WO1998041654A1 - In vitro evolution of oligonucleotides - Google Patents
In vitro evolution of oligonucleotides Download PDFInfo
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- WO1998041654A1 WO1998041654A1 PCT/IL1997/000283 IL9700283W WO9841654A1 WO 1998041654 A1 WO1998041654 A1 WO 1998041654A1 IL 9700283 W IL9700283 W IL 9700283W WO 9841654 A1 WO9841654 A1 WO 9841654A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C12N15/1048—SELEX
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
- C12N2310/124—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes based on group I or II introns
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
Definitions
- the present invention is generally in the field of in vitro evolution of oligonucleotides. More specifically, the present invention concerns a method wherein in vitro evolution is utilized to obtain oligonucleotides featuring a desired property, i.e. binding to a specific agent or having a certain desired catalytic activity.
- in vitro evolution refers to a method of generating and selecting nucleic acid sequences (which may be DNA or RNA sequences, or sequences comprising both dNTP's and rNTP's, comprising naturally or non-naturally occurring nucleotides) having desired characteristics, without a priori knowing the exact construct of the selected nucleic acid sequence. Typically, it entails production of a huge number of random, or partially random, nucleic acid sequences, then providing the conditions required for selection of those sequences which feature a specific property for example, adding a protein and selecting only those nucleic acid sequences which bind with some affinity to the protein.
- the selected nucleic acid sequences are then amplified, for example, by polymerase chain reaction (PCR), and the selection and amplification steps are repeated over many cycles, e.g. ranging from 10 to 100, resulting in an enrichment of the reaction mixture by those nucleic acid sequences which feature the desired property. It is at times useful to progressively raise the criteria for selection in each round of a selection and amplification. For example, as the steps of the in vitro evolution proceeds, only species having progressively higher affinity to the desired protein are selected.
- PCR polymerase chain reaction
- Ribozymes are typically RNA molecules which have enzyme-like catalytic activities usually associated with cleavage, splicing or ligation of nucleic acid sequences.
- RNA molecules The typical substrates for ribozymes catalytic activities are RNA molecules, although ribozymes may catalyze reaction in which DNA molecules) are the substrates.
- ribozymes There are many known ribozymes ranging with respect to the type of their catalytic activity, with respect to the co-factors which are needed for their catalytic activity as well as with respect to the substrates on which their activity is exerted.
- In vitro evolution is a useful method for preparing and selecting specific types of ribozymes.
- a method for preparing ribozymes which are active in the presence of an assayed agent, which serves as their co-factor, has been described in co-pending Israel Patent Application No. 119135.
- Such ribozymes which feature catalytic activity in the presence of an assayed agent, may at times also exert catalytic activity in the presence of a non-assayed agent although at a lower rate or lower affinity. Such non-specific activity may be undesirable for various applications of the ribozymes, e.g. where such ribozymes are used to diagnose the presence of the assayed agent in a medium wherein such nonspecific activity may give rise to false positive results.
- the potential incomplete specificity of nucleic acid sequence obtained by in vitro evolution which are presumed to be specific, is also a problem for nucleic acids with other specific properties, e.g. such where the specific activity is binding to a specific agent.
- Oligonucleotide a sequence of nucleotides. May be composed entirely of dNTP's, rNTP's or a combination of both and may comprise non-naturally occurring nucleotides such as 2'-0'-methyl nucleotides or a combination of the above.
- Oligonucleotide specie - a plurality of oligonucleotide molecules in a mixture featuring essentially the same nucleotide sequence and produced by the amplification step (see below). Each oligonucleotide species has a different sequence.
- Desired property - a biological property of the oligonucleotide, may be binding to a macromolecule or may be a catalytic activity (see below).
- SS conditions Selected set of conditions in which the desired property of the oligonucleotide should be evident.
- examples of such conditions are the presence of an assayed agent (see below), high or low temperature, high or low pH, high or low ionic strength and the like.
- the purpose of the in vitro evolution method is to produce oligonucleotides featuring the desired property only under SS conditions.
