Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-13
2003-11-04
Wang, Andrew (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S006120, C435S007100, C435S007600, C435S091500, C435S091500, C435S091500, C435S091500
Reexamination Certificate
active
06642014
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a library of catalysts of interest coupled to a substrate by an exchangeable linker pair, X and Y, and a selection method that uses multiple catalytic turnover events to isolate the more active of the catalysts in said library.
BACKGROUND OF THE INVENTION
In the past, novel biopolymer (i.e. DNA, RNA or polypeptide) based catalysts have been created in several different ways. The following paragraphs describe some of these selection schemes.
(1). Binding to Transition State Analogs
Catalytic RNA, DNA and protein (particularly antibodies) have been isolated by this approach. It has mostly been applied to the isolation of catalytic antibodies by immunization of mice with transition state analogs (TSA), also antibodies displayed on phage as well as RNA and DNA libraries have been challenged with TSA. The idea is that a molecule (protein, RNA or DNA) that binds a given TSA is likely to bind the substrate and stabilize the geometry and/or energetics of the transition state. This may result in catalysis.
The method does not select for catalytic activity per se, but rather for binding to a transition state analog (TSA). However, it has been included here as it is currently one of the most used methods to isolate novel catalysts. Problems encountered with this approach include: i) Detailed mechanistic knowledge of the target reaction is required (in order to design an appropriate TSA); ii) In many cases a TSA that adequately resembles the transition state is unobtainable or unstable; iii) It is not possible to mimic the structural and electronic dynamics of the reaction coordinate.
Consequently, a rather limited set of reaction types have been successfully targeted by this approach. In most cases the isolated catalysts have poor turn-over numbers.
(2). Functional Tagging of Active Catalysts
This selection scheme has been applied to protein and nucleic acids. The substrate is designed so that a reactive product is formed during the reaction (the substrate is called “suicide substrate” or “inhibitor analog”). The reactive product is likely to react with the catalyst that produced it, to form a covalent bond. As a result, active catalysts can be separated from inactive ones by way of the attached label. Catalytic antibodies displayed on phage have been isolated by this method, and it was shown in a model system that catalytically active and inactive proteins could be separated using this approach. The method should allow the isolation of rare catalysts.
Important limitations with this approach include: i) For many reactions it is not possible to design an appropriate suicide substrate. ii) Successful catalysts need only perform one turn-over during the selective process/round, which is typically on the order of minutes. Hence, there is no selective advantage for efficient catalysts.
(3). Continuous Evolution (RNA)
RNA libraries have been designed that contain both the substrate and the potentially catalytic domain in the same molecule. RNAs capable of performing the desired reaction (typically ligation) will “activate” themselves for amplification (reverse transcription followed by RNA polymerase transcription). By adequate dilutions and additions of nucleotide precursors this continuous selection can be maintained over several hours, and then analyzed.
The method has two important limitations: i) Both the substrate and the catalyst must be a nucleic acid; ii) As the catalyzed reaction and the amplification of successful enzymes is not separated, the time of the selective step is the sum of the turn-over time of the target reaction and the time of amplification of the “activated” molecules. Thus, as the amplification is on the order of seconds, there is no selective advantage for an efficient catalyst.
(4). Substrate-Enzyme-Linked Selection (SELS)
Recently, methods have been described, involving the attachment of the substrate of the target reaction to a protein with potential catalytic activity towards the attached substrate (Pedersen et al., Proc. Natl. Acad. Sci., US, 1998, vol. 95, pp. 10523-10528; Jestin et al., 1999, Angew. Chem. Int. Ed., vol. 38, pp. 1124-1127; Demartis et al., 1999, JMB, vol. 286, pp. 617-633; Neri et al., 1997, WO 97/40141). Upon intramolecular conversion of the substrate, the active catalyst can be isolated by means of the attached product.
This scheme is very general. However, since successful catalysts need only perform one turn-over during the selective process/round, which is typically in the order of minutes, there is no selective advantage for efficient catalysts. For the same reason, it presumably is not possible to distinguish enzymes with slightly different specific activity with this selection scheme.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide a method for in vitro selection, from a library of catalyst molecules, of a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest, as compared to the rest of the catalyst molecules within said library, and wherein said in vitro selection method is characterised by that it allows multiple catalytic activity turn-overs (i.e. substrate to product catalytic activity turn-overs), by the catalyst molecule of interest, before it is finally collected.
The solution is based on using a novel sample comprising a number of individual units in said in vitro selection method.
A summary of the characteristics of said novel sample is given immediately below.
Said novel sample comprises a library of catalyst molecules provided in the form of individual units, wherein the individual units comprise a first type individual unit having the following general structure:
C—XY—S,
wherein C denotes a catalyst molecule, XY an XY exchange pair, and S a substrate which is capable of being catalysed into a product by at least one catalyst comprised within said library of catalyst molecules and thereby providing the possibility of obtaining a second type individual unit comprising the general structure:
C—XY—P,
wherein C and XY has the meaning defined above and P is the product molecule resulting from the catalytic conversion of the substrate S of the first type individual unit. See
FIG. 1
for a graphic illustration of a suitable example of such an individual unit.
Said novel sample, is then characterised by that it comprises following functionally defined features:
Feature 1:
The substrate S is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit; and
the nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product molecule.
For illustration reference is made to
FIG. 1
, where is shown a suitable example of such an individual unit comprising feature 1 above.
Feature 2:
Said sample comprising a number of individual units and comprising feature 1 above is further characterised by that said XY exchange pair allows an asymmetric exchange of the Y-moiety with another Y-moiety (i.e. Y exchanges with Y, not with X); whereby
said XY exchange pair then allows an exchange reaction between the unit structure:
a catalyst—an XY exchange pair—a product and a “Y—substrate” component
and thereby generating the unit structure
a catalyst—an XY exchange pair—a substrate.
For illustration reference is made to
FIG. 2
, where is shown a suitable example of such an individual unit comprising feature 2 above.
Using such a novel sample in a in vitro selection method as described herein (vide infra), provides then the possibility of selecting a catalyst molecule of interest essentially only based on a characteristic of the product molecule which has been generated by the catalyst molecule of interest (Feature 1 allows this; see
FIG. 1
for an illustration);
and it further pr
Hölder Swen
Kjems Jørgen
Lund Mette
Pedersen Henrik
Friend Tomas
Garbell Jason
Lambiris Elias
Novozymes A/S
Wang Andrew
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