WO2009001135A2

WO2009001135A2 - Combinatorial libraries and related methods

Info

Publication number: WO2009001135A2
Application number: PCT/GB2008/050495
Authority: WO
Inventors: Steven Nicholas Kilenyi
Original assignee: CYTHERA TECHNOLOGIES Ltd
Current assignee: CYTHERA TECHNOLOGIES Ltd
Priority date: 2007-06-25
Filing date: 2008-06-25
Publication date: 2008-12-31
Anticipated expiration: 2009-12-25
Also published as: WO2009001135A3; GB0712245D0

Abstract

There is disclosed a method of synthesizing a library of molecular constructs, comprising a plurality of rounds of synthesis, wherein during at least two of the plurality of rounds of synthesis, tag elements from a group of tag elements are combinatorially attached to molecular constructs to form tags, each of which comprises a plurality of said tag elements, to form a library of more diverse molecular constructs such that, after completion of the said plurality of rounds of synthesis, different molecular constructs within the resulting library have different tags attached thereto, the method further comprising the step of separating molecular constructs from a mixture of the resulting molecular constructs using a plurality of specific binding elements each of which specifically binds a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements, to form a combinatorial library of molecular constructs. There is also disclosed a method of screening a library of molecular constructs in which molecular constructs are separated with spatially separate specific binding regions and then remain spatially separated during screening.

Description

COMBINATORIAL LIBRARIES AND RELATED METHODS

Field of the invention

The invention relates to the field of combinatorial libraries comprising a plurality of different molecular constructs, as well as methods for making and screening such libraries.

Background to the invention

Combinatorial libraries were developed to address the problem of identifying molecules which have specific properties, for example, molecules which bind to, or have a particular effect on, a drug target. Traditional synthetic chemistry focuses on the rational design of molecules and their individual preparation by the methods of synthetic chemistry. Combinatorial libraries, which contain a plurality of different molecular constructs formed using different permutations of the same building blocks, facilitate large scale and potentially high-throughput screening methods.

One method of creating a combinatorial library is the split and mix method. A suitable amount of a molecular scaffold, which may, for example, be attached to a microscopic bead, is divided into a plurality of separate portions. Each of these separate portions is then subject to a different chemical procedure, such that a different molecular component (referred to as a diversity element) is attached to the molecular scaffold in each separate portion. If n separate portions each undergo a reaction with a different molecular component, the result will be a library of n different molecular constructs. The various portions are then combined to form a mixture which, after thorough mixing, is again divided into separate portions, each of which undergoes a further chemical reaction with a different molecular component. If there are m separate portions at this stage, this results in the formation of a library of up to n x m different molecules. The process can be repeated to produce large libraries.

The result of this procedure is a plurality of separate mixtures, which are equal in number to the number of separate portions in the final synthetic stage, each of which comprises a diverse array of molecules. Each portion which is used in subsequent screening therefore comprises a diverse mixture of molecular constructs. Thus, a disadvantage of this approach is that, if a particular portion is found to have a desired activity, it is then necessary to establish which of the many different molecular constructs within that portion has the relevant activity. Furthermore, the molecular constructs within each mixture may have different, competing activities, their effects may be compromised by the relatively low concentration of each molecular construct, and effects due to the formation of dimers or other polymers of molecular constructs may be lost.

Various methods are known for facilitating the identification of molecular constructs within a combinatorial library, after a screening step, in order to establish which molecular construct within a mixture has had a desired effect. For example, it is known to tag beads to which molecular construct are attached with a tag which denotes the molecular components which have been incorporated into the molecular construct on that bead, to facilitate the identification of molecular constructs, after a screening step. A disadvantage of this approach is that, although it is relatively easy to tag a bead, there are many circumstances where it would be preferable to use a library of molecular constructs which are in the liquid phase, rather than attached to a solid phase, such as a bead, which can affect their chemical properties.

It is known to create a library using a bi-functional scaffold which has a first reactive group, or groups, through which a plurality of alternative molecular components can be attached to form a combinatorial library, and a second reactive group, or groups, which can be used to create a customised tag which uniquely identifies the steps through which the molecular scaffold has passed. For example, the second reactive group may comprise the 3' or 5' terminus of DNA, or a linker for attachment to a DNA sequence. A custom DNA sequence which encodes the molecular components incorporated into a specific molecular constructs is build up by the successive addition of additional nucleotide bases during each round of split and mix synthesis. It is known for such combinatorial libraries to be made in the liquid phase. However, although the inclusion of DNA tags facilitates the analysis of a selected species from the library, there is again the problem that a mixture of compounds is used in any subsequent screening procedure.

It has been proposed to tag molecular scaffolds with one of a plurality of fluorous groups, and then carry out further rounds of combinatorial library synthesis. The resulting portions can then be separated using a chromatography column comprising a resin which has a different affinity for fluorous groups which differ in the number of fluorine atoms which they contain. However, because a single scaffold is used to bind the different fluorous groups, there is a limit to the number of unique tags which could be created using this method.

It has also been proposed to form combinatorial libraries using a plurality of micro- reactors, each of which encloses molecular constructs during the procedure of forming a combinatorial library. Individual micro-reactors are tagged to indicate the successive molecular constructs which have been added to the scaffold. This allows liquid phase molecular constructs to be prepared. However, this procedure is complex and the encapsulation of the molecular constructs by micro-reactors reduces the range of chemical modifications which can be made to the molecular constructs.

Combinatorial libraries have also been created using parallel synthesis. It is known to carry out parallel synthesis in which a plurality of different molecular constructs, such as molecular scaffolds, undergo the same combination of reactions in order to efficiently form a combinatorial library. This parallel synthesis approach does not usually increase the diversity of molecular constructs which act as a starting point, but does facilitate efficient preparation of libraries of more complex molecular constructs. Parallel synthesis has been used to facilitate the provision of combinatorial libraries in which different molecular constructs are spatially separated, by starting with spatially separated diverse scaffolds. However, conventional parallel synthesis does not facilitate the provision of libraries of molecular constructs in the liquid phase. It would be advantageous to provide a method of parallel synthesizing combinatorial libraries in which different molecular constructs could be formed within the same body of liquid and in which the resulting molecular constructs could be liquid phase. Some aspects of the invention aim to provide improved or alternative methods of forming combinatorial libraries, and the libraries which are thereby formed. Some aspects of the invention extend to improved or alternative methods for carrying out parallel synthesis of a combinatorial library, such as methods in which at least some rounds of chemical synthesis take place on diverse molecular constructs within the same body of liquid.

It has been proposed, for example in WO 02/099078 (Winssinger et al.) to carry out screens using a mixture of diverse molecular constructs in solution before separating out molecular constructs which have undergone a particular interaction and then separating the screened constructs on a polynucleotide array. For example, where the screen is a binding assay, molecular constructs which bind a target macromolecule can be separated out using a size exclusion filter and then immobilised on a polynucleotide array. A disadvantage of this strategy is that molecular constructs of each individual composition are at a very low concentration during the screening step. Any dimers or other multimers of molecular constructs are unlikely to be homogenous. Accordingly, some aspects of the present invention aim to provide improved or alternative methods of preparing and/or screening combinatorial libraries, which mitigate these disadvantages.

Summary of the invention

According to a first aspect of the invention there is provided a method of synthesizing a library of molecular constructs, comprising a plurality of rounds of synthesis, wherein during at least two of the plurality of rounds of synthesis, tag elements from a group of tag elements are combinatorially attached to molecular constructs to form tags, each of which comprises a plurality of said tag elements, to form a library of diverse molecular constructs, such that, after completion of the said plurality of rounds of synthesis, different molecular constructs within the resulting library have different tags attached thereto, the method further comprising the step of separating molecular constructs from a mixture of the resulting molecular constructs using a plurality of specific binding elements each of which specifically binds a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements, to form a combinatorial library of molecular constructs. Thus, by associating different molecular constructs within a combinatorial library of molecular constructs with different tags, each of which comprises a different combination of tag elements from the group of different tag elements, and separating a mixture of molecular constructs using the plurality of specific binding elements, different molecular constructs from the library may be separated from each other and so used separately in subsequent applications of the library. This avoids or mitigates the problems discussed above in relation to procedures in which a diverse (or more diverse) mixture of molecular constructs is used for screening purposes and then molecular constructs which have specific properties are separated from, or identified within, the mixture of molecular constructs. Accordingly, the resulting library of molecular constructs which have been separated on the basis of tag elements may be used in screening procedures subsequently to the separation step which determines whether or not specific molecular constructs have a particular property. After the separation stage, tags (comprising tag elements) may be cleaved from the molecular constructs. Tags (comprising tag elements) may be cleaved from the molecular constructs after the separation stage and before the tags are used in a screening procedure. The tags (comprising tag elements) may initially be connected to the remainder of the molecular construct via a cleavable linker which may be cleaved to cleave the tags from the remainder of the molecular constructs.