- Non-selected set of conditions non-SS conditions
- the purpose of the in vitro evolution method is to remove any oligonucleotides that featured the desired property under these non-selected set of conditions.
- Functional sequence a sequence of the oligonucleotide which imparts a desired property of the oligonucleotides (examples of desired properties are binding to a macromolecule, or a catalytic activity).
- Variable sequence - a sequence in the oligonucleotide which is a candidate for evolving into the functional sequence (after carrying out the in vitro evolution method of the invention).
- the in vitro evolution method of the invention is performed on an initial mixture comprising a plurality of different oligonucleotide sequences, each of which has a different variable sequence.
- the variable sequence can be prepared utilizing a DNA synthesizer and may be completely random or alternatively may be semi- random or "dopped", i.e. each sequence features X% identity to a sequence of a known dinozyme while (100- X)% of each sequence is random.
- the variable sequence may constitute a small part, a large part or at times essentially all of the oligonucleotide.
- Tag sequence a random sequence of oligonucleotides which is not part of said variable sequence and is added to the oligonucleotide for screening purposes only.
- the randomness of the nucleotide sequence of the tag sequence renders each tag to be dissimilar to a tag sequence of another species of molecules.
- the tag sequence and the variable sequence are linked via a cleavable sequence (see below).
- Cleavable sequence - a sequence in the oligonucleotide linking the tag and variable sequence capable of being cleaved, for example, by an enzyme (such as a restriction enzyme) or ribozyme.
- an enzyme such as a restriction enzyme
- ribozyme a sequence in the oligonucleotide linking the tag and variable sequence capable of being cleaved, for example, by an enzyme (such as a restriction enzyme) or ribozyme.
- Positive selection step - a step in the in vitro evolution method in which molecules featuring a specific property under the SS conditions are collected.
- Negative selection step - a step in the in vitro evolution method in which molecules featuring a desired property under non-selected SS conditions are removed.
- Amplification step - a step in the in vitro evolution method in which each oligonucleotide molecule is amplified several folds in order to obtain several copies of each molecule.
- the copies may be identical to the parent oligonucleotides, or may have some modifications which were inserted due to the amplification process (for example mutations inserted during the reverse transcription). This non-perfect accuracy of the amplification process is in fact an advantage in the in vitro evolution method.
- Examples of amplification techniques are PCR, NASBA, 3SR, SDA, etc.
- Hybridization under stringent conditions - hybridization under conditions which will give rise to hybridization in the case of homology of the two hybridizing nucleic acid sequences but will not permit hybridization in a case of even a single base mismatch between two sequences.
- Assayed agent - a molecule the presence of which is to be detected in the sample.
- molecules include, for example: proteins; peptides; oligopeptides; antibiotics; phosphate nucleotides such as ATP, GTP, cyclic AMP, and others; carbohydrates; lipids; nucleic acid sequences (DNA or RNA sequences); etc.
- the present invention is directed to a novel method for obtaining oligonucleotides which feature a desired property under a selected set of conditions, which makes use of in vitro evolution.
- the novelty of the present invention resides in a unique combination of positive and negative selection steps.
- oligonucleotides which feature the desired property under the selected set of conditions (the "SS conditions") are selected.
- the negative selection step involves removal of oligonucleotides which feature the desired property under conditions wherein the oligonucleotides are not intended to have their desired properties, i.e. under any condition which is different than the selected condition (the "non- SS conditions").
- the positive selection step will involve collecting oligonucleotides which feature the desired catalytic activity in the presence of the assayed agent (i.e. said SS conditions are the presence of the assayed agent).
- the negative selection step will involve removing oligonucleotides which feature the desired catalytic activity also in the presence of an agent other than said specific agent (i.e. said non-SS conditions are the presence of a non-assayed agent).
- the problem intended to be solved by the present invention is the difficulty in separating between oligonucleotides which feature the desired property only under the SS conditions (for example, only in the presence of a specific assayed agent) and those which feature the desired property both under the SS conditions, as well as under non-SS conditions (e.g. feature the desired property also in the presence of non-assayed agents).
