WO2003076943A1 - Encoded self-assembling chemical libraries (esachel) - Google Patents
Encoded self-assembling chemical libraries (esachel) Download PDFInfo
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- WO2003076943A1 WO2003076943A1 PCT/EP2002/004153 EP0204153W WO03076943A1 WO 2003076943 A1 WO2003076943 A1 WO 2003076943A1 EP 0204153 W EP0204153 W EP 0204153W WO 03076943 A1 WO03076943 A1 WO 03076943A1
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- C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
- C40B50/10—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support involving encoding steps
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
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- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/10—Detection mode being characterised by the assay principle
- C12Q2565/101—Interaction between at least two labels
Definitions
- the organic molecules are linked to an oligonucleotide which mediates the self-assembly of the library and/or pro- vides a code associated to each binding moiety.
- the resulting library can be very large (as it originates by the combinatorial self-assembly of smaller sub-libraries).
- the "binding code” can be "decoded” by a number of experimental techniques (e.g., hybridization on DNA chips or by a modified polymerase chain reac- tion (PCR) technique followed by sequencing).
- specific binding molecules e.g., organic molecules
- drugs approved by the U.S. Food and Drug Administration are specific binders of biological targets which fall into one of the following categories: enzymes, receptors or ion channels.
- the specific binding to the biological target is not per se sufficient to turn a binding molecule into a drug, as it is widely recognized that other molecular properties (such as pharmacokinetic behaviour and stability) contribute to the performance of a drug.
- the isolation of specific binders against a relevant biological target typically represents the starting point in the process which leads to a new drug [Drews J. Drug dis- covery: a historical perspective. Science (2000) 287: 1960-1964].
- the ability to rapidly generate specific binders against the biological targets of interest would be invaluable also for a variety of chemical and biological applications.
- the specific neutralization of a particular epitope of the intra- cellular protein of choice may provide information on the functional role of this epitope (and consequently of this protein).
- the use of monoclonal antibodies specific for a given epitope may provide the same type of information [Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR. Making antibodies by phage display technology. Annu Rev Immunol. (1994) 12:433-455].
- most antibodies do not readily cross the cell membrane and have to be artificially introduced into the cell of interest.
- intracellular antibodies can also be expressed into target cells by targeted gene delivery (e.g., by cell transfection with DNA directing the expression of the antibody).
- the antibody often does not fold, as the reducing intracellular milieu does not allow the formation of disulfide bonds which often contribute in an essential manner to antibody stability [Desiderio A, Franconi R, Lopez M, Villani ME, Viti F, Chiaraluce R, Con- salvi V, Neri D, Benvenuto E. A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold. J Mol Biol. (2001) 310: 603-615]. High affinity binding molecules amenable to chemical synthesis may provide a valuable alternative to antibody technology.
- phage display produces parti- cles in which a phenotype (typically the binding properties of a protein, displayed on the surface of filamentous phage) is physically coupled to the corresponding genotype (i.e., the gene coding for the protein displayed on phage) [Winter, 1994], allowing the facile amplification and identification of library binding members with the desired binding specificity.
- a phenotype typically the binding properties of a protein, displayed on the surface of filamentous phage
- genotype i.e., the gene coding for the protein displayed on phage
- biomacromolecules can provide very useful binding specificities, their scope is essentially limited to repertoires of polypeptides or of nucleic acids [Brody EN, Gold L. Aptamers as therapeutic and diagnostic agents. J Biotechnol. (2000) 74:5-13].
- large biomacromolecules are not ideal. For example, they are often unable to efficiently cross the cell membrane, and may undergo hydrolytic degradation in vivo.
- the authors postulated the combinatorial synthesis of polymeric chemical compounds on a solid support (e.g., a bead), where a step in the combinatorial synthesis would be followed by the synthesis (on the same bead) of a DNA sequence, to be used as a "memory tag" for the chemical reactions performed on the bead.
- DNA-encoded beads would be incubated with a target molecule (e.g., a protein of pharmaceutical relevance).
- a target molecule e.g., a protein of pharmaceutical relevance
- the nature of the polymeric chemical structure bound to the receptor could be decoded by sequencing the nucleotide tag.
- the ECL method has the advantage of introducing the concept of "coding" a par- ticular polymeric chemical moiety, synthesized on a bead, with a corresponding oligonucleotidic sequence, which can be "read” and amplified by PCR.