The rounds of synthesis may comprise at least one, and typically a plurality of, rounds of synthesis in which a mixture comprising a plurality of diverse molecular constructs undergoes the same synthetic procedure. Thus, a plurality of diverse molecular constructs may be subject to the same synthetic procedure and thereby subject to the same or equivalent chemical changes. That is to say, a plurality of diverse molecular constructs may undergo parallel synthesis.

The rounds of synthesis may comprise at least one round, and typically a plurality of, rounds of synthesis in which diversity elements from a group of diversity elements are combinatorially attached to molecular constructs to form a more diverse group of molecular constructs.

At least two rounds, and optionally each round, of synthesis in which tag elements are attached to molecular constructs may occur prior to at least one round, and optionally each round, of synthesis in which diversity elements are attached to molecular constructs. Different molecular constructs may be spatially separated prior to a mixing step prior to the separation step. For example, different molecular constructs may be attached at spatially separate locations to one or more supports throughout the rounds of synthesis and cleaved from the or each support to form a mixture prior to the separation step. The molecular constructs may each be attached to the same support, such as a planar support. Different molecular constructs may be attached to different supports, such as microcapsules, beads etc.

Advantageously, in some embodiments, different molecular constructs, with different tags, may be provided within the same body of liquid (typically at spatially separate liquids within the same body of liquid) during each round of synthesis in which diversity elements are attached to molecular constructs, and optionally during each round of synthesis. Each round of synthesis in which diversity elements are attached to molecular constructs may take place in respect of each molecular construct within the same vessel such that, at least once during each round of synthesis, each molecular construct is within the same continuous body of liquid.

The rounds of synthesis in which tag elements are attached to molecular constructs and the rounds of synthesis in which diversity elements are attached to molecular constructs may take place simultaneously, or alternately.

The method may comprise a plurality of rounds of split and mix synthesis in which a liquid containing molecular constructs is split into a plurality of portions, each of which is reacted with a different diversity element from the group of diversity elements, such as to attach the respective diversity element to the molecular constructs in the portion and, if there is to be a further round of split and mix synthesis, the portions are combined to form a further liquid containing molecular constructs for use in further rounds of split and mix synthesis, wherein, in at least one round of split and mix synthesis, each portion which is reacted with a different diversity element from a group of different diversity elements is reacted with a different tag element from the group of different tag elements.

The invention extends in a second aspect to a method of synthesizing a library of molecular constructs, comprising at least one round of split and mix synthesis in which a liquid containing molecular constructs is split into a plurality of portions, each of which is reacted with a different diversity element from a group of diversity elements, such as to attach the respective diversity element to the molecular constructs in the portion and, if there is to be a further round of split and mix synthesis, the portions are combined to form a further liquid containing molecular constructs for use in further rounds of split and mix synthesis, wherein, in at least one round of split and mix synthesis, each portion which is reacted with a different diversity element from a group of different diversity elements is reacted with a different tag element from a group of different tag elements, the method further comprising the step of separating molecular constructs from a mixture of molecular constructs using a plurality of specific binding elements each of which specifically binds a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements, to form a combinatorial library of molecular constructs.

The mixture of molecular constructs from which molecular constructs are separated may comprise one of the portions from the final round of split and mix synthesis. In some embodiments, tag elements will be attached to each molecular construct during each round of split and mix synthesis except for the final round of split and mix synthesis. This is because the portions which have had different diversity elements attached thereto in the final synthetic round will be separate to each other, unless they are subsequently mixed.

The step of splitting a liquid comprising molecular constructs into a plurality of portions during a round of split and mix synthesis typically splits the portions into distinct liquids which are not in liquid communication with each other, for example discrete wells of a microtitre plate or discrete test-tubes. However, the portions may comprise spatially separate regions within a continuous liquid, for example discrete locations on the surface of a solid support, or portions associated with separate solid phase supports, such as beads, or separate chambers within a liquid, such as distinct microcapsules.

The invention extends in a third aspect to a method of synthesizing a library of molecular constructs, comprising forming a solution comprising diverse molecular constructs, wherein each different molecular construct within the solution has a different tag comprising a unique combination of tag elements selected from a group of tag elements, carrying out at least one round of parallel synthesis in which equivalent modifications are made to diverse molecular constructs within the solution, and then separating molecular constructs from the resulting mixture of molecular constructs using a plurality of specific binding elements each of which specifically binds a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements, to form a combinatorial library of molecular constructs. Preferably, the method comprises a plurality of rounds of parallel synthesis.

By "equivalent modifications" we include modifications which are made by equivalent chemical procedures under the same reaction conditions. Typically, the same modification may be made to each molecular construct within the solution, although there may be circumstances where different molecular constructs will undergo different chemical changes under the same reaction conditions.

The diverse molecular constructs may be intimately mixed, for example, they may be free in solution. Alternatively, the diverse molecular constructs within the solution may be spatially separated. For example, they may be immobilised to different supports, such as beads or microcapsules, or at different locations in the same support, such as in an array on a generally planar surface. The solutions comprising diverse molecular constructs may comprise diverse scaffolds which can be modified during the method to create molecular constructs of greater molecular weight.

In each aspect, the step of separating molecular constructs, using a plurality of specific binding elements, may comprise separation into discrete liquid portions. The step of separating molecular constructs, using a plurality of specific binding elements, may comprise spatial separation within the same body of liquid, for example, onto discrete locations on a surface or onto or into discrete carriers, such as a beads or microcapsules. In order to achieve this, the specific binding elements may be immobilised at separate locations on a surface or at or in discrete carriers, such as beads or microcapsules. Thus, in some embodiments, the specific binding elements are located in an array on a surface such that, following separation, the molecular constructs are immobilised in a corresponding array on the surface. The specific binding elements may be located in an array on a surface and eluted into a corresponding array of separate liquid retaining containers, such as wells on a multi- well plate. The specific binding elements may be arranged as protuberances in an array on a surface. Molecular constructs which bound to each specific binding element in the array may be eluted simultaneously into respective wells of a multi- well plate.

The method may use a group of specific binding elements, each of which is specific to a different combination of tag elements which could be present in the combinatorial library. This arrangement may be helpful, for example, when forming an array of molecular constructs using an array of different specific binding elements, each of which is specific to a different combination of tag elements.

However, the method may use a group of specific binding elements, each of which is specific to a different tag element, so that the group of specific binding elements may be used to separate molecular constructs tagged with more different combinations of tag elements than there are specific binding elements in the group of specific binding elements. These options are discussed further below.

The specific binding elements may each be part of a resin or other solid phase support which, in use, retains molecular constructs including the corresponding tag element in a mixture of molecular constructs passed through or mixed with the resin or other solid phase support and from which molecular constructs including the corresponding tag element can subsequently be eluted.

In some embodiments, each tag element comprises a polynucleotide sequence, such as a peptide nucleic acid (PNA) sequence. The specific binding elements may comprise complementary sections of polynucleotide, such as PNA. By "polynucleotide" we include DNA, RNA, PNA and variants and derivatives thereof which can be used for the purposes of the present invention, such as variants which include unnatural nucleotides. Polynucleotides are useful tag elements and components of specific binding elements because a group of tag elements each of which can be specifically bound by a corresponding specific binding element (comprising a complementary sequence of polynucleotide or derivative thereof) can be readily created. Where the tag elements are polynucleotide sequences, the tag sequences which are added to the molecular constructs in each successive round of split and mix synthesis may be ligated to each other, for example using a ligase enzyme or a chemical procedure. Preferably, each tag element comprises at least two polynucleotide bases.

