reaction product may also comprise one or more further reactive groups. These reactive groups may be been protected during the formation of the reaction product and may subsequently be de-protected, so that the reaction product can participate in a further reaction. Alternatively, these further reactive groups may be orthogonal to the first reactive group.
A protecting group can be used to protect the functional groups which are not required to participate in a particular reaction. Protecting a functional group prevents it from reacting.
The choice of a protective group depends on the functional group which is to be protected. Suitable protective groups and techniques for de-protecting are well known in the art (see, e.g. [10]).
Suitable protecting groups include a boc protecting group (tert-Butyl carbamates) and an f-moc protecting group (9-fluorenylmethyloxycarbonyl).
Further suitable groups include 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4,4′-dimethoxytrityl (all can be used for —OH and NH2 groups and are protected, for example, with 3% TCA in DCM), 4-nitroveratryloxy carbonyl (NVOC, for amines, deprotection by UV-light), allyloxy carbonyl (Alloc, deprotection with palladium(0)), N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) (DDE, hydrazinolysis), cyclic acetals (for aldehydes, removal with acidic water). Further examples of protective groups are known in the art, see for example [10].
A protected reactive group may be de-protected so that it can then participate in a further reaction.
Techniques for de-protection are well known in the art. For example a boc group may be removed by trifluoroacetic acid (TFA) and an f-moc group may be removed using a secondary amine base like piperidine.
A binding member is used to remove the unreacted and capped reactants from the reaction members. The binding member binds to the capture group on the capping molecule. When the unreacted reactants are attached to the capping molecule, contacting the reactants with the binding member will bind the unreacted reactants to the binding member, through the binding between the capture group and binding member. This binding may be covalent (for example an azide tagged capping molecules could react with strained alkynes on a solid support) or non-covalent. The binding is generally non-covalent as explained below.
In some embodiments, the binding member is immobilised, for example on a solid support.
Solid supports are generally well known in the art and include beads, columns, arrays, multiwell plates, resins, polymers or other surface substrates.
Suitable solid supports are well known in the art and include cross-linked dextran such as Sephadex™ (Pharmacia Fine Chemicals (Piscataway, N.J.); agarose, borosilicate, polystyrene or latex beads about 1 micron to about 5 millimeters in diameter, polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose or nylon-based webs such as sheets, strips, paddles, plates multiwell plate wells and other insoluble matrices.
Binding members can be immobilized on solid supports by physical adsorption, covalent coupling or other methods that are well known in the art.
Selection of the immobilization method depends on parameters such as the reaction conditions needed for coupling between the capture group and binding member, and the type of reactive groups present. When the binding member is immobilised, reactants binding to the immobilized binding member via the capture moiety can be readily separated from the nucleic acid conjugate products, which do not bind to the binding member and may, for example remain present in the liquid phase. In other words, the unreacted reactants are removed by selective immobilization.
In some embodiments, the binding member may comprise a biotin-binding protein. The biotin-binding protein may be streptavidin or avidin.
Preferably, when the binding member comprises a biotin-binding protein, the capture group comprises biotin or a biotin derivative, for example a biotin derivative as described below.
In other embodiments, the binding member may comprise biotin or a biotin derivative and the capture group may comprise a biotin-binding protein.
The capping molecule comprises a capture group which is capable of binding to the binding member. Nucleic acid or nucleic acid conjugate reactants which have reacted with the capping molecule (which attaches the reactants to the capping molecule) can be removed from the reaction products by their binding to the binding member.
The binding between the capture group and binding member is generally achieved through non-covalent intermolecular forces such as ionic bonds, hydrogen bonds and van der Waals forces, which are generally reversible. The binding may occur through covalent bonding, which is generally irreversible. Suitable covalent bonds are well known in the art.
Exemplary non-covalent binding pairs suitable for use as a capture group or binding member include the following high affinity pairs: biotin-biotin-binding protein, protein A-Fc receptor, ferritin-magnetic beads, antibody/immunogenic epitope, glutathione-S-transferase/glutathione, chitin-binding protein/chitin (e.g. chitin resin), maltose binding protein/amylose, polyhistidine/nickel or cobalt, and the like. Other suitable binding pairs which may be used in the capture group or binding member are well known in the art.