- the ECL method has a number of drawbacks. First, a general chemistry is needed which allows the alternating synthesis of polymeric organic molecules (often with different reactivity properties) and DNA synthesis on a bead. Second, the synthe- sis, management and quality control of large libraries (e.g., > 1 million individual members) remains a daunting task. In fact, the usefulness of the ECL method has yet to be demonstrated with experimental examples. _
- an encoded combinatorial chemical library which comprises a plurality of bifunctional molecules according to the formula A-B-C, where A is a polymeric chemical moiety.
- B is a linker molecule operatively linking A and C, consisting of a chain length of 1 to about 20 atoms and preferably comprising means for attachment to a solid support.
- C is an identifier oligonucleotide comprising a sequence of nucleotides that identifies the structure of the chemical moiety.
- the attachment to a solid support is especially preferred when synthe- sizing step by step the chemical moiety (a polymer built of subunits X ⁇ - ⁇ ) and the oligonucleotide (built of nucleotides Z ⁇ -n which code for and identify the structure of the chemical subunits of the polymer). Also described are the bifunctional molecules of the library, and methods of using the library to identify chemical structures within the library that bind to biological active molecules in preselected binding interactions.
- a target molecule e.g. a biological target
- an oligonucleotide or functional analogue thereof which chemical compound does not need to be individually synthesized in order to build up a chemical library.
- a chemical compound comprising a chemical moiety (p) capable of performing a binding interaction with a target molecule (e.g. a biological target) and further comprising an oligonucleotide (b) or functional analogue thereof which is characterized in that the oligonucleotide (b) or functional analogue comprises at least one self-assembly sequence (bl) capable of performing a combination reaction with at least one self-assembly sequence (bl 1 ) of a complementary oligonucleotide or functional analogue bound to another chemical compound comprising a chemical moiety (q).
- a target molecule e.g. a biological target
- oligonucleotide (b) or functional analogue comprises at least one self-assembly sequence (bl) capable of performing a combination reaction with at least one self-assembly sequence (bl 1 ) of a complementary oligonucleotide or functional analogue bound to another chemical compound comprising
- a chemical compound comprising a chemical moiety (p) capable of performing a binding interaction with a target molecule (e.g. a biological target) and further comprising an oligonucleotide (b) or functional analogue thereof, which comprises a coding sequence (bl) coding for the identi- fication of the chemical moiety (p), and which is characterized in that the chemical compound further comprises at least one self-assembly moiety (m) capable of performing a combination reaction with at least one self-assembly moiety (m 1 ) of a another chemical compound comprising a chemical moiety (q).
- a target molecule e.g. a biological target
- b oligonucleotide
- bl coding sequence
- the chemical compound further comprises at least one self-assembly moiety (m) capable of performing a combination reaction with at least one self-assembly moiety (m 1 ) of a another chemical compound comprising a chemical moiety
- the present invention provides a chemical compound comprising a chemical moiety of any kind capable of performing a binding interaction with a target molecule (e.g. a biological target) and further comprising an oligonucleotide or functional _
- oligonucleotide analogue thereof which can be synthesized separately and then coupled together.
- the resulting chemical derivative(s) of the oligonucleotide can further assemble with other similar compounds to generate higher order structures and encoded libraries of compounds.
- FIG. 1 For illustrative purposes, one particular embodiment of our invention is depicted in Figure 1.
- Two chemical libraries are synthesized by chemical modification of the 3' end and the 5' end, respectively, of oligonucleotides capable of duplex formation and which carry distinctive "sequence tags" (associated with [and therefore "coding for”] the chemical moiety attached to their extremity).
- the resulting encoded self-assembled chemical library (ESACHEL) can be very large (as it originates from the combinatorial self-assembly of two smaller libraries) and can be screened for binding to a biological target (e.g., a protein of pharmaceutical interest).
- Those members of the library which display suitable binding specifi- cities can be captured with the target of interest (for example, using a target immobilized on a solid support). Their genetic code, encoding the chemical entity responsible for the binding specificity of interest, can then be retrieved using a number of ingenious methods, which are described in the section "Description of the Invention" (see below).
- This may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s).
- specificity is associated with a significant difference in binding affinity, relative to "non-specific" targets.
- the term is also applicable where e.g. a binding member is specific for a particular surface on the target molecule (hereafter termed as " epitope"), in which case the specific binding member with this specificity will be able to bind to various target molecules carrying the epitope.
- Figure 1 A simple embodiment of ESACHEL technology:
- ESACHEL encoded self-assembled chemical library
- Those members of the library which display suitable binding specificities can be captured with the target of interest (for example, using a target immobilized on a solid support). Their genetic code, encoding the chemical entity re- sponsible for the binding specificity of interest, can then be retrieved using a number of ingenious methods
- the main ingredients of ESACHEL technology are chemical compounds, com- prising an oligonucleotidic moiety (typically, a DNA sequence) linked to an oligomerization domain [capable of mediating the (homo- or hetero-) dimeri- zation, trimerization or tetramerization of the chemical compounds], linked to a chemical entity, which may be involved in a specific binding interaction with a target molecule.