Typically, the same group of diversity elements will be used in two or more rounds of synthesis (e.g. two or more rounds of split and mix synthesis). However, different groups of diversity elements could be used in different rounds of synthesis.

Each tag element from the group of tag elements may denote that a specific diversity element from the group of diversity elements is present in the molecular construct. In embodiments comprising at least two rounds of split and mix synthesis, each tag element may denote that a specific diversity element from the group of diversity elements was included in the molecular construct in a particular round of split and mix synthesis.

Each tag element may denote that a specific diversity element from the group of diversity elements is present in the molecular construct at a particular location. The particular location may be a particular location on the molecular scaffold (e.g. a location which is initially populated by a reactive group). The particular location may represent the position in a chain of diversity elements which are attached to each other, in which case, when the method includes at least two rounds of split and mix synthesis, a tag element may denote both the location and the round of split and mix synthesis during which a specific diversity element from the group of diversity elements was included in the molecular construct. This facilitates the separation of molecular constructs on the basis not only of the diversity elements from which they have been constructed, but also on the basis of the location of a specific diversity element. By using a sufficiently large group of tags, each tag may denote that a specific diversity element is present in the molecular construct at a specific location on the molecular scaffold.

For example, each tag element may comprise a polynucleotide sequence (such as PNA) selected from a group of polynucleotide sequences. The groups of polynucleotide sequences used as tag elements in successive rounds of synthesis in which a tag element is attached to a molecular construct (e.g. a round of split and mix synthesis) may be selected so that they do not comprise any sequences in common. The same groups of diversity elements, different groups of diversity elements including at least some diversity elements in common, or entirely different groups of diversity elements may be used in each round of synthesis. Therefore, each polynucleotide sequence can be related not only to a diversity element which is a part of the molecular construct but the round of synthesis (e.g. the round of split and mix synthesis, where appropriate) in which the diversity element was incorporated into the molecular construct. If each round of synthesis in which a tag element is attached to a molecular construct (e.g. a round of split and mix synthesis) incorporates diversity elements at different locations within the molecular construct, the polynucleotide sequence can be related to the location of a specific diversity element within the molecular construct. Where each tag element is a polynucleotide sequence, the supergroup comprising the groups of tag elements to be used during each round of synthesis in which a tag element is attached to a molecular construct (e.g. a round of split and mix synthesis) may be selected so that tag elements within the supergroup do not include any sequences of polynucleotides which might be formed by the overlap between two or more tag elements within the supergroup which might be ligated to each other during the method. This is to avoid a specific binding element binding not to a polynucleotide sequence which is to function as a tag element, but to a polynucleotide sequence which overlaps consecutive tag elements.

The same group of tag elements, or groups of tag elements which have tag elements in common, may be used in two or more rounds of synthesis in which a tag element is attached to a molecular construct (e.g. a round of split and mix synthesis). The combinatorial library may comprise molecular constructs tagged with more different combinations of tag elements from the group of tag elements than there are tag elements in the group of tag elements. In some applications, for example, the formation of a micro-array using an array of different specific binding elements a different specific binding element may be used to specifically bind each combination of tag elements which could be present in the combinatorial library. In other applications, each specific binding element may be selected to specifically bind a specific tag element and the step of separating molecular constructs from a mixture of molecular constructs may include a plurality of separation stages, each of which uses fewer different specific binding elements than the number of combinations of tag elements which could be attached to the molecular constructs in the combinatorial library, wherein at least some (and preferably all) of the specific binding elements in a group of specific binding elements each of which binds specifically to a tag element from the group of tag elements, is used in two or more of the separation stages to enable molecular constructs tagged with each combination of tag elements which could be present in the combinatorial library to be individually separated. This procedure facilitates the separation of a mixture of molecular constructs which comprises more molecular constructs than there are specific binding elements.

These procedures reduce the number of different tag elements and specific binding elements which must be synthesized in comparison to an arrangement in which a different specific binding element was required to separate each different molecular construct because each specific binding element binds specifically to a different combination of tag elements. Furthermore, this allows a greater freedom of choice when selecting tag elements and corresponding specific binding elements, because it is not necessary to identify as many different pairs of tag elements and specific binding elements which bind specifically to each other.

In order to facilitate the separation of a mixture of molecular constructs prepared using the same group of tag elements, or groups of tag elements which have tag elements in common, in two or more rounds of split and mix synthesis, the method may comprise the step of blocking tag elements which were added to molecular constructs in a round of split and mix synthesis before adding further tag elements in a subsequent round of split and mix synthesis. Alternatively, the tag elements which are added to a molecular construct during a round of synthesis in which a tag element is attached to a molecular construct (e.g. a round of split and mix synthesis or the preparation of a molecular construct for use in a solution comprising diverse molecular constructs for use in parallel synthesis) may already be blocked when they are added to the molecular constructs.

This allows separation of a larger group of different molecular constructs than would be possible with a given group of tag elements and corresponding specific binding elements.

By "blocking" we include reversible chemical procedures which prevent a tag element from being specifically bound by and separated using the complementary specific binding element until an unblocking step has been carried out. Accordingly, step of separating molecular constructs may comprise the step of using specific binding elements to separate molecular constructs on the basis of tag elements, or combinations of tag elements, which have not been blocked, without separation dependent on the presence of blocked tagged elements, followed by the step of unblocking blocked tag elements and separation of molecular constructs using the specific binding elements which are specific to the now unblocked tag elements. This procedure may be repeated to allow further separation on the basis of further tag elements. In order to facilitate this procedure, tag elements may be cleaved from the molecular constructs, reblocked, or otherwise amended such that they no longer bind to the corresponding specific binding elements, prior to unblocking further tag elements and separation of the basis of the now unblocked tag elements. In some embodiments, tags are blocked by acylation and unblocked using a deacylation step. In order to enable several rounds of separation to be performed using the same specific binding elements, more than one different blocking chemistry may be used, such that specific tag elements, or tag elements which were attached to the molecular constructs during a specific round of synthesis may be unblocked.

Typically, the molecular constructs will comprise a molecular scaffold, although the method may be used to create a scaffold-free combinatorial library in which there is no element which is common to each molecular construct in the library. For example, in the case of split and mix synthesis, the first round of split and mix synthesis may comprise mixing a plurality of different molecular constructs to which diversity elements may be attached, each of which has a different label. In the case of parallel synthesis, the diverse molecular constructs may comprise different molecular scaffolds, each of which is tagged with a different combination of tag elements.

The molecular constructs may comprise a molecular scaffold. Diversity elements may be attached directly to the molecular scaffold (i.e. not through other diversity elements) or indirectly, through other diversity elements. Diversity elements may be attached to different locations on the molecular scaffold during subsequent rounds of synthesis in which diversity elements are attached to molecular constructs (e.g. rounds of split and mix synthesis).

Tag elements may be attached directly to each molecular construct. Where each molecular construct comprises a molecular scaffold, the tag elements may be attached directly to the molecular scaffold. Tag elements may be attached to the molecular scaffold through a linker, which may be cleavable for removing the tag elements after separation using specific binding elements. In some embodiments, tag elements may be attached to the molecular scaffold other than through a solid phase support such as a bead. Tag elements may be attached consecutively to other tag elements to form a chain. Tag elements may be removed sequentially from the chain in the opposite order to the order in which they were attached to the chain. The attachment of a further tag element to a previously attached tag element may function to block the previous tag element and the subsequent removal of the further tag element may unblock the previous tag element.

In some embodiments, during one or more (and optionally all) of the rounds of synthesis in which tag elements are attached to molecular constructs (e.g. during one or more rounds of split and mix synthesis), each tag element may be attached to the molecular construct through a corresponding diversity element. Where each molecular construct comprises a molecular scaffold, each tag element may be attached to the molecular scaffold through a corresponding diversity element. Each tag element in the group of tag elements may be pre-attached to the corresponding diversity element from the group of diversity elements. This may in some circumstances, reduce the number or complexity of synthetic steps, as only a single connection (sometimes only a single chemical bond) need be formed between the diversity element which is being added and the body molecular construct, or molecular scaffold where present. Typically, each tag element will be pre-attached to a corresponding diversity element using a cleavable bond so that the tag element may be cleaved from the molecular construct after it has been used to separate the molecular constructs.