- the molecules removed during the negative selection step may be collected, and may be used to "fish out", by hybridization, other identical molecules of the same species which should have been removed, but due to the short incubation time did not have a chance to feature the desired property, (for example, catalytic activity).
- sequences of oligonucleotides which feature catalytic activity both under SS and non-SS conditions (to be removed), and the sequences of the oligonucleotides which feature the desired property only under SS conditions (to be maintained), may be very similar, the difference in sequence being very small relative to the large size of the oligonucleotide, so that it would be practically impossible to distinguish between the two by hybridization.
- the solution to this problem in accordance with the invention is the addition of a random tag sequence to each of the oligonucleotides in the initial oligonucleotide mixture, so that after amplification all oligonucleotides of the same species (originating from the same parent oligonucleotide) have the same random tag sequence.
- This tag sequence does not form part of the functional sequence of the nucleotide which is the part which imparts the desired property.
- the tag sequence is linked to the variable sequence (the candidate for evolving to the functional sequence). While the variable sequence of different oligonucleotide species may be similar (for example since all variable sequences were "doped" from an original known sequence) the tag sequence is completely different from one oligonucleotide species to the other.
- oligonucleotides featuring the desired property under non-SS conditions are collected, it is possible to cleave the cleavable sequence, and thus collect the tag sequences of the undesired oligonucleotides.
- These tag sequences which are random, have a very high probability, depending on the size of the tag, of being different in each species of oligonucleotide molecules, and thus may be used to effectively "fish out” complementary tag sequences of other members of the undesired oligonucleotide species. Since the "fishing out", i.e.
- the present invention is suitable for the production of oligonucleotides which feature a desired property under SS conditions and do not feature the desired property under non- SS conditions, wherein the initial pool of oligonucleotides, on which the in vitro evolution takes place is already known to possess the desired property under SS conditions. All that is necessary, in accordance with said first embodiment is to remove from said initial pool of oligonucleotides those oligonucleotides which feature the desired property also under non- SS conditions.
- An example of the utilization of the first embodiment of the invention is when starting with a pool of oligonucleotides which are derived (for example by doping or by changing a specific region), from an oligonucleotide having a known desired property, for example, derived from a ribozyme having catalytic activity under SS conditions. It is assumed that the derived oligonucleotides in the original pool (which vary slightly from one another) already have catalytic activity under the SS conditions, so that all that is necessary is to remove from the pool those oligonucleotides having the catalytic activity also under non-SS conditions. Even if as a result that catalytic activity under SS conditions is lowered, this is still tolerable.
- the method comprises only negative selection steps.
- the third selected mixture of oligonucleotides can be amplified to obtain a plurality of copies thereof. It is possible to add an amplification step to amplify the oligonucleotides of the first selected mixture. Such an amplification step may yield both oligonucleotides with a sense sequence ("sense oligonucleotides”) as well as oligonucleotides having a complementary, anti- sense sequence ("an -sense oligonucleotides").
- the amplification of the second group of oligonucleotides may also yield both groups of oligonucleotides (sense oligonucleotides and anti-sense oligonucleotides) and similarly, the collected tag sequences (step (d)) will comprise of both sense tag sequence and anti-sense tag sequences. If the first selected mixture has not been amplified, the hybridization will be between the anti-sense tag sequences and the tag sequences in the original sense oligonucleotides.
- both sense tag sequences and anti-sense tag sequences may be hybridized to complementary sequences in the oligonucleotides of the amplified first selected mixture (to anti-sense and sense oligonucleotides, respectively).
- the tag sequence may be a terminal sequence of the oligonucleotides, or the tag sequence may be a non-terminal sequence, constructed in a mid portion of the oligonucleotide.
- the oligonucleotides featuring the desired properties only under SS conditions and not under non-SS conditions are evolved from a pool of oligo- nucleotides which are not known, a priori, to have the desired property and therefore should be screened also by positive selection steps.