Thus, for example, the capture group may be biotin and the binding member may be avidin or streptavidin, the capture group may be protein A and the binding member may be Fc receptor, the capture group may be ferritin and the binding member may be magnetic beads and the like, or vice versa. The binding member may be in the solid phase as is well known in the art.
Preferably, the capture group is biotin or a biotin derivative. Suitable biotin derivatives include desthiobiotin and/or iminobiotin and the like.
The capture group may be joined to the capping molecule directly or via a linker. For example, when the capture group is biotin, the biotin may be joined to the reactive group by a linker. Suitable linkers are well known in the art.
Preferably, the capping molecule may consist or comprise biotin isobutyloxy anhydride (compound A) or biotin N-hydroxysuccinimidyl ester (compound B):
Further exemplary capping molecules include activated esters, including pentafluorophenoxy, symmetric and asymmetric anhydrides. Other amine-reactive groups could be used in principle as well. The activated esters can be formed prior to the capping reaction or in situ by the addition of an activating agent. Methods for forming activated esters are known in the art.
Optionally, the purity of the population of nucleic acid conjugate products may be further increased by repeating the steps of:
Optionally, one or more purification steps may be carried out to remove unreacted chemical moieties. Chemical moieties may be removed by, for example, washing if the DNA is attached to a solid support during synthesis or alternatively by ethanol precipitation, extraction (e.g., chloroform) or elution from a C12-cartridge.
The present method generally avoids the need for additional HPLC purification.
The chemical moiety may be any moiety of interest. Suitable chemical moieties include small organic molecules, amino acid residues or other amino-containing moieties (optionally with appropriate amino protection); and peptides or globular proteins (including antibody domains). Where a reactant or product comprises more than one chemical moiety, the chemical moieties may be different (i.e. not identical in structure) from each other, or may be the same.
In some embodiments, a chemical moiety may have a molecular weight of 300 Da or less, for example about 100 to 300 Da.
Suitable populations of chemical moieties which may be linked to nucleic acids are well known in the art (see [1] to [9]).
A chemical moiety may be conjugated to the nucleic acid strand directly or indirectly, for example via a linker. Suitable linkers, such as alkyl chains or polyethylene glycol (PEG), are well known in the art.
Generally the addition of chemical moieties to a nucleic acid is sequential, so that in a first reaction, a first chemical moiety is conjugated to a nucleic acid strand to form a nucleic acid conjugate product. In a further reaction, a second chemical moiety may be conjugated to the nucleic acid conjugate to form a nucleic acid conjugate product. The further reaction may be repeated to add further chemical moieties to the nucleic acid conjugate product.
In some embodiments, in a first reaction, a first chemical moiety is conjugated to a nucleic acid conjugate to form a nucleic acid conjugate product. In a further reaction, a second chemical moiety may be conjugated to the nucleic acid conjugate product to form a further nucleic acid conjugate product. The second reaction may be repeated to add further chemical moieties to the nucleic acid conjugate product.
The sequential addition of chemical moieties may be part of a split and pool method which are well known in the art, see for example [1], [8] and [9].
Where one or more chemical moieties are conjugated to a nucleic acid strand, each chemical moiety may be conjugated to the nucleic acid strand, or one or more of the chemical moieties may be conjugated to one or more other chemical moieties.
Chemical moieties may be coupled to a nucleic acid strand via other chemical moieties. For example, each of the chemical moieties coupled to a nucleic acid strand may be covalently bonded to other chemical moieties and one of the chemical moieties may be coupled to the nucleic acid strand. Suitable methods for covalently bonding chemical moieties are well known in the art.
Where the chemical moieties form a pharmacophore, the pharmacophore may be formed from a single compound comprising the covalently bound chemical moieties coupled to a nucleic acid strand. In other embodiments, the pharmacophore may be formed from two or more chemical moieties which are covalently bonded to each other, and a further one or more chemical moieties which are not covalently bonded to the covalently bonded moieties.
For example, two or more chemical moieties may be covalently linked together, and to the nucleic acid strand, and a further one or more chemical moieties may be independently linked to the nucleic acid strand. This may occur when the two or more chemical moieties and further one or more chemical moieties are linked to a nucleic acid strand through different reactive groups on the nucleic acid strand. One or more reactive groups on the nucleic acid strand may be protected or orthogonal, as described herein, to prevent an undesired reaction occurring.