By "specifically binds a tag element from the group of tag elements" we mean that each specific binding element binds one tag element from the group of tag elements under appropriate conditions with sufficient affinity and specificity to separate molecular constructs containing that tag element from molecular constructs which contain other tag elements from the group of tag elements but which do not contain that tag element.

By "specifically binds... a specific combination of different tag elements from the group of different tag elements", we means that each specific binding element binds one combination of tag elements from the group of different tag elements under appropriate conditions with sufficient affinity and specificity to separate molecular constructs containing that combination of tag elements group molecular constructs with different combinations of tag elements, but not the same combination of tag elements. Typically, in these circumstances, the specific binding elements will be specific to a combination of tag elements from the group of different tag elements in a specific order. Typically, each specific binding element will comprise a plurality of identical specific binding molecules, so that one specific binding element can bind to many tags.

Each round of synthesis may involve one or more synthetic steps.

A single diversity element may be attached to each molecular construct during a round of synthesis in which a diversity element is attached to a molecular construct, (e.g. during a round of split and mix synthesis). However, a plurality of (typically identical) diversity elements may be attached to each molecular construct during a round of split and mix synthesis in which a diversity element is attached to a molecular construct (e.g. during a round of split and mix synthesis). Thus, the method may be used to create a library of different dendrimers.

By a "molecular scaffold" we refer to a common structural element to which diversity elements can be attached, and derivatives or variants thereof which occur during the methods of the invention. The molecular scaffold will typically be common to each molecule in the library, although in some embodiments the molecular scaffold will be selected from a group of different molecular scaffolds. By "first round of synthesis" we imply a round of synthesis which takes place before subsequent rounds of synthesis but do not intend to exclude the possibility that rounds of synthesis may have occurred before the first round of synthesis. The molecular scaffold may be attached to a support which may be a solid phase support, such as a bead. In this case, the molecular scaffold may be cleaved from the solid phase support after the separation of molecular constructs using specific binding elements. Preferably, the molecular constructs in the resulting library are dissolved in an appropriate solvent (typically an aqueous solvent).

By a "diversity element" we mean a structural element which is not present in the same location in each molecular construct within the resulting library of molecular constructs, and variants or derivatives thereof which occur during the method of the present invention. Molecular constructs within a combinatorial library include different permutations of diversity elements selected from one or more groups of diversity elements. In some libraries, diversity elements may be included at more than one possible location with the molecular constructs of the library.

The liquid containing molecular constructs may be a solution of molecular constructs. The liquid containing molecular constructs may be a mixture or may initially comprise identical molecular constructs, such as a molecular scaffold which is common to each molecular construct in the completed library.

The method may further comprise the step of cleaving the tags from molecular constructs within the library after the step of separating molecular constructs from a mixture of molecular constructs.

According to a fourth aspect of the present invention, there is provided a library of molecular constructs obtainable by the method of first .second or third aspect. Preferably, the molecular constructs are obtained by the method of the first, second or third aspect.

According to a fifth aspect of the present invention, there is provided a kit of parts comprising a library of molecular constructs according to the fourth aspect of the present invention and a plurality of specific binding elements, which are typically immobilised, each of which specifically binds a different tag element from the group of tag element, or a specific combination of different tag elements from the group of tag elements. The specific binding elements may be attached in an array to a surface. The specific binding elements may each be attached to a separate resin. The kit may further comprise a multi-well plate comprising a plurality of wells which are arranged in a corresponding formation to the array of specific binding elements.

According to a sixth aspect of the present invention, there is provided a method of screening a library of diverse molecular constructs, comprising the steps of:

(i) providing a library comprising a mixture of diverse molecular constructs, different molecular constructs within the library having different tags attached thereto;

(ii) separating molecular constructs from the mixture using a plurality of spatially separate specific binding elements, which specifically bind different tags; and

(iii) screening the separated molecular constructs for a desired activity, while the separated molecular constructs remain spatially separated.

Thus, in contrast to methods in which a mixture of diverse molecular constructs is screened for a desired activity, before different molecular constructs are separated from the mixture of diverse molecular constructs, the method enables different molecular constructs to be screened separately for a desired activity. Thus, the activity of molecular constructs of different structures can be investigated separately. The method may enable molecular constructs with a given structure to be screened at a higher concentration than would be practical in a library comprising a mixture of diverse molecular constructs and may enable molecular constructs which form multimers in solution to be screened as homogenous multimers. The spatially separate specific binding elements may be located on separate supports (e.g. beads or pins). The spatially separate specific binding elements may be located at discrete locations within, or on the surface of a support. For example, the spatially separate specific binding elements may be located at different regions of the surface of a planar micro-array.

The library comprising a mixture of diverse molecular constructs may be synthesised by a method according to the first, second or third aspect of the invention, or by another method, such as using known split and mix synthesis methods, or known parallel synthesis methods.

The steps of providing a library comprising a mixture of diverse molecular constructs, and separating molecular constructs from the mixture may be a method of synthesizing a library according to the first, second or third aspect of the invention.

The separated molecular constructs may be screened for a desired activity while they remain spatially separated from each other, while remaining attached to the spatially separate specific binding elements. The separated molecular constructs may be screened for a desired activity in a procedure where the separated molecular constructs are initially attached to the spatially separate specific binding elements, but molecular constructs which have, or (alternatively) which do not have, a desired activity are removed from the spatially separate specific binding elements during the screening procedure.

The separated molecular constructs may be removed from the spatially separated specific binding elements before they are screened for a desired activity. This can enable them to be screened in solution, where they may have a different activity to their activity whilst they are bound to specific binding elements.

Where the spatially separate specific binding elements are at discrete locations on the same solid support, molecular constructs which are attached to a first specific binding elements may be removed from the solid support before molecular constructs which are attached to a second specific binding element are removed from the solid support. This procedure may be repeated, so that molecular constructs are sequentially removed from a plurality of spatially separate specific binding elements in turn. For example, where the solid support is a micro-array, and the spatially separate specific binding elements are at different locations on the surface of the micro-array, the location from which molecular constructs are removed may be swept sequentially across the surface of the micro-array, to remove molecular constructs in turn from the surface of the array. Molecular constructs which are removed from the surface of a solid support may be drawn away from the solid support, for example by being drawn into a conduit, thereby remaining spatially separated.

The separated molecular constructs may be removed from the surface of the array, separately to each other, before they are screened for a desired activity. For example, the molecular constructs which are bound to first specific binding elements located at a first location on the surface of the array, may be removed from the surface of the array, before the molecular constructs which are bound to a second specific binding element and/or at a second location on the surface of the array, are removed. Molecular constructs may be drawn away from the surface of the array, for example, drawn into a conduit, as they are removed from the surface of the array, thereby remaining spatially separated. At the location on the surface from which molecular constructs are removed, may be swept sequentially across the surface of the array, to remove many of molecular constructs in turn from the surface of the array.

In order to remove the molecular constructs from the surface of the array, the binding of specific binding elements to corresponding tags may be disrupted, for example, by the introduction of one or more chemical species which selected to disrupt binding. Alternatively, in order to remove the molecular constructs from the surface of the array, one or more bonds within the molecular constructs, or specific binding elements, may be cleaved, thereby causing the molecular constructs to be removed from the surface of the array.

As the separated molecular constructs are screened for a desired activity after they have been spatially separated, molecular constructs which are or were attached to different tags can be screened separately. Molecular constructs may be cleaved from their tags after they have been separated and before they are screened for the desired activity. Preferably, the molecular constructs are not screened for the desired activity until after they have been separated using the plurality of spatially separate specific binding elements.

Typically, the tags comprise polynucleotides, such as DNA, RNA or peptide nucleic acids. Typically also, the specific binding elements comprise a plurality of polynucleotide molecules, such as DNA, RNA or peptide nucleic acid molecules which are complementary to respective tags. The tags may comprise a plurality of tag elements, each of which is selected from a group of different tag elements. The specific binding elements may specifically bind a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements.