- a positive selection step comprising applying said selected set of conditions and separating between the oligonucleotides which feature said desired property and those which do not, to obtain a first selected mixture comprising a first group of oligonucleotides featuring said desired property under said selected set of conditions;
- an amplification step comprising amplifying said first group of oligonucleotides in said first selected mixture to produce a plurality of copies of each of said first group of oligonucleotides to obtain a first amplified mixture;
- a negative selection step comprising: (bca) applying a second set of conditions, being different than said selected set of conditions, and separating between a second group of oligonucleotides which do not feature the desired property under said second set of condi- tions and a third group of oligonucleotides featuring said desired property under said second set of conditions, to obtain a second selected mixture comprising said second group of oligonucleotides and a third selected mixture comprising said third group of oligonucleotides; (bcb) amplifying said third group of oligonucleotides in said third selected mixture to produce a plurality of copies of each of said third group of oligonucleotides to obtain a second amplified mixture;
- the collected tag sequences which are contacted with the second selected mixture may comprise, similarly as pointed out above, of both sense and anti-sense tag sequences which then hybridize to anti- sense or sense oligonucleotides respectively, of the second selected mixture.
- Anti-sense oligonucleotides may be present in the second selected mixture, again similarly as above, following an optional amplification step of the second selected mixture.
- the positive selection step should precede the negative selection step. It is preferable that in the positive selection step of each consecutive cycle, the conditions become more and more stringent, for example, shorter assay times, harsher sample preparation conditions etc.; while in the negative selection step conditions become less stringent allowing even those oligonucleotides with a very small activity in the conditions of the negative selection step to exert their activity (and thus to be removed), for example by utilizing longer incubation times, conditions which more closely resemble those in the positive selection step, etc. Where the negative selection step is repeated a plurality of times, each step or several steps may be performed under a different set of non-SS conditions. For example, where said SS conditions include the presence in the medium of a certain protein, the non-SS conditions which are applied in the negative selection steps may be the presence of different proteins, each one of a plurality of different proteins being present in a different negative selection step.
- a specific example for said desired property is catalytic activity, similar to catalytic activity featured by ribozymes.
- Another desired property is binding of the oligonucleotide to a protein for the preparation of aptamers.
- the SS conditions may for example be temperature, ionic strength, acidity, and preferably is the presence of a certain agent in the medium. The latter is useful particularly for diagnostic purposes.
- Said assayed agent may for example be a nucleic acid sequence, a protein or any other macromolecule.
- the variable sequence may be random but in accordance with a preferred embodiment of the invention, the variable sequence is doped, i.e. having a certain degree of similarity (for example 70%) to a sequence having a known desired property, for example, to the sequence of a ribozyme.
- Separation between various groups of oligonucleotides for example between those having a catalytic activity in the SS conditions and those not having such catalytic activity may be carried out by any of a variety of known ways.
- the separation may be based on separating between soluble and immobilized oligonucleotides.
- the catalytic activity is for example cleavage
- all oligonucleotides in the initial mixture of different oligonucleotides can be, a priori, immobilized onto a solid support and those which are catalytically active and are able to perform cleavage in cis are then released into the medium.
- oligonucleotides which, when catalytically active are able to ligate to a substrate oligonucleotide which is immobilized on a solid support.
- active oligonucleotides will become immobilized, and inactive oligonucleotides will remain soluble.
- Aptamers can be separated utilizing their binding properties to immobilized substrates, such as to immobilized antibodies.
- Separation between oligonucleotides that hybridize to the collected tag sequences and those which do not, can be performed, for example, by immobilizing the tag sequences on a solid support and then separating between the immobilized and unimmobilized oligonucleotides.
- the separation may be based on the some designed properties of the tag sequences. For example, it is possible to construct a tag sequence which is partially random but has a site, which when double-stranded, forms a restriction site.
- a restriction enzyme is added and then only the oligonucleotides which hybridize with a tag sequence (the hybridization is either between an anti- sense oligonucleotide to a sense tag sequence or between a sense oligonucleotide and an anti-sense tag sequence) will have a restriction site and will be restricted by the enzyme. Cleavage of these oligonucleotides will avoid their amplification in subsequent steps since the primer sequences at both ends of the oligonucleotides have been separated by the cleavage.