A nucleic acid strand or nucleic acid conjugate may comprise a first reactive group. In a nucleic acid conjugate comprising a nucleic acid strand conjugated to at least one chemical moiety, the conjugated chemical moiety may comprise a first reactive group. A further chemical moiety which reacts with the first reactive group may be reacted with the first reactive group on the nucleic acid strand or conjugated chemical moiety to produce reaction members comprising nucleic acid conjugate products that contain the chemical moiety.
In a further aspect, the present invention provides a method of making a nucleic acid chemical library comprising:
In some embodiments, each nucleic acid strand comprises a coding sequence that encodes the chemical moiety that is conjugated thereto.
The coding sequence is an identifier which uniquely identifies the chemical moiety or moieties conjugated to that nucleic acid strand. The coding sequence (or coding region) can be any sequence of nucleic acid bases that is uniquely associated with a particular chemical moiety. This allows the identity of the chemical moiety to be determined by sequencing or otherwise ‘reading’ (i.e., determining the information content) of the coding sequence.
Methods for encoding and decoding chemical moieties, including suitable encoding sequences, are well known in the art (see [1] to [9]).
In some embodiments, a method of making a nucleic acid chemical library as described herein further comprises:
Steps (a) to (d) may be repeated to produce purified libraries of nucleic acid conjugates comprising different combinations of chemical moieties and multiple additional chemical moieties.
A method of making a nucleic acid chemical library as described herein may comprise:
or
A reaction pool comprises one or more reactants. A reaction pool may also contain any necessary fluid phase or catalyst for the reaction to occur.
Initially a reaction pool may comprise nucleic acid strands each comprising a first reactive group. One or more further reactants may subsequently be added to the pool before a reaction occurs. For example, a chemical moiety capable of reacting with the first reactive group of the nucleic acid strands to form covalent bonds.
Where there are a plurality of reaction pools, the reactants in each pool may be different.
For example, a plurality of nucleic acid strands (reactants) comprising a first reactive group may be added to each of a plurality of reaction pools. Each reaction pool may then be contacted with a different chemical moiety (reactant) comprising a further reactive group. Following the reaction between reactive groups, each pool will then contain nucleic acid reaction members covalently bonded to a different chemical moiety. As the reaction is unlikely to go to completion, each reaction pool may also contain unreacted reactants, which will reduce the purity of the population of reaction members in the pool.
In order to remove the unreacted reactants, a capping molecule comprising a capture group may be added to the reaction pool. The reaction members in each pool may be contacted with a capping molecule separately. Alternatively, the different reaction pools may be combined before the capping molecule is added. The capping molecule reacts with the first reactive groups of unreacted nucleic acid strands to form covalent bonds. The unreacted, capped reactants may then be removed using a binding member that binds to the capture moiety.
In some embodiments, the method may further comprise the steps of:
Optionally, steps (e) to (j) may be repeated to add one or more additional chemical moieties to produce a purified library comprising a diverse population of combinations of chemical moieties and two or more additional chemical moieties, each combination being conjugated to a nucleic acid strand.
Optionally, any of the methods described herein may further comprise an encoding step, whereby the identity of the chemical moiety is encoded into the nucleic acid conjugate product. Suitable encoding methods are known in the art, see for example, [10]-[9].
In a further aspect the present invention provides a chemical library obtained by any one of the methods described herein.
The chemical library may be a library of DNA conjugates, such as a DNA encoded chemical library.
Another aspect of the invention provides a kit for use in a method of producing a purified population of nucleic acid conjugate reaction products as described above, comprising;
A kit may include one or more other reagents used in the method, such as buffer solutions, washing solutions and other reagents. A kit may include one or more articles and/or reagents for performance of the method, including suitable binding members and/or chemical moieties as described herein
Other aspects of the invention provide for the use of a capping molecule as described herein in the production of a purified population of nucleic acid conjugate reaction products, including nucleic acid chemical library members, as described above,
Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.
It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.
Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such these are within the scope of the present invention.
All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.