- oligonucleotidic moiety will be uniquely associated with the chemical entity (therefore acting as a "code”).
- the oligomerization domain and the code can be distinct portions of the same molecule (typically an oligonucleotide).
- n of different chemical compounds carrying a thiol-reactive moiety (e.g., a maleimido or a io- doacetamido group), are reacted (in separate reactions) with n different DNA oligonucleotides, carrying a thiol group at the 3' end.
- a thiol-reactive moiety e.g., a maleimido or a io- doacetamido group
- a number m of differ ⁇ ent chemical compounds, carrying a thiol-reactive moiety are reacted (in separate reactions) with m different DNA oligonucleotides, carrying a thiol group at the 5' end.
- a thiol-reactive moiety e.g., a maleimido or a iodoacetamido group
- the corresponding pool of m conjugates is indicated in the Figure as "pool B”.
- the resulting self-assembled library members will correspond to m x n combinations.
- DNA sequences are known to be capable of forming stable trimeric complexes or stable tetrameric complexes.
- Hoogsten pairing of DNA triplexes could allow the self-assembly of Pools A x B x C, containing n, m and I members, respectively.
- the tetrameric assembly of DNA-(chemical moiety) con- jugates would allow even larger library sizes, starting from sub-libraries A, B, C and D of small dimension.
- the oligonucleotides of sub-library A bear chemical entities at the 3' end. To- wards the 3' extremity, the DNA sequence is designed to hybridize to the DNA sequences at the 5' extremity of oligonucleotides of sub-library B.
- the hybridization region is interrupted by a small segment. In sub-library A, this small segment is conveniently composed of phosphodiester backbone without bases (termed d-spacer in the Figure); in sub-library B, the corresponding short seg- ment will have unique sequence for each member of the sub-library (therefore acting as "code" for the sub-library B).
- oligonucleotides of sub-library A have their distinctive code towards the 5' extremity.
- oligonucleotides of sub-library B remain stably annealed to oli- gonucleotides of sub-library A, and can work as primers for a DNA polymerase reaction on the template A.
- the resulting DNA segment, carrying both code A and code B, can be amplified (typically by PCR), using primers which hybridize at the constant extremities of the DNA segment.
- Figure 6 A general method of ESACHEL decoding:
- binders isolated from sub-libraries A and B carrying chemical moieties at the extremities of partially-annealing oligonucleotides, is established by hybridization with target oligonucleotides immobilized on one or more chips.
- chips preferably are made from silicon wafers with attached oligonucleotide fragments.
- chip A will allow the reading of the identity (and frequency) of members of sub-library A, rescued after a biopanning experiment.
- chip B will allow the reading of the identity (and frequency) of members of sub-library B.
- the decoding method depicted in the Figure will not provide information about the pairing of code A and code B within specific binding members.
- decoding on chip A and B will suggest candidate components of sub-libraries A and B, to be re-annealed and screened in a successive round of bio-panning.
- Increasingly stringent binding to the target will be mirrored by a reduction in the number of A and B members, identified on the chip.
- the possible combinations of the candidate A and B members will be assembled individually (or in smaller pools), and assayed for binding to the target.
- Figure 7 A PCR-based method for ESACHEL deconvolution: Sub-libraries A and B form a heteroduplex, flanked by unique sequences coding for the different library members and by constant DNA segments at the termini. After biopanning, suitable pairs of primers allow the PCR amplification of the two strands, yielding PCR products whose sequence can be identified using standard methods (e.g. by concatenation of the PCR products, followed by subcloning and sequencing.
- a deconvolution procedure may be applied (consisting of one or more rounds of panning, followed by sequencing and by the choice of a restricted set of sub-library components for the next ESACHEL screening), restricting the number of candidate ESACHEL members capable of giving specific binders after self-assembly.
- chemical derivatives of self-assembling oligonucleotides will be isolated at the end of one or more rounds of panning.
- it will be desirable to covalently link together the chemical moieties, responsible for the interaction with the target molecule of interest.
- the length, rigidity, stereoelectronic chemical properties and solubility of the linker will influence the binding affinity and performance of the resulting molecule.
- Figure 9 Chemical equilibria contributing to the chelate effect: The diagram shows the possible states of the interactions between a bidentate ligand (A-B) binding to a target molecule. In state nl, both A and B moieties are bound to their respective binding pockets. In state nil and nlll only moiety A or B are bound, respectively. In state nIV, the compound A-B is dissociated from the target.