- the tag sequences which are collected in the negative selection step are complementary to the tag sequences in the mixture from which oligonucleotides should be "fished out".
- the complementary tag sequence used for fishing out is preferably a DNA sequence.
- the oligonucleotide to be fished out is a DNA oligonucleotide, e.g. being a catalytic DNA molecule or an aptamer
- the complementary tag sequence used for fishing out of such oligonucleotides is an RNA sequence.
- a specific utility of the method of the invention is in preparing ribozymes which are capable of featuring their catalytic activity only when they have a complete structure (hereinafter: “full form "), and cannot feature the activity when nicked (i.e. the sequence is non-continuous), when having a missing portion or when attached to a redundant sequence (which is a sequence that should be removed in order to render the ribozyme active) (hereinafter: “incomplete form ").
- Such ribozymes can be used as part of a ribozyme amplification cascade, for example, as that described in PCT Application No. PCT/US96/02380 and corresponding Israel Patent Applications Nos. 112799 and 115772 the context of which are being incorporated herein by reference.
- a first, initiation ribozyme once a first, initiation ribozyme has been produced, for example due to the presence of an assayed agent in the medium, it can activate other, initially inactive ribozymes, which in turn activate further inactive ribozymes, in a self-amplificatory positive feed-back manner, which produces in a very short time an easily detectable signal.
- the initially inactive ribozyme is either nicked, has a missing portion or comprises a nick and a redundant sequence, and only upon ligation, gap filling or splicing, respectively, it becomes catalytically active, and may in turn activate other ribozymes in the amplification cascade.
- the invention provides in accordance with this embodiment an in vitro evolution method for obtaining oligonucleotides which feature a catalytic activity, but which do not feature the catalytic activity when being defective by having a nick or missing a portion in their sequence, the method comprising: (a) preparing a mixture of different oligonucleotides each of which comprises a tag sequence and at least one variable sequence, the variable sequence being a candidate sequence for evolving into a sequence which together with a conserved sequence, identical in all oligonucleotides in the mixture, will feature the catalytic activity; the variable sequence and the tag sequence being different in different oligonucleotides in the mixture, the variable sequence and the tag sequence being linked by at least one cleavable sequence;
- step (b) processing the oligonucleotide mixture of step (a), in two different manners so as to give rise, together with an oligonucleotide which comprise said conserved sequence, two oligonucleotide constructs, for each of the oligonucleotides in said mixture, the constructs consisting of:
- step (ba) a first oligonucleotide construct comprising the oligonucleotide obtained in step (a) or a transcription product thereof and an oligonucleotide comprising said conserved sequence, the construct being incomplete by having a nick or a missing portion, and
- a second oligonucleotide construct comprising the oligonucleotides of step (a) or a transcription product thereof and an oligonucleotide comprising said conserved sequence without a nick or a missing portion;
- a negative selection step comprising: (ca) contacting said first oligonucleotide constructs with a substrate for catalytic activity under conditions enabling said catalytic activity and removing oligonucleotide constructs which do not feature said catalytic activity thereby obtaining a first selected mixture; (cb) amplifying said first selected mixture to obtain a first amplified mixture comprising a plurality of copies of each oligonucleotide constructs;
- step (e) amplifying said second selected mixture to obtain a second amplified mixture comprising a plurality of copies of each oligonucleotide construct; (f) contacting the collected tag sequences obtained from step (cc) with the second amplified mixture under stringent conditions of hybridization and removing hybridized oligonucleotide constructs from other oligonucleotide constructs of the amplified selected mixture, thereby obtaining a third selected mixture of oligonucleotide constructs featuring said catalytic activity and being essentially devoid of oligonucleotides which feature catalytic activity when present in nicked form, when missing a portion, or containing a nick together with a redundant portion.
- the catalytic activity of the oligonucleotides according to this specific embodiment may be either ligation, splicing, cleavage, gap filling, kinase-like activity, biotynilation and any other catalytic activity.