The desired activity may, for example, be that a molecular construct binds to a specific molecule or molecules, or binds to a specific molecule or molecules with more than a predetermined affinity, or does not bind to a specific molecule or molecules, or binds to a specific molecule or molecules with less than a predetermined affinity or that a molecular construct catalyses a particular reaction, or is a reactant in a specific reaction.

Preferred and optional features of the molecular constructs, tags, tag elements, specific binding elements, and supports correspond to those discussed above in relation to the first five aspects of the invention.

Description of the Drawings

An example embodiment of the present invention will now be illustrated with reference to the following Figures in which:

Figure 1 is a flow chart of a first example method of synthesizing a combinatorial library;

Figure 2 is a schematic diagram of the first example method of synthesizing a combinatorial library;

Figure 3 is a schematic diagram of a reaction scheme to form a molecular construct comprising a scaffold, linker and tag;

Figure 4 is a schematic diagram of a reaction scheme using molecular constructs according to Figure 3;

Figure 5 is a schematic diagram of a second example method of synthesizing a combinatorial library; and Figure 6 is a schematic diagram of apparatus for sequentially removing spatially separate molecular constructs from the surface of a micro-array.

Detailed Description of Example Embodiments

Example One

With reference to Figures 1 and 2, a split and mix method of synthesizing a combinatorial library begins with a solution 1 of identical molecular scaffolds 2 (functioning as molecular constructs). The solution is split 100 into three separate portions. The molecular scaffold within each portion is reacted 102 with a different tag T₁, T₂ or T₃ from a group of possible tags and a different first diversity element R₁, R₂ or R₃ from a group of possible first diversity elements. This results in three portions of different molecular constructs 4A, 4B and 4C.

The three portions of different molecular constructs are then mixed 104 to form a diverse mixture 6 of molecular constructs in which each construct has a tag element which is related to the diversity element which has been added to that construct. The mixture is then split 106 into three separate portions 8A, 8B and 8C, each of which is an identical mixture of three different molecular constructs. Each separate portion is then reacted 108 with a different second diversity element R₄, R₅ or R₆ from a group of different second diversity elements. The results is three separate portions 10A, 10B and 10C, each of which comprises molecular constructs with three different combinations of first and second diversity elements.

Each portion is then separated 1 10 using three specific binding elements S₁, S₂ and S₃ , each of which specifically binds a respective tag, T₁, T₂ or T₃ respectively, thereby forming nine separate portions 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, 121, each of which comprises molecular constructs with a different combination of first and second diversity elements. In order to achieve separation, each specific binding element is attached to a different resin. A portion is then passed through each resin, whereupon the molecular constructs within the portion will adhere to the resin including the specific binding element which specifically binds the tag element on each individual molecular construct. The molecular constructs can be eluted separated from each resin, yielding separate portions of different molecular constructs. This process is repeated for each portion. The tags may then be cleaved from the molecular constructs in each resulting portion and the resulting combinatorial library of separate molecular constructs may be used for further procedures, such as screening procedures to determine whether any of the molecular constructs in the library have a specific chemical activity and/or biological effect. The resulting molecular constructs can be used in the liquid phase in subsequent procedures.

Groups of any number of different diversity elements may be used in the first and second round of split and mix synthesis. Where there are N different diversity elements in the group of diversity elements used for the first round and M different diversity elements in the group of diversity elements used for the second round, the procedure may create and separate up to N x M different molecular constructs. The same group of different diversity elements may be used in each round of split and mix synthesis.

One skilled in the art will appreciate that many different chemical species could be used as the scaffold, diversity elements, tags and specific binding elements. In the example illustrated in Figures 3 and 4, 2-benzyl-4-piperidone is used as a scaffold. Some derivatives of 2-benzyl-4-piperidone are known to be pharmacologically useful. For example, 2-benzyl-4-piperidone, and similar molecules, are at the core of a family of Tachykinin and Substance P receptor antagonists disclosed in WO 97/16440 and WO 97/24350 (Janssen Pharmaceutica N.V.). Accordingly, 2-benzyl-4-piperidone is a suitable scaffold for building a combinatorial library of small molecule drug candidates.

A two base pair sequence of peptide nucleic acid (PNA) functions as a tag element. There are sixteen possible two base pair sequences, giving sixteen possible tag elements, each of which could be used to label a different diversity element. The tag element is attached to the scaffold via a UV-cleavable linker, 4-propynyloxy-2- aminobenzaldehye by reductive amination using sodium cyanoborohydride. A terminal azide on the two base pair PNA sequence facilitates the attachment of the PNA sequence to the linker by copper-catalysed azide-alkyne ligation.

Diverse amines (functioning as diversity elements) may be attached to the scaffold by reductive amination. The ketone of the 2-benzyl-4-piperidone is mixed with a primary or secondary amine selected from a group of such amines and reduced with sodium cyanoborohydride, forming a secondary or tertiary amine respectively. The resulting molecules can be further derivatised to introduce additional diversity elements.

The two base pair sequences of PNA can be separated using specific binding elements comprising complementary sequences of PNA, DNA or RNA. In order to facilitate separation, each complementary sequence may be attached to a separate resin to which molecular constructs including the complementary tag element can be attached and subsequently eluted.

Example Two

With reference to Figure 5, a second example method of preparing a combinatorial library of molecular constructs comprises three round of synthesis in which diversity elements are attached to the molecular constructs. In a first round of split and mix synthesis, a portion of molecular scaffold is divided into N separate portions, each of which is derivatised with a different first diversity element R_N and tagged with a different first tag element T_N. In this example, the first tag element is blocked (denoted T^* _N). This may be achieved by attaching a tag element to the molecular scaffold and then blocking it, or by attaching a tag element which is already blocked to the molecular scaffold. Where the tag elements are PNA sequences, they may be blocked by, e.g. attaching trityl protecting groups to the nitrogen atoms of the PNA peptide backbone.

The various portions 14 are then mixed and split to form M separate portions 16, each of which is derivatised with a different second diversity element R_M and tagged with a different second tag element T_M. The first and second tag elements together form a tag. The various portions are then mixed and split to form L separate portions 18, each of which is derivatised with a different third diversity element R_L. The result is a library comprising up to N x M x L different molecular constructs.

The molecular constructs can be separated from each other using a two-stage procedure. In a first stage, each of the resulting L portions is separated using M different specific binding elements S_M, each of which binds a respective second tag element T_M. This gives up to L x M separate portions. Because the first tag elements are blocked, molecular constructs will be separated solely on the basis of the second tag elements, even if some or all of the second tag elements and some or all of the corresponding second specific binding elements are the same as some or all of the first tag elements and some or all of the corresponding first specific binding elements.

The first tag elements are then unblocked. In some embodiments, the second tag elements must first be cleaved. The up to L x M portions are each separated again into N portions, giving a total of up to L x M x N portions, using specific binding elements S_N, each of which binds a respective first tag element T_N. The first tag elements may then be cleaved, leaving the final molecular constructs 20.

Where the groups of specific binding elements and tag elements used in each successive round are the same, or have elements in common, this procedure has facilitated the tagging and separation of a number of different molecular constructs which is significantly or substantially larger than the number of different tag elements/specific binding elements. Thus, diverse combinatorial libraries may be prepared using a limited number of different tag elements and corresponding specific binding elements.

This procedure may be implemented with additional rounds in which further diversity elements are attached to the molecular constructs along with further tag elements. In each round, previously attached tag elements may be blocked prior to the attachment of further tag elements.

Example Three

A third example provides a method of carrying out parallel synthesis in a single reaction vessel. Diverse molecular constructs are prepared in separate liquid samples and individually tagged using a unique combination of tag elements, such as the PNA sequences discussed above. The diverse molecular constructs may be based on a single scaffold with different diversity elements attached thereto, or may be diverse scaffold molecules.