- A-B bidentate ligand
- a computer program has been written for the approximate evaluation of the contribution of the chelate effect to the residence time of A-B on the target in irreversible dissociation conditions, as a function of kinetic association and dissociation constants of the moieties A and B towards their respective binding pockets, and of the linker length between A and B.
- the probability that one moiety dissociates per time unit is indicated as poff.
- the probability that one moiety binds to the target per time unit is indicated as pon.
- Figure 10 Assembly of molecule p with molecular repertoire Q: The diagram shows heteroduplex formation between an oligonucleotide, coupled to a low-affinity binder p, and a second class of oligonucleotides, which bear chemical moieties q and distinctive codes, capable of identifying the molecules q which synergise with p for binding to a target molecule (e.g., a protein target).
- a target molecule e.g., a protein target.
- the main ingredients of ESACHEL technology are chemical compounds, comprising an oligonucleotidic moiety (typically, a DNA sequence) linked to an oligomerization domain [capable of mediating the (homo- or hetero-) dimeri- zation, trimerization or tetramerization of the chemical compounds], linked to a chemical entity, which may be involved in a specific binding interaction with a crizosin, acridinucleic acid, or oligomerization domain
- oligonucleotidic moieties in ESACHEL technology is best understood by the description of the "coding" system, provided in the sections below and in the Examples.
- a unique oligonucleotidic sequence e.g., a sequence of DNA or DNA analogues
- one provides that chemical entity with a unique code which can be "read” in a variety of ways (sequencing, hybridization to DNA chips, etc.) and which may be amenable to amplification (e.g., by the use of the polymerase chain reaction [PCR]).
- the code of one particular chemical compound may become physically linked to the code of other chemical compound(s), when these chemical compounds are associated by means of an oligomerization domain.
- Suitable DNA sequences can be considered as possible oligomerization domains.
- a number n of different chemical compounds, carrying a reactive moiety are reacted (in separate reactions) with n different DNA oligonucleotides, carrying a reactive moiety (e.g. a thiol group at the 3' end).
- a reactive moiety e.g. a thiol-reactive maleimido or a iodoacetamido group
- n different DNA oligonucleotides carrying a reactive moiety (e.g. a thiol group at the 3' end).
- the corresponding pool of n conjugates is indicated in the Figure as "pool A".
- the oligonucleotides of pool A are designed to have: - one portion of the DNA sequence which can hybridize to compounds of pool B (see Figure 3 and comments below)
- a number m of different chemical compounds, carrying a thiol-reactive moiety are reacted (in separate reactions) with m different DNA oligonucleotides, carrying a thiol group at the 5' end.
- a thiol-reactive moiety e.g., a maleimido or a iodoacetamido group
- the oligonucleotides of pool B are designed to have:
- the partially complementary strands of the DNA conjugates of pool A and pool B can easily heterodimerize in solution, with comparable efficiency within the different n members of Pool A and the m members of Pool B. If suitable stoichi- ometric ratios of the compounds of Pool A and Pool B are used, the n different types of compounds of Pool A will heterodimerize with the m different types of compounds of Pool B, yielding a combinatorial self-assembled chemical library of dimension m x n. For example, two libraries of thousands of compounds would yield millions of different combinations. Furthermore, the resulting self- assembled m x n combinations will carry unique DNA codes, corresponding to the non-covalent but stable association (heterodimerization) of the DNA code of the member of Pool A with the DNA code of the member of Pool B.
- oligo- nucleotides carrying a phosphodiester bond at one extremity forming chemical structures such as -O-P(O) 2 -O-(CH 2 )n-NH-CO-R, where R may correspond to a number of different chemical entities, and n may range between 1 and 10).
- a DNA portion is used as heterodimerization domain, and thioether bond formation is used for the coupling of DNA oligonucleotides to chemical entities of the library.
- oligomerization domains could be considered, as well as other chemical avenues for the coupling of chemical entities to DNA.
- DNA sequences are known to be capable of forming stable trimeric complexes [Strobel, 1991] or stable tetrameric complexes [Various authors, 2000- 2001].
- Hoogsten pairing of DNA triplexes could allow the self- assembly of Pools A x B x C, containing n, m and I members, respectively ( Figure 4).
- the tetrameric assembly of DNA-(chemical moiety) conjugates would al- low even larger library sizes, starting from sub-libraries A, B, C and D of small dimension.
- the decoding of binding interactions can, in some cases, be more difficult for trimeric and/or tetrameric self-assembled encoded libraries, as compared to dimeric libraries.