- Fig. 1 shows a schematic representation of the method of the invention for preparing an oligonucleotide which features catalytic activity only in the presence of an assayed agent (Fig. 1 A shows the initial steps of the method and Fig. IB subsequent steps); and
- Fig. 2 shows a schematic representation of a method for preparing oligonucleotides which feature catalytic activity only when present in a full, i.e. non-defective form.
- DNA sequences are represented by a straight line and RNA sequences by a wavy line.
- Two complementary sequences are marked by the same letters one of them also marked with an apostrophe ('"").
- Example 1 In vitro evolution where the selected condition is the presence of an assayed agent i. Preparing random panel of nucleic acid sequences (1)
- a panel of DNA oligonucleotides is prepared on a standard nucleic acid synthesizer using a program for generating desired sequences.
- a typical oligonucleotide is shown in Fig. 1, (1) and comprises a promoter (P) of about 20 bases, a substrate sequence S which can be cleaved in cis by a catalytically active oligonucleotide, a first primer for PCR amplification (PCR1), a first restriction site C x of about 4-8 bases, a random tag (TAG) sequence of about 15 bases, a second restriction site C 2 of about 4-8 bases (both and C 2 sequences are constant and not random), a variable sequence (V) of 50-8000 b.p. averaging at 100, the variable sequence being a candidate for evolving to a functional sequence and a second PCR primer sequence (PCR2).
- P promoter
- substrate sequence S which can be cleaved in cis by a catalytically active oligonucleotide
- PCR1 first primer for PCR amplification
- TAG random tag
- V variable sequence
- V variable sequence of 50-8000 b.
- variable sequence may be a sequence derived from a sequence known to have the desired function, e.g. catalytic activity. For example, it may be a sequence obtained by doping the known sequence, at a certain degree of randomness, with other nucleic acid residues than in the original sequence. If the variable sequence is obtained by doping of a functional sequence, there may be some degree of similarity in the variable sequence between different oligonucleotides in a mixture of such oligonucleotides.
- DNA sequences of i. above are transcribed, using a primer with biotin (B) at the 5' end (2).
- the biotin is then allowed to react with avidin which is present on a solid support, such as Strepravidin beads (SA), so that each oligonucleotide of the panel becomes immobilized onto a solid support (3).
- SA Strepravidin beads
- the specific assayed agent which is a protein (Pro) is then added to the reaction mixture (4(a)).
- Magnesium and/or other co-factors required for catalytic activity are then added to the reaction mixture in order to allow for the catalytic cis cleavage activity of the variable sequences of the oligonucleotides on substrate sequence S.
- oligonucleotide sequences in the medium may be divided into three classes, as shown (4(b)):
- the last step of the positive selection comprises collecting of those oligonucleotides which were freed from the immobilized beads (Class i and Class ii).
- protein attached to the oligonucleotides can be removed by denaturation through heating or by phenol extraction followed by chloroform extraction.
- step 4 Sequences collected in step 4 above are reversed transcribed and amplified by PCR. Since substrate sequence S was cleaved (step 4b) this sequence should be reconstructed by using, both in the case of reverse transcription and of PCR, primers which contain the sequence of the cleaved substrate and thus the amplified and reconstructed product is again identical to (1).
- the amplified products are transcribed, bound to biotin and attached to a solid support as specified above (step 6).
- the amplified and immobilized oligonucleotides of step 6 are subjected to a negative selection step.
- the catalytic activity is determined either with no assayed agent, or in the presence of a non- assayed agent, which, although not identical, closely resembles the assayed agent (preferably such a non-assay agent may be one which can be encountered by the oligonucleotide in the assayed conditions); the non- assayed agent illustrated here is a protein - Pro' (step 7a); magnesium and/or other co-factors required for catalytic activity are added (step 7b).
- the oligonucleotides released to the medium by cleavage of the substrate sequence S belong to three classes:
- Both Classes i. and ii. are collected (step 7c) and the protein is removed (step 7d).
- the sequences are reversed transcribed and amplified by PCR in order to produce a double-stranded construct (step 8).
- Suitable restriction enzymes are added in order to cleave sequences and C 2 , one cleavage site forming a blunt end and one a sticky end (step 9).