The separate liquid samples are then mixed to form a single liquid sample containing diverse molecular constructs, each of which has a unique tag. The molecular constructs in the single liquid sample are then subject to multiple rounds of chemical synthesis which lead to the same or equivalent chemical reactions being carried out in respect of each of the diverse molecular constructs. After the multiple rounds of chemical synthesis have been carried out, the resulting combinatorial library typically has the same number of product molecular constructs are there were diverse molecular constructs before the multiple rounds of chemical synthesis. The product molecular constructs can then be readily separated by using specific binding elements which specifically bind to individual tag elements as before. Some tag elements may be blocked before the separation stage, as discussed above.

Thus, the method has enabled parallel synthesis of diverse product molecular constructs within the same reaction vessel. This is an efficient method of synthesizing a library of molecular constructs which include common elements. Although the multiple rounds of chemical synthesis do not generally change the diversity of the library of molecular constructs, this method preserves the diversity of the uniquely labelled molecular constructs and enables the product molecular constructs to be separated.

One skilled in the art will appreciate that rounds of parallel synthesis and split and mix synthesis may be applied in either order or alternated. For example, a library of individual tagged molecular constructs may be created using the split and mix methods described herein. The molecular constructs in the resulting library may then be subjected to further rounds of synthesis in parallel, for example within a single reaction vessel. Further rounds of split and mix synthesis may then be employed to further increase the diversity of the library while continuing to uniquely tag each different molecular construct in the library.

Alternative Methods of Creating a Combinatorial Library

A number of variations on the above examples are set out below.

(a) The tag elements may be selected to encode not just the corresponding diversity element, but the location of that diversity element, or the round of synthesis in which that diversity element was attached to the molecular constructs. Accordingly, the same diversity element may be associated with more than one different tag element depending on the location, or round of synthesis, in which the diversity element was attached to the molecular construct. For example, twenty-seven different diversity elements may be used in each of four rounds of synthesis. Each of the twenty-seven different diversity elements may be encoded by a different tag element in each round. In the first round, each of the twenty-seven possible combinations of the three bases A, C and G (i.e. each combination excluding the base T) may be employed in a tag element. In each subsequent round, each of the twenty- seven possible combinations of three of the four bases A, C, G and T may be employed in a tag element, such that each base is omitted in one of the four rounds of synthesis.

(b) The tag elements may be attached consecutively in a chain, to form a tag. This method is typical where the tag elements are polynucleotide sequences, such as PNA sequences. However, the tag elements may alternatively be attached at different locations on the molecular constructs. Similarly, the diversity elements may be attached consecutively in a chain, or at different locations on the molecular construct.

(c) In the examples shown above, each molecular construct comprises a molecular scaffold. However, the methods can be readily adapted to include no scaffold which is common to every molecular construct. For example, the molecular constructs used at the beginning of the methods may be diverse. Different starting constructs may have different tag elements attached thereto at the beginning of the library synthesis procedure. In a parallel synthesis approach, the diversity elements may be attached directly to the tag elements.

(d) The specific binding elements may be arranged in an array. For example, they may be arranged as an array of discrete pins on a backboard. A mixture of molecular constructs may then be applied to the array of specific binding elements, which will then be separated according to the tag element or combination of tag elements which are specifically bound by each individual specific binding element. Each pin may then be located in a different well of a multi-well plate, enabling the molecular constructs which bound to each individual specific binding element to be eluted into different wells of a multi- well plate. This is a convenient method of creating a spatially separated library of molecular constructs in the liquid phase which can be readily automated using known technology for working with multi-well plates of standard configurations and sizes. The resulting spatially separate library of molecular constructs can be used in screening methods, as discussed further below. (e) Instead of separating the molecular constructs into different portions of liquid, they may alternatively be immobilised on discrete supports (such as beads or microcapsules) or at discrete locations on a solid support (e.g. the surface of a micro-array), by using specific binding elements, which are themselves immobilised on the discrete supports or at discrete locations on a solid support, which specifically bind different combinations of tag elements. For example, where the tag elements comprise sequences of PNA, or another polynucleotide, the specific binding elements may comprise longer sequences of DNA, or another polynucleotide, which are complementary to combinations of tag elements (forming the whole or part of the tags) which may be formed by the methods of the invention. Thus, the different molecular constructs may be attached to the surface of a support. For example, the molecular constructs could be attached to the surface of a micro-array which has different nucleotide sequences adhered at discrete locations thereon, to form an array of different molecular constructs. The resulting spatially separate library of molecular constructs can be used in screening methods, as discussed further below.

Screening Methods

In a further example, a combinatorial library is created by a method according to the first or second example method described above, except that the final separation stage is carried out using a micro-array, in the form of a planar substrate with a plurality of different spots (functioning as spatially separated specific binding elements). The PNA within each different spot has a different sequence, so that it will bind a different tag found in the library of molecular constructs. The creation of micro- arrays having many discrete regions with polynucleotides with different sequences is well known to one who is skilled in the field.

Thus, a mixture of diverse molecular constructs is separated so that molecular constructs having different structure (and therefore different tags encoding those structures) are immobilised at different locations on the surface of the micro-array and are thereby spatially separate.

A labelled macromolecule, such as a protein modified to include a fluorescent label, such as fluorescein, is then brought into contact with the micro-array, whilst different molecular constructs remain spatially separate by virtue of their having bound to spatially separate spots of PNA. The surface of the micro-array is washed, and the distribution of the label on the surface of the micro-array is analysed, to determine to which molecular constructs the macromolecule bound. Thus, in this example, diverse molecular constructs from a library of diverse molecular constructs are each screened separately for the activity of binding to the labelled macromolecule, whilst they remain immobilised on the surface of the micro-array.

In a further example of a screening method, different molecular constructs are separated by immobilisation to different spatially separate spots of PNA on the surface of a micro-array, as before. However, before they are screened for a desired activity, they are sequentially removed from the surface of the micro-array, by apparatus illustrated in Figure 6. The apparatus, shown generally as 200, comprises a tube 202, which is scanned across the surface of micro-array 204 by a robotic arm (not shown) so that it passes different spots 206a, 206b, 206c in turn. Each spot has immobilised PNA of a different sequence, to which molecular constructs labelled with a complementary tag are bound.

A pump 208, draws liquid from the vicinity of the surface of the micro-array into the tube and then out through an outlet conduit 210. Furthermore, the tube is associated with a second conduit 212, through which DMSO is introduced to the surface of the micro-array, where it disrupts the bonds between the PNA tags which are attached to the molecular constructs, and the PNA molecules which are attached to the surface of the micro-array. Thus, molecular constructs which are eluted from each spot in turn are drawn consecutively up into the tube and so the liquid which is drawn up through the tube comprises spatially separate regions of liquid including molecular constructs of different structure. The resulting liquid can then be introduced to other components of an assay, to allow the separated molecular constructs to be screened for a desired activity whilst they remain spatially separate.

Instead of introducing a chemical agent to the surface of the micro-array, to remove the molecular constructs from the surface of the micro-array, a photolabile linker may be incorporated into the molecular constructs, or between the molecular constructs and the tags, or within the tags, or within the immobilised PNA sequences. A beam of light having a wavelength which is selected to cleave the photolabile linker may therefore be scanned across the surface of the micro-array, where it will cause molecular constructs to be removed from the surface of different spots in turn. At the same time, liquid in the vicinity of the surface of the micro-array is removed continuously, e.g. using a pump. As molecular constructs which have been bound to different spots, and which therefore had different tags and therefore different structures, are removed from the surface in turn, they will remain spatially separate in the liquid which is removed from the vicinity of the surface of the micro-array, provided that they are not mixed or left for a sufficiently long period of time that they diffuse together.

One who is skilled in the art will appreciate that corresponding methods can be employed to screen libraries comprising a mixture of diverse molecular constructs which have different structures to those which can be prepared by the methods which are described above, or which have been made in different ways, provided that the molecular constructs which are initially mixed together are each attached to a tag which encodes the structure of the respective molecular construct, and which can be selectively bound by a corresponding specific binding element.

Further variations and modifications may be made within the scope of the invention herein disclosed.

Claims

1. A method of screening a library of diverse molecular constructs, comprising the steps of:

2. A method according to Claim 1 , wherein the spatially separate specific binding elements are located at discrete locations within, or on the surface of a support.