- linkers between the DNA strands and the chemical moieties displayed at their ex- tremity may either facilitate or hinder the identification of specific binding members of the encoded self-assembled chemical (ESACHEL) library.
- a certain degree of flexibility may allow suitable chemical moieties to find complementary pockets on the target molecule ( Figure 4).
- the affinity contribution of the chelate effect is expected to decrease with linker length.
- trimeric ESACHEL libraries would contain 10 6 members, while tetrameric ESACHEL libraries would contain 10 8 members. It is easy to calculate the resulting library size, starting from sub-libraries of different dimension.
- the large combinatorial com- plexity of encoded self-assembling chemical compounds may allow the identification of specific binding members, which have so far escaped identification using conventional combinatorial chemical methods.
- An analogy can be drawn from the field of antibody phage technology, where it was demonstrated that library size plays a crucial role in the isolation of high-affinity antibodies.
- oligonucleotidic sequences typically, DNA sequences
- DNA sequences provide chemical entities with a unique code. How many different se- quences do we need, in order to identify members of a library?
- ESACHEL the key components of ESACHEL technology are chemical compounds, comprising an oligonucleotidic moiety (typically, a DNA sequence) linked to an oligomerization domain, in turn linked to a chemical entity.
- the oligonucleotidic moiety will also provide the oligomerization domains.
- ESACHEL components will be chemical entities coupled to judiciously chosen DNA oligonucleotides.
- such oligonucleotides will contain a constant part and a variable part (uniquely characteristic for each member of the library).
- a sub-library "A” (containing n compounds attached at the 3' extremity of DNA oligonucleotides) is assembled to a sub-library “B” (containing m compounds attached at the 5' extremity of oligonucleotides).
- Sub-library A can be represented by a DNA sequence of x bases, where 4 X is greater or equal to n.
- Sub-library B can be represented by a DNA sequence of y bases, where 4 Y is greater or equal to m.
- identification of the code of sub-library members also provides information about which sub-library a par- ticular code (and therefore a particular compound) belongs to.
- sub-library A this small segment is conveniently composed of phosphodiester backbone without bases (termed d-spacer in the Figure); in sub-library B, the corresponding short segment will have unique sequence for each member of the sub-library (therefore acting as "code” for the sub-library B).
- code for the sub-library B.
- oligonucleotides of sub- library A have their distinctive code towards the 5' extremity.
- Oligonucleotides of sub-library B remain stably annealed to oligonucleotides of sub-library A, and can work as primers for a DNA polymerase reaction on the template A.
- the resulting DNA segment, carrying both code A and code B, can be amplified (typically by PCR), using primers which hybridize at the constant extremities of the DNA segment ( Figure 5).
- the decoding method of Figure 6 will not provide information about the pairing of code A and code B within specific binding members.
- decoding on chip A and B will suggest candidate components of sub-libraries A and B, to be re-annealed and screened in a successive round of bio-panning.
- Increasingly stringent binding to the target will be mirrored by a continuous reduc- tion in the number of A and B members, identified on the chip.
- the possible combinations of the candidate A and B members will be assembled individually (or in smaller pools), and assayed for binding to the target. We refer to this iterative strategy as deconvolution.
- the decoding method of Figure 6 is valid also for ESACHEL, when libraries self-assemble to form trimeric or tetrameric complexes (e.g. using DNA triplexes or quadruplexes for the oligomerization of compounds). In these cases, 3 or 4 chips may be used, respectively, which carry distinctive target oligonucleotides for decoding.
- DNA of selected binding moieties of Figure 6 may be PCR amplified prior to chip hybridization.
- oligonucleotide design will resemble the one described in the next paragraph (see also Figure 7).
- FIG. 7 Another possible decoding method is illustrated in Figure 7. Sub-libraries A and B form a heteroduplex, flanked by unique sequences coding for the different library members and by constant DNA segments at the termini. After biopanning, suitable pairs of primers allow the PCR amplification of the two strands, yielding PCR products whose sequence can be identified using standard methods (e.g. by con- ⁇ ⁇
- a de- convolution procedure may be applied (consisting of one or more rounds of panning, followed by sequencing and by the choice of a restricted set of sub-library components for the next ESACHEL screening), restricting the number of candidate ESACHEL members capable of giving specific binders after self-assembly.
- ESACHEL library construction is facilitated not only by the large dimension that can be achieved by self-assembly of sub-libraries, but also by the facile generation and purification of chemical derivatives of DNA oligonucleotides.