- the sticky end is completed with a biotinated nucleotide and then the separated TAG sequence is immobilized on a solid support (through an immobilized avidin) and denaturated so that there remains a single-stranded denaturated immobilized tag sequence.
- the immobilized tag sequences of step 10 are brought into contact with oligonucleotides collected of step 4(b) (Class i and Class ii) (step 11). Molecules of said group which hybridize with the immobilized TAG sequence are removed (step 12) so there remain only molecules which feature cleavage in the presence of an assayed agent (Pro) and do not feature cleavage activity without any agent or in the presence of a non- assayed agent (Pro').
- oligonucleotides obtained in step 11 are subjected again to all preceding steps for 2-1000 cycles, preferably 10-100 cycles, most preferably 20-30 cycles.
- Fig. 2 shows another embodiment of the in vitro evolution method of the invention. The purpose of the method disclosed in Fig. 2 is to produce oligonucleotides which are devoid of catalytic activity in an incomplete form and are capable of catalytic activity only when in a complete form.
- An oligonucleotide which is in an incomplete form includes an oligonucleotide with a nick in its sequence, an oligonucleotide with a missing nucleotide segment, an oligonucleotide with a nick and with a redundant sequence, etc.
- a mixture of different DNA sequences 1 is prepared (step a) featuring (from 5' ⁇ 3'): immobilization moiety (such as a bead), linked to a promoter (PROM), a first PCR primer (PCR1'), a variable sequence V,', a cleavage sequence Cj, tag sequence (TAG), a cleavable sequence C 2 , another variable sequence V 2 ' and a second PCR primer (PCR2').
- V x ' and V 2 ' are both sequences which are complementary to doped sequences from an original Group I ribozyme (hereinafter "known ribozyme") and comprise together only a part (for example, half) complementary to that known ribozyme.
- the TAG sequence between V, and V 2 ' is present within a sequence coding for an intron of the known ribozyme and thus does not interfere with the activity of the transcript.
- DNA sequence 1 is processed by two routes: First by Route I (left) which brings about production of nicked oligonucleotides for negative selection purposes and second by Route II (right) which brings to production of complete oligonucleotides.
- RNA oligonucleotide 3 comprising a sequence denoted "CONS " which is a conserved sequence of the known ribozyme required to complete the doped parts V x and V 2 (transcribed from V/ and V 2 ') to a complete ribozyme, (so that V x +V 2 +CONS constitute together a construct resembling that of the known ribosome wherein the 5 '-end part (V,+V 2 ) is doped (as compared to a known ribozyme) and the 3'-end part (CONS) is identical to a known ribozyme).
- CONS a conserved sequence of the known ribozyme required to complete the doped parts V x and V 2 (transcribed from V/ and V 2 ') to a complete ribozyme, (so that V x +V 2 +CONS constitute together a construct resembling that of the known ribosome wherein the 5 '-end part
- Transcript 2 and oligonucleotide 3 hybridize to form together construct 4, (step c) which is essentially similar to the known ribozyme (with doped V, and V 2 sequences and conserved CONS) but having a nick 5.
- the purpose of the method of the invention according to this embodiment is to remove all species of oligonucleotides which feature catalytic activity despite having a nick - i.e. to remove all species represented by construct 4.
- Oligonucleotides which serve as substrates for ligation and which are immobilized to a solid support 6 are then added to construct 4, and the two are allowed to ligate (step d).
- the construct 4 fall into two classes (step e): Class (1) constructs which were able to ligate to the immobilized substrate and are thus collected and Class (2) constructs which were not able to ligate, remain soluble and are thus removed. Separation can also be carried out, mutatis mutandis, utilizing cleavage activity, gap filling, splicing activities, etc.
- the collected constructs are reverse transcribed and amplified utilizing PCR primers PCR1 and PCR2 (the CONS sequence is thus not amplified) and the TAG sequence are removed therefrom, for example by restriction enzymes (step f).
- the TAG is then immobilized to a solid support to yield immobilized tag 11, which will be used to "fish out” species which should be removed (step g).