In
The desired DNA conjugates may be generated by the condensation of an amino modified DNA with an activated carboxylic acid to form an amide bond. As a general method, DEAE sepharose (Whatman, GE Healthcare) is washed with 10 mM aq. AcOH (2×) and water (2×). DNA-C12NH2 (1 nmol) is immobilized on the anion exchange resin by incubation of an aqueous solution of the DNA for 5 min, followed by washing with 10 mM eq. AcOH (2×) and DMSO (2×). A solution of an acid (50 mM), EDC (50 mM) and HOAt (5 mM) in DMSO (500 μL) is preincubated for 5 min and added to the immobilized DNA. The reaction mixture is gently shaken for 2 h.
The resin is rinsed with DMSO (2×). The coupling and washing steps are repeated (2×). Then, the resin is washed with DMSO (2×).
Capped nucleic acid conjugates were generated according to the following exemplary methods:
Method 1
A solution of biotin (free carboxylic acid) (50 mM), 1-Ethyl-3-(3-dimetylaminopropyl) carbodiimide (EDC, 50 mM) and 1-Hydroxy-7-azatriazole (HOAt, 5 mM) in DMSO (500 uL) is preincubated for 5 min and added to the immobilized DNA. The reaction mixture is shaken overnight (16 h). The resin is washed with DMSO (2×) and 10 mM aq. AcOH (2×). The DNA is eluted with AcOH buffer (3.0 M, pH 4.75) and precipitated with EtOH. Affinity capture of capped DNAs is performed as described below.
Method 1 was used in examples 1-4 (
Method 2
Preparation of the Capping Molecule Compound A (Biotin Isobutyloxy Anhydride) and Capping of Unreacted Amine-DNA Conjugates
Diisopropylethylamine (DIPEA, 1.5 mmol) is added to a solution of carboxylic acid biotin (1 mmol) in dry DMSO (8 ml) under argon and stirred at 0° C. followed by the dropwise addition of isobutyl chloroformate (0.8 mmol). The reaction mixture is stirred for 1 h forming Compound A (biotin isobutyloxy anhydride) Freshly prepared Compound A is immediately added to the immobilized DNA (see above) and shaken overnight (16 h).
The resin is washed with DMSO (2×) and 10 mM aq. AcOH (2×). The DNA is eluted with AcOH buffer (3.0 M, pH 4.75) and precipitated with EtOH. Affinity capture of capped DNAs is performed as described below.
Method 2 was used in examples 5-9 (
Method 3
A solution of biotin N-hydroxysuccinimidyl ester (compound B; 200 mM) and 4-dimethylaminoaniline (20 mM) in DMSO (200 uL) was added to the immobilized DNA (1-2 nmol). The reaction mixture is shaken for 4 h at 37° C. The resin is washed with DMSO (2×) and 10 mM aq. AcOH (2×). The DNA is eluted with AcOH buffer (3.0 M, pH 4.75) and precipitated with EtOH. Affinity capture of capped DNAs is performed as described below.
Method 3 was used in examples 10-13.
Affinity Capture:
Streptavidin Sepharose High Performance (Amersham Biosciences) is washed with H2O (2×) and PBS (2×). DNA solution (1.8 nmol) in PBS is added on the streptavidin resin and incubated for 15 min, then centrifuged (2×). DNA conjugates are analysed directly by HPLC or further processed by EtOH precipitation.
Table 1 shows the chemical structures of chemical moieties 1-5 used in examples 5-9 (
1. Example 1 was carried out according to method 1. Chemical moiety A352 (structure shown in
2. Example 2 was carried out according to method 1. Chemical moiety A540 (structure shown in
3. Example 3 was carried out according to method 1. Chemical moiety A548 (structure shown in
4. Example 4 was carried out according to method 1. Chemical moiety A625 (structure shown in
Examples 5-9 were carried out according to method 2. In examples 5-9, respectively, chemical moieties 1-5 from Table 1 were reacted with amino modified DNA. Unreacted amino-modified DNA was capped with a capping molecule (Compound A) and removed by affinity capture. The purity of the reaction product before and after affinity capture is shown in Table 1.
Example 10 was carried out according to method 3. Chemical moiety A335 (structure shown in
Examples 11-13 were carried out according to method 3. In examples 11-13, respectively, chemical moieties shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 1404552.0 | Mar 2014 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/054768 | 3/6/2015 | WO | 00 |