- DNA oligonucleotides bearing a thiol group at their 3' or 5' end
- DNA oligonucleotides bearing a thiol group at their 3' or 5' end
- reagents bearing reactive groups such as iodoacetamido moieties or maleimido moieties
- iodoacetamido moieties or maleimido moieties
- several methods are available in the literature for the chemical modification of 3' or 5' extremities of DNA oligonucleotides, for example during solid phase synthesis procedures.
- DNA Chemical derivatives of DNA (or some DNA analogues) have the characteristic property of being highly negatively charged at neutral pH. This facilitates the development of general purification strategies of the DNA derivatives. For example, anion exchange chromatography allows the non-covalent (but stable) immobilization of DNA oligonucleotides (and their derivatives) on a resin, while other components of a reaction mixtures can be washed away. DNA derivatives can then be eluted by buffer change. Alternatively, other purification methods (e.g. reverse phase chromatography, hydrophobic interaction chromatography, hy- droxyapatite chromatography etc.) could be considered. __
- ESACHEL components amenable to robotization [for example using a TECAN Genesys 200-based workstation (TECAN, Mannedorf, Switzerland), equipped with liquid handling system and a robotic manipulation arm]. Robotization may be necessary, in order to create ESACHEL sub-libraries containing several hundred different compounds.
- Biopanning experiments The use of ESACHEL for the identification of specific binders relies on the incubation of ESACHEL components with the target molecule (e.g., a protein of pharmacological interest), followed by the physical separation of the resulting complex from the ESACHEL components which have not bound to the target.
- ESACHEL biopanning experiments are analogous to biopanning experiments which can be performed with phage libraries and/or ribosome display libraries, for which an extensive literature and several experimental protocols are available [Winter, 1994;; Viti, 2000; Schaffitzel, 1999].
- physical separation of the complex between ESACHEL members and the target molecule, from the pool of non-bound ESACHEL members could be achieved by immobilizing the target molecule of a solid support (e.g. a plastic tube, a resin, magnetic beads, etc.).
- a solid support e.g. a plastic tube, a resin, magnetic beads, etc.
- chelate effect Some of the contributions of ESACHEL technology for the identification of specific binders are related to a chemical process, termed the "chelate effect".
- the term chelate was first applied in 1920 by Sir Gilbert T. Morgan and H.D.K. Drew [J. Chem. Soc, 1920, 117, 1456], who stated: "The adjective chelate, derived from the great claw or chela (chely- Greek) of the lobster or other crustaceans, is sug- gested for the caliper-like groups which function as two associating units and fasten to the central atom so as to produce heterocyclic rings.”
- the chelate effect can be seen by comparing the reaction of a chelating ligand and a metal ion with the corresponding reaction involving comparable monoden- tate ligands. For example, comparison of the binding of 2,2'-bipyridine with pyri- dine or 1,2-diaminoethane (ethylenediamine) with ammonia. It has been known for many years that a comparison of this type always shows that the complex resulting from coordination with the chelating ligand is much more thermody- namically stable.
- the chelate effect has been shown to be able to contribute to high-affinity binding not only in the case of multidentate metal ligands, but in many other chemical situations, including binding interactions with macromolecules (e.g., multidentate DNA binding, chelating recombinant antibodies) [Neri D, Momo M, Pros- pero T, Winter G. High-affinity antigen binding by chelating recombinant anti- bodies (CRAbs). J Mol Biol. (1995) 246:367-73].
- macromolecules e.g., multidentate DNA binding, chelating recombinant antibodies
- ESACHEL self- assembled molecules into analogues, in which the chemical entities responsible for the binding are covalently linked.
- the length, rigidity, stereoelectronic chemical properties and solubility of the linker will influence the binding affinity and performance of the resulting molecule [Shuker SB, Hajduk PJ, Meadows RP, Fesik SW. Discovering High-Affinity Ligands For Proteins - Sar by Nmr. Science (1996) 274:1531-1534] (see also Example 4).
- Example 1 As mentioned in previous sections, one strength of ESACHEL technology is its compatibility with a variety of different chemical moieties, including peptides and globular proteins (e.g., antibody domains).
- HH10VL_Hind_ba TCA ATC TGA TTA AGC TTA GTG ATG GTG ATG GTG ATG ACA TCC ACC TTT TAT TTC CAG CTT GGT CCC CCC
- the resulting PCR products are subcloned, using standard molecular biology procedures, into the EcoRl/Hindl ⁇ l sites of plasmid pQE12 (Qiagen, Germany).
- the resulting plasmids code for V domains, which carry the following sequence at their C-terminus: -Gly-Gly-Cys-His-His-His-His-His-His-His-His.