3. A method according to Claim 2, wherein the spatially separate specific binding elements are located at different regions of the surface of a planar micro- array.

4. A method according to any one preceding Claim, wherein the separated molecular constructs are screened for a desired activity while they remain spatially separated from each other, while remaining attached to the spatially separate specific binding elements.

5. A method according to any one of Claims 1 to 3, wherein the separated molecular constructs are screened for a desired activity in a procedure where the separated molecular constructs are initially attached to the spatially separate specific binding elements, but molecular constructs which have, or which do not have, a desired activity are removed from the spatially separate specific binding elements during the screening procedure.

6. A method according to any one of Claims 1 to 3, wherein the separated molecular constructs are removed from the spatially separated specific binding elements before they are screened for a desired activity.

7. A method according to Claim 6, wherein the spatially separate specific binding elements are discrete locations on the same solid support and molecular constructs which are attached to a first specific binding element are removed from the solid support, before molecular constructs which are attached to a second specific binding element are removed from the solid support.

8. A method according to Claim 7, wherein the solid support is a micro-array, and the spatially separate specific binding elements are at different locations on the surface of the micro-array and the location from which molecular constructs are removed is swept sequentially across the surface of the micro- array, to remove molecular constructs in turn from the surface of the array.

9. A method according to Claim 8, wherein molecular constructs which are removed from the surface of a solid support are drawn away from the solid support, thereby remaining spatially separated.

10. A method according to any one of Claims 6 to 9, wherein the binding of specific binding elements to corresponding tags is disrupted by the introduction of one or more chemical species which selected to disrupt binding.

1 1. A method according to any one of Claims 6 to 9, wherein one or more bonds within the molecular constructs, or specific binding elements, is cleaved, thereby causing the molecular constructs to be removed from the surface of the array.

12. A method according to any one preceding Claim, wherein the tags comprise polynucleotides and the specific binding elements comprise a plurality of polynucleotide molecules which are complementary to respective tags.

13. A method according to any one preceding Claim, wherein the library comprising a mixture of diverse molecular constructs is synthesised by a method according to any one of Claims 15 to 68

14. A method according to any one preceding Claim, wherein the steps of providing a library comprising a mixture of diverse molecular constructs, and separating molecular constructs from the mixture are carried out by a method of synthesizing a combinatorial library according to any one of Claims 15 to 68.

15. A method of synthesizing a library of molecular constructs, comprising a plurality of rounds of synthesis, wherein during at least two of the plurality of rounds of synthesis, tag elements from a group of tag elements are combinatorially attached to molecular constructs to form tags, each of which comprises a plurality of said tag elements, to form a library of more diverse molecular constructs such that, after completion of the said plurality of rounds of synthesis, different molecular constructs within the resulting library have different tags attached thereto, the method further comprising the step of separating molecular constructs from a mixture of the resulting molecular constructs using a plurality of specific binding elements each of which specifically binds a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements, to form a combinatorial library of molecular constructs.

16. A method according to Claim 15, wherein the plurality of rounds of synthesis comprises at least one round of synthesis in which diversity elements selected from a group of diversity elements are combinatorially attached to molecular constructs to form a library of more diverse molecular constructs.

17. A method according to Claim 15 or Claim 16, wherein at least two rounds of synthesis in which tag elements are attached to molecular constructs occur prior to at least one round of synthesis in which diversity elements are attached to molecular constructs.

18. A method according to Claim 16 or Claim 17, wherein different molecular constructs are spatially separated prior to a mixing step prior to the separation step.

19. A method according to any one of Claims 16 to 18, wherein different molecular constructs, with different tags, are provided within the same body of liquid during each round of synthesis in which diversity elements are attached to molecular constructs.

20. A method according to any one of Claims 16 to 21 , wherein the rounds of synthesis in which tag elements are attached to molecular constructs and the rounds of synthesis in which diversity elements are attached to molecular constructs take place simultaneously or alternately.

21. A method according to Claim 20, comprising a plurality of rounds of split and mix synthesis in which a liquid containing molecular constructs is split into a plurality of portions, each of which is reacted with a different diversity element from the group of diversity elements, such as to attach the respective diversity element to the molecular constructs in the portion and, if there is to be a further round of split and mix synthesis, the portions are combined to form a further liquid containing molecular constructs for use in further rounds of split and mix synthesis, wherein, in at least one round of split and mix synthesis, each portion which is reacted with a different diversity element from a group of different diversity elements is reacted with a different tag element from the group of different tag elements.

22. A method according to Claim 15, wherein the rounds of synthesis comprise at least one round of synthesis in which different molecular constructs in a mixture comprising a plurality of diverse molecular constructs undergo the same synthetic procedure.

23. A method according to any one of Claims 15 to 22, wherein the step of separating molecular constructs, using a plurality of specific binding elements, comprises separating the molecular constructs into discrete liquid portions.

24. A method according to Claim 23, wherein the step of separating molecular constructs using a plurality of specific binding elements, comprises spatially separating the molecular constructs using specific binding elements located in an array on a surface such that, following separation, the molecular constructs are immobilised in a corresponding array on the surface.

25. A method according to Claim 23 or Claim 24, wherein the specific binding elements are located in an array on a surface and eluted into a corresponding array of separate liquid retaining containers.

26. A method according to Claim 25, wherein the specific binding elements are arranged as protuberances in an array on a surface and molecular constructs which bound to each specific binding element in the array are eluted simultaneously into respective wells of a multi-well plate.

27. A method according to any of Claims 15 to 26, in which molecular constructs are separated using a group of specific binding elements, each of which is specific to a different combination of tag elements which could be present in the combinatorial library.

28. A method according to any one of Claims 15 to 26, in which molecular constructs are separated using a group of specific binding elements, each of which is specific to a different tag element, so that the group of specific binding elements may be used to separate molecular constructs tagged with more different combinations of tag elements than there are specific binding elements in the group of specific binding elements.

29. A method according to any one of Claims 15 to 28, wherein the specific binding elements are each part of a resin or other solid phase support which, in use, retains molecular constructs including the corresponding tag element in a mixture of molecular constructs passed through or mixed with the resin or other solid phase support and from which molecular constructs including the corresponding tag element can subsequently be eluted.

30. A method according to any one of Claims 15 to 29, wherein each tag element comprises a polynucleotide sequence.

31. A method according to Claim 30, wherein each tag element comprises a peptide nucleic acid (PNA) sequence.

32. A method according to any one of Claims 29 to 31 , wherein each tag element comprises a polynucleotide sequence and the supergroup comprising the groups of tag elements to be used during each round of synthesis in which a tag element is attached to a molecular construct is selected so that tag elements within the supergroup do not include any sequences of polynucleotides which might be formed by the overlap between two or more tag elements within the supergroup which might be ligated to each other during the method.

33. A method according to any one of Claims 15 to 32, wherein the tags are cleaved from the molecular constructs after the separation step.

34. A method according to any one of Claims 15 to 33, wherein the resulting combinatorial library comprises molecular constructs tagged with more different combinations of tag elements from the group of tag elements than there are tag elements in the group of tag elements.

35. A method according to any one of Claims 15 to 34, wherein each specific binding element is selected to specifically bind a specific tag element and the step of separating molecular constructs from a mixture of molecular constructs includes a plurality of separation stages, each of which uses fewer different specific binding elements than the number of combinations of tag elements which could be attached to the molecular constructs in the combinatorial library, wherein at least some of the specific binding elements in a group of specific binding elements is used in two or more of the separation stages to enable molecular constructs tagged with each combination of tag elements which could be present in the combinatorial library to be individually separated.

36. A method according to Claim 35, wherein the method comprises the step of blocking tag elements which were added to molecular constructs in a round of split and mix synthesis before adding further tag elements in a subsequent round of synthesis., or adding tag elements to a molecular construct during a round of synthesis in which a tag element is attached to a molecular construct which is already blocked when it is added to the molecular constructs.

37. A method according to Claim 35, wherein the step of separating molecular constructs comprises the step of using specific binding elements to separate molecular constructs on the basis of tag elements, or combinations of tag elements, which have not been blocked, without separation dependent on the presence of blocked tagged elements, followed by the step of unblocking blocked tag elements and the step of separating molecular constructs using specific binding elements on the basis of the now unblocked tag elements.