- step (f) of route II (which is identical to DNA sequence 1) or the DNA sequence 1 itself is brought into contact with DNA oligonucleotide 7 having PCR2 sequence complementary to PCR2', CONS' of coding for a conserved sequence as described above and PCR3 sequence which complements to a third PCR primer (step h).
- the two are allowed to hybridize and in the presence of DNA polymerase and nucleotides give rise to double-stranded DNA construct 8 (step i) which in the presence of T 7 polymerase brings to the production of transcript 9 (step j).
- Transcript 9 is in essence identical to construct 4 but it does not have a nick.
- Oligonucleotide substrate 6 is then added. Those transcripts 9 which were able to ligate to substrate 6 are maintained (class 1) and those which were not able to are removed (class 2) (step k). The maintained transcripts are reverse transcribed and PCR amplified to give transcripts 10 (step 1).
- Transcripts 10 are contacted with immobilized TAG 11 obtained in step g in a negative selection step (step m). Those transcripts 10 which hybridize with the TAG are then removed (step n).
- step m a negative selection step which removes oligonucleotides which are catalytically active when present with a nick and a positive selection step which selects for oligonucleotides showing catalytic activity without a nick.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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AU38621/97A AU3862197A (en) | 1997-03-17 | 1997-08-26 | (in vitro) evolution of oligonucleotides |
JP54028898A JP2001515358A (en) | 1997-03-17 | 1997-08-26 | In vitro evolution of oligonucleotides |
EP97935737A EP0970241A1 (en) | 1997-03-17 | 1997-08-26 | (in vitro) evolution of oligonucleotides |
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IL12046697A IL120466A0 (en) | 1997-03-17 | 1997-03-17 | In vitro evolution of oligonucleotides |
IL120466 | 1997-03-17 |
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WO1998041654A1 true WO1998041654A1 (en) | 1998-09-24 |
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PCT/IL1997/000283 WO1998041654A1 (en) | 1997-03-17 | 1997-08-26 | In vitro evolution of oligonucleotides |
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EP (1) | EP0970241A1 (en) |
JP (1) | JP2001515358A (en) |
AU (1) | AU3862197A (en) |
IL (1) | IL120466A0 (en) |
WO (1) | WO1998041654A1 (en) |
Cited By (1)
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JP2011206056A (en) * | 1999-01-07 | 2011-10-20 | Medical Research Council | Optically sorting method |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994009158A1 (en) * | 1992-10-14 | 1994-04-28 | University Research Corporation | Method for selecting nucleic acids on the basis of structure |
WO1996027026A1 (en) * | 1995-02-27 | 1996-09-06 | Intelligene Ltd. | Detection of biomolecules |
-
1997
- 1997-03-17 IL IL12046697A patent/IL120466A0/en unknown
- 1997-08-26 JP JP54028898A patent/JP2001515358A/en active Pending
- 1997-08-26 EP EP97935737A patent/EP0970241A1/en not_active Withdrawn
- 1997-08-26 AU AU38621/97A patent/AU3862197A/en not_active Abandoned
- 1997-08-26 WO PCT/IL1997/000283 patent/WO1998041654A1/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994009158A1 (en) * | 1992-10-14 | 1994-04-28 | University Research Corporation | Method for selecting nucleic acids on the basis of structure |
WO1996027026A1 (en) * | 1995-02-27 | 1996-09-06 | Intelligene Ltd. | Detection of biomolecules |
Non-Patent Citations (1)
Title |
---|
KLUG S J ET AL: "ALL YOU WANTED TO KNOW ABOUT SELEX", MOLECULAR BIOLOGY REPORT, vol. 20, no. 2, 1994, pages 97 - 107, XP000617781 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011206056A (en) * | 1999-01-07 | 2011-10-20 | Medical Research Council | Optically sorting method |
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JP2001515358A (en) | 2001-09-18 |
AU3862197A (en) | 1998-10-12 |
EP0970241A1 (en) | 2000-01-12 |
IL120466A0 (en) | 1997-07-13 |
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