- the plasmids, encoding the cysteine-tagged V-domains, are electroporated into E.coli cells (preferentially, in the Origami strain of Novagen, which have a slightly oxidizing cytoplasmic redox potential), expressed and purified, using metal chelate chromatography on NiNTA resin (Qiagen, Germany).
- oligonucleotides carrying a thiol group at the 3' end or at the 5' end, are ordered from a commercial supplier (e.g., Microsynth, Balgach, Switzerland). Individual DNA oligonucleotides with the thiol group at the 3' end are used for coupling to individual VH domains. Individual DNA oligonucleotides with the thiol group at the 5' end are used for coupling to individual VL domains.
- a commercial supplier e.g., Microsynth, Balgach, Switzerland.
- Individual DNA oligonucleotides with the thiol group at the 3' end are used for coupling to individual VH domains.
- Individual DNA oligonucleotides with the thiol group at the 5' end are used for coupling to individual VL domains.
- Anti-GST (from ETH-2 library):
- the purified thiol-containing DNA oligonudotides are reacted with a molar excess of bismaleimido-hexane (Pierce, Belgium) in PBS + DMSO, following the manufacturer's instructions.
- the resulting derivatives are purified from unreacted bismaleimido-hexane using anion exchange chromatography, then reacted with slight molar excess of purified VH-cys or VL-cys, respectively, at a domain concentration > 0,1 mg/ml.
- the resulting (V domain)-DNA reaction products are separated from unreacted V-domain by anion exchange chromatography.
- the resulting bead preparation is then used as template for two separate PCR reactions, which amplify the (L19_5SH, HyHellO_5SH, GST_5SH) and (L19_3SH, HyHellO_3SH, GST_3SH) oligonucleotides (see above), using oligos:
- a sub-library "A” is created, by coupling n compounds to the 3' extremity of n different DNA oligonucleotides.
- n compounds to the 3' extremity of n different DNA oligonucleotides.
- a convenient one is represented by the coupling of iodoacetamido- or maleimido-derivatives of n chemical entities to individual DNA oligonucleotides, which carry a thiol group at the 3' end.
- the thiol-containing oligonucleotides can be purchased from commercial suppliers. Each of them contains a constant sequence portion
- DNA sequence portion XXXXX at the 5' end is (at least in part) different in each member of the sub-library A, therefore acting as a code.
- a sub-library "B” is created, by coupling m compounds to the 5' extremity of m different DNA oligonucleotides. Coupling of iodoacetamido- or maleimido-derivatives of m chemical entities to individual DNA oligonucleotides, which carry a thiol group at the 5' end, is performed similar to what described for sub-library "A".
- Such oligonucleotides can be purchased from commercial suppliers. Each of them contains a constant sequence portion
- the DNA sequence portion YYYYY at the 3' end is (at least in part) different in each member of the sub-library B, therefore acting as a code.
- Assembly of sub-library A members with sub-library B members is carried out by mixing the sub-libraries in PBS, heating the mixture at 70 degrees centigrade for 1 minute (if compatible with the stability of the chemical entities used in sub-library construction), followed by equilibration at room temperature.
- the resulting ESACHEL library contains n x m members, and can be used in biopanning experiments, followed by decoding of the binding members.
- the decoding strategy is based on the principle that, after biopanning of desired ESACHEL binding specificities, PCR fragments are generated, each of which carries the code of pairs of sub-library members, whose combination was rescued in the biopanning experiment, there- fore allowing the identification of the corresponding heterodimerized chemical entities.
- One pool of oligonucleotides carries the chemical entities at the 3'-end (pool A), whereas the other pool carries the chemical entity at the 5'-end (pool B).
- oligonucleotides of pool B allow the specific dimerization of any individual member of pool B with any in- dividual member of pool A.
- oligonucleotides from pool B contain a "code" region, which codes for the chemical entity at the 5'-end.
- Oligonucleotides of pool A contain a sufficient number of desoxyri- bose backbone elements without bases (d-Spacer), to prevent any undesired pairing to the bases of code B.
- Oligonucleotides of sub-library A have their distinctive code towards the 5' extremity.
- Oligonucleotides of sub-library B remain stably annealed to oligonucleotides of sub-library A, and can work as primers for a DNA polymerase reaction on the template A.
- the resulting DNA segment, carrying both code A and code B, can be amplified (typically by PCR), using primers which hybridize at the constant extremities of the DNA segment ( Figure 5).
- model oligonucleotides A and B which can be used for the generation of a PCR product, which carries both code A and B, is provided below:
- typeA_oligo and type_B oligo are mixed in approx. equimolar amounts.
- the resulting mixture is heated up to 70°C, and cooled to room temperature, allowing the heterodimerization of typeA_oligo and type_B oligo.