38. A method according to Claim 36, wherein the step of unblocking tag elements and separating molecular constructs on the basis of the thereby unblocked tag elements procedure is repeated to facilitate further separation on the basis of further tag elements.

39. A method according to any one of Claims 15 to 38, wherein the molecular constructs comprise a molecular scaffold.

40. A method according to any one of Claims 15 to 39, wherein the tag elements are attached to the molecular scaffold through a cleavable linker.

41. A method according to any one of Claims 15 to 40, wherein, during one or more of the rounds of synthesis in which tag elements are attached to molecular constructs each tag element is attached to the molecular construct through a corresponding diversity element.

42. A method of synthesizing a library of molecular constructs, comprising at least one round of split and mix synthesis in which a liquid containing molecular constructs is split into a plurality of portions, each of which is reacted with a different diversity element from a group of diversity elements, such as to attach the respective diversity element to the molecular constructs in the portion and, if there is to be a further round of split and mix synthesis, the portions are combined to form a further liquid containing molecular constructs for use in further rounds of split and mix synthesis, wherein, in at least one round of split and mix synthesis, each portion which is reacted with a different diversity element from a group of different diversity elements is reacted with a different tag element from a group of different tag elements, the method further comprising the step of separating molecular constructs from a mixture of molecular constructs using a plurality of specific binding elements each of which specifically binds a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements, to form a combinatorial library of molecular constructs.

43. A method according to Claim 42, wherein the mixture of molecular constructs from which molecular constructs are separated comprises one of the portions from the final round of split and mix synthesis.

44. A method according to Claim 42 or Claim 43, wherein the step of splitting a liquid comprising molecular constructs into a plurality of portions during a round of split and mix synthesis splits the portions into distinct liquids which are not in liquid communication with each other.

45. A method according to any one of Claims 42 to 44, wherein the step of separating molecular constructs, using a plurality of specific binding elements, comprises separating the molecular constructs into discrete liquid portions.

46. A method according to Claim 45, wherein the step of separating molecular constructs using a plurality of specific binding elements, comprises spatially separating the molecular constructs using specific binding elements located in an array on a surface such that, following separation, the molecular constructs are immobilised in a corresponding array on the surface.

47. A method according to any one of Claims 42 to 46, in which molecular constructs are separated using a group of specific binding elements, each of which is specific to a different combination of tag elements which could be present in the combinatorial library.

48. A method according to any one of Claims 42 to 46, in which molecular construct are separated using a group of specific binding elements, each of which is specific to a different tag element, so that the group of specific binding elements may be used to separate molecular constructs tagged with more different combinations of tag elements than there are specific binding elements in the group of specific binding elements.

49. A method according to any one of Claims 42 to 48, wherein the specific binding elements are each part of a resin or other solid phase support which, in use, retains molecular constructs including the corresponding tag element in a mixture of molecular constructs passed through or mixed with the resin or other solid phase support and from which molecular constructs including the corresponding tag element can subsequently be eluted.

50. A method according to any one of Claims 42 to 49, wherein each tag element comprises a polynucleotide sequence.

51. A method according to Claim 50, wherein each tag element comprises a peptide nucleic acid (PNA) sequence.

52. A method according to any one of Claims 49 to 51 , wherein each tag element comprises a polynucleotide sequence and the supergroup comprising the groups of tag elements to be used during each round of synthesis in which a tag element is attached to a molecular construct is selected so that tag elements within the supergroup do not include any sequences of polynucleotides which might be formed by the overlap between two or more tag elements within the supergroup which might be ligated to each other during the method.

53. A method according to any one of Claims 42 to 50, wherein the resulting combinatorial library comprises molecular constructs tagged with more different combinations of tag elements from the group of tag elements than there are tag elements in the group of tag elements.

54. A method according to any one of Claims 42 to 53, wherein each specific binding element is selected to specifically bind a specific tag element and the step of separating molecular constructs from a mixture of molecular constructs includes a plurality of separation stages, each of which uses fewer different specific binding elements than the number of combinations of tag elements which could be attached to the molecular constructs in the combinatorial library, wherein at least some of the specific binding elements in a group of specific binding elements is used in two or more of the separation stages to enable molecular constructs tagged with each combination of tag elements which could be present in the combinatorial library to be individually separated.

55. A method according to Claim 54, wherein the method comprises the step of blocking tag elements which were added to molecular constructs in a round of split and mix synthesis before adding further tag elements in a subsequent round of split and mix synthesis., or adding tag elements to a molecular construct during a round of synthesis in which a tag element is attached to a molecular construct which are already blocked when they are added to the molecular constructs.

56. A method according to Claim 55, wherein the step of separating molecular constructs comprises the step of using specific binding elements to separate molecular constructs on the basis of tag elements, or combinations of tag elements, which have not been blocked, without separation dependent on the presence of blocked tagged elements, followed by the step of unblocking blocked tag elements and the step of separating molecular constructs using specific binding elements on the basis of the now unblocked tag elements.

57. A method according to Claim 56, wherein the step of unblocking tag elements and separating molecular constructs on the basis of the thereby unblocked tag elements procedure is repeated to facilitate further separation on the basis of further tag elements.

58. A method according to any one of Claims 42 to 57, wherein the molecular constructs comprise a molecular scaffold.

59. A method according to any one of Claims 42 to 58, wherein the tag elements are attached to the molecular scaffold through a cleavable linker.

60. A method according to any one of Claims 42 to 59, wherein, during one or more of the rounds of synthesis in which tag elements are attached to molecular constructs each tag element is attached to the molecular construct through a corresponding diversity element.

61. A method of synthesizing a library of molecular constructs, comprising forming a solution comprising diverse molecular constructs, wherein each different molecular construct within the solution has a different tag comprising a unique combination of tag elements selected from a group of tag elements, carrying out at least one round of parallel synthesis in which equivalent modifications are made to diverse molecular constructs within the solution, and then separating molecular constructs from the resulting mixture of molecular constructs using a plurality of specific binding elements each of which specifically binds a different tag element from the group of different tag elements, or a specific combination of different tag elements from the group of different tag elements, to form a combinatorial library of molecular constructs.

62. A method according to Claim 61 , comprising a plurality of rounds of parallel synthesis.

63. A method according to Claim 61 or Claim 62, wherein the diverse molecular constructs within the solution are spatially separated.

64. A method according to any one of Claims 61 to 63, wherein the solutions comprising diverse molecular constructs comprise diverse scaffolds which can be modified during the method to create molecular constructs of greater molecular weight.

65. A method according to any one of Claims 61 to 64, wherein the step of separating molecular constructs, using a plurality of specific binding elements, comprises separating the molecular constructs into discrete liquid portions.

66. A method according to Claim 64, wherein the step of separating molecular constructs using a plurality of specific binding elements, comprises spatially separating the molecular constructs using specific binding elements located in an array on a surface such that, following separation, the molecular constructs are immobilised in a corresponding array on the surface.

67. A method according to Claim 65 or Claim 66, wherein the specific binding elements are located in an array on a surface and eluted into a corresponding array of separate liquid retaining containers.

68. A method according to Claim 65, wherein the specific binding elements are arranged as protuberances in an array on a surface and molecular constructs which bound to each specific binding element in the array are eluted simultaneously into respective wells of a multi-well plate.

69. A library of molecular constructs obtainable by the method of any one of Claims 15 to 68.

70. A library of molecular constructs according to Claim 69, wherein the molecular constructs are obtained by the method of any one of Claims 15 to 68.

71. A kit of parts comprising a library of molecular constructs according to Claim 69 or Claim 70, and a plurality of specific binding elements, each of which specifically binds a different tag element from the group of tag element, or a specific combination of different tag elements from the group of tag elements.

72. A kit of parts according to Claim 71 , wherein the specific binding elements are attached in an array to a surface.

73. A kit of parts according to Claim 72, further comprising a multi-well plate comprising a plurality of wells which are arranged in a corresponding formation to the array of specific binding elements.