- the resulting mixture is mixed with a suitable buffer for PCR reaction, dNTPs (250 ⁇ M per nucleotide,
- affinity gain contribution of the chelate effect will depend on the length, rigidity, stereoelectronic chemical properties and stability of the linkage between the two (or more) chemical entities, in contact with the target antigen. Furthermore, the affinity gain will directly depend on the magnitude of the association and dissociation rate constants (k on and k off ) of the individual chemical entities, binding to the target.
- nl Both A and B bound to their binding pocket
- nil A bound to its binding pocket
- nlll B bound to its binding pocket
- nIV Both A and B not bound to their binding pockets
- the kinetic parameters k on A, k 0 ff A , k onB and k 0 ff B describing the binding properties of the individual chemical entities A and B to the corresponding binding pockets, are known. From these constants, it is possible to determine probabilities for a bound molecule to go off the binding pocket (poff), and for an unbound molecule to bind to its binding pocket (pon)
- the half life of binding can be expressed as:
- Equation [3] can be approximated to the probability that a molecule B binds to its pocket in the time increment ⁇ t.
- writeln ('molecules A and B are connected with a linker '); writeln;
- tl2B ln(2)/koffB
- rad: linkerA*le-10
- conc: l/((2/3*Pi*rad*rad*rad)*6.01e26);
- nl : nl + deltal
- nil : nll + deltall
- nIII: nIII + deltalll
- nIV : nIV + deltalV
- n: nl+nll+nlll
- ESACHEL technology can be used as follows, i.e. omitting the "code" oligonucleo- tide sequence from the binder to be optimized.
- a target molecule e.g., a protein
- the chemical moiety p will be coupled to the 5' end of oligonucleotide 5' - 5'- GGA GCT TCT GAA TTC TGT GTG CTG -3'. It will then be convenient to chemically couple, in individual reactions, many different chemical moieties q at the 3' end of oligonucleotides, of general sequence 5' - XX XX - Y - CAG
- Y represents a biotinylated base analogue; - the sequence 5' - CAG CAC ACA GAA TTC AGA AGC TCC - 3' will be identical in all cases, allowing the heteroduplex formation with the sequence 5' - GGA GCT TCT GAA TTC TGT GTG CTG - 3', chemically coupled to p, for all members of the ensamble of molecules q.
- the resulting library will pair p with molecules q, each of which bears a distinctive oligonucleotide code.
- the self-assembled library can be submitted to biopanning, under conditions of suitable stringency.
- the binders rescued at the end of the biopanning procedure will be identified by means of their code.
- the codes of the molecules q, which together with p give rise to high-affinity binders for the target molecule, can be read by hybridization to an oligonucleotide chip, in which the different positions are covered with oligonucleotides, which are complementary to the sequences XX XX of the members of the sub-library
- biotin moiety on members of sub-library Q will allow the detection of the binding events on the chip.
- Candidate chemical moieties q will then be chemically linked to p, and the resulting conjugate will be used as a specific binder for the target molecule of interest.
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AU2002360272A1 (en) * | 2001-10-10 | 2003-04-22 | Superarray Bioscience Corporation | Detecting targets by unique identifier nucleotide tags |
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Also Published As
Publication number | Publication date |
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US20060154246A1 (en) | 2006-07-13 |
KR100916889B1 (en) | 2009-09-09 |
CA2478203A1 (en) | 2003-09-18 |
US8673824B2 (en) | 2014-03-18 |
US20040014090A1 (en) | 2004-01-22 |
US9783843B2 (en) | 2017-10-10 |
EA200401107A1 (en) | 2005-04-28 |
DE60231436D1 (en) | 2009-04-16 |
US8642514B2 (en) | 2014-02-04 |
US20180245141A1 (en) | 2018-08-30 |
ATE424561T1 (en) | 2009-03-15 |
EP1483585B1 (en) | 2009-03-04 |
DK1483585T3 (en) | 2009-06-22 |
AU2002310846A1 (en) | 2003-09-22 |
CA2478203C (en) | 2011-06-14 |
KR20050002846A (en) | 2005-01-10 |
EA006965B1 (en) | 2006-06-30 |
US20140128290A1 (en) | 2014-05-08 |
ES2321067T3 (en) | 2009-06-02 |
AU2002310846B2 (en) | 2008-03-20 |
US20110319278A1 (en) | 2011-12-29 |
US10895019B2 (en) | 2021-01-19 |
US20100184611A1 (en) | 2010-07-22 |
HK1070427A1 (en) | 2005-06-17 |
EP1483585A1 (en) | 2004-12-08 |
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