Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-04-29
2003-08-12
McGarry, Sean (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100
Reexamination Certificate
active
06605713
ABSTRACT:
The present invention relates to methods for identifying and producing L-nucleic acids that interact with a target molecule having a natural configuration, as well as to the L-nucleic acids produced by means of this method. Furthermore, the invention relates to the use of the D-nucleic acids binding to the optical antipode of the target molecule as a matrix for producing L-nucleic acids with an identical sequence, as well as to pharmaceutical compositions, kits, diagnostic agents and sensor systems containing the L-nucleic acids of the invention.
In the past few years, new technologies have been established in order to use nucleic acids in a manner previously unanticipated. Among these are, e.g., the use of such molecules as catalysts, inhibitors or stimulators of biochemical reactions that take place within or outside of the cell. There is hardly any doubt that these technologies will in the future play a dominant role in the fields of medicine, pharmaceutical diagnostics, biotechnology and agriculture.
An essential part of some of the new DNA and RNA techniques is in vitro selection or evolution (cf. for example the review article of J. W. Szostak, TIBS 17 (1992), 89 to 93, Famulok and Szostak, Angew. Chemie 104 (1992), 1001-1011 and Gold et al., Annu. Rev. Biochem. (1995)). These techniques are based on the working methods of biological systems. Thereby, novel DNA and RNA molecules with desired properties may be obtained from a combinatorial library of heterogeneous nucleic molecules by means of variation, selection and replication. Thus, such an in vitro system contains all factors present in biological evolution. It may rightfully be referred to as an in vitro evolution which many times accelerates the speed of natural methods. As long as no additional variation steps occur in this system, it is no in vitro evolution, but merely an in vitro selection method.
From a group of up to 10
18
different RNA species, the RNA molecules with highly affine binding properties or catalytic properties may be isolated by means of a cyclic process of the polymerase chain reaction (PCR), transcription, selective binding and reverse transcription; cf. Gold et al., loc. cit. One of the essential advantages of this method is the fact that it is not necessary to know the structure of the molecule to be selected; molecules with the “correct” structure are filtered out from the starting population by means of a selection step and, if desired, they may subsequently be sequenced. The principle of this selection or evolution process is schematically depicted in FIG.
1
.
Examples for such in vitro selection or evolution methods have been provided by Tuerk and Gold, Science 249 (1990), 505-510, Berzal-Herranz et al., Genes & Development 6 (1992), 129-134 and by Robertson and Joyce, Nature 344 (1990), 467-468. These work groups successfully selected functional ribonucleic acids cleaving or binding a predetermined nucleic acid differing from the substrate by means of slightly varying experimental approaches. In a more recent study, Lehmann and Joyce have shown that a ribozyme's metal ion specificity may be changed from Mg2+ to Ca2+ by such an in vitro evolution process (Nature 361 (1993), 182-185).
Highly affine RNA, but also DNA, molecules may not only be constructed for the purpose of interaction with other nucleic acids, but primarily for their interacting with proteins, with other, smaller molecules of the cell or with synthetic compounds. Attempts may also be made for interactions with the cellular receptors or with viral particles. Usually, such interactions of highly affine nucleic acids have the purpose of inhibiting or stimulating a biological function or of prompting a signal in sensor systems.
The advantage of nucleic acid libraries when compared to combinatorial libraries of other oligomers or polymers may be found in the dual nature of nucleic acids. The molecules possess a genotype (a sequence capable of propagation) and a phenotype (a functional structure). This enables the amplification of functional molecules from very large combinatorial libraries and their identification by means of sequencing. The additional labeling of the molecule library in order to identify functional variants e.g. by “tagging” (Janda, Proc. Natl. Acad. Sci. USA 91 (1994), 10779-10785) and the technical problems associated therewith (Gold et al., loc. cit.; Gold, J. Biol. Chem. 270 (1995), 13581-13584) may be avoided. By using combinatorial phage libraries in order to identify peptide motifs (Scott and Smith, Science 249 (1990), 386-390, Devlin et al., Proc. Natl. Acad. Sci. USA (1990), 6378-6382), which also involves the combination of genotype and phenotype, other disadvantages occur. Whereas oligonucleotides with only 25 nucleotides may already form very stable structures, comparable oligopeptides possess large conformational liberties (Gold et al., loc. cit.). The structural liberty of peptides and the entropic disadvantages in the interaction with target structures resulting therefrom limit the possibilities of using peptides, as long as high affinities and specificities are necessary for the application of the molecules. This limitations also occur in the selection of biologically stable D-peptides by means of phage libraries (Schumacher et al., Science 271 (1996); 1854-1857). The use of cyclic peptides may not offset these basic disadvantages (Gold et al., loc. cit.).
The particular disadvantage in using combinatorial nucleic acid libraries instead of other oligomers or polymers is the low stability of nucleic acids in biological liquids.
However, all selection and evolution processes known so far are only capable of producing highly affine or catalytic RNAs or DNAs in natural form, i.e. with D-ribose or D-deoxyribose as a basic component. During use in a biological environment these molecules are degraded by enzymes. The degradation leads to a short term of effect of these highly affine or catalytic nucleic acids.
Although it is possible after the selection of unmodified nucleic acids to introduce a targeted modification in order to slow down the enzymatic degradation, the influence of this modification on the structure and thereby on the functionality of the nucleic acids may, however, not be predicted. Furthermore, altered, undesired properties cannot be anticipated. In addition, the degradation of chemically modified DNAs or RNAs leads to products that may influence the cell metabolism in a serious and disadvantageous manner in the form of analogues of nucleosides, nucleotides or oligonucleotides.
It is furthermore possible to integrate into the process modified nucleoside triphosphates which increase the stability of the nucleic acids. Examples for this procedure have been described by Jellinek et al., Biochemistry 34 (1995), 11363-11372. and Eaton and Pieken, Annu. Rev. Biochem. 64 (1995), 837-863. As the nucleoside triphosphates must be compatible to the polymerases used, the range of possible modifications is very limited. Furthermore, it has to be expected that the degradation of these modified nucleic acids leads to particularly toxic effects.
Thus, the technical problem underlying the present invention was to provide processes, by means of which highly affine nucleic acid molecules can be produced via in vitro selection or evolution, which do not exhibit the above-described disadvantages mentioned in the prior art. This technical problem is solved by the embodiments characterized in the claims. Thus, the present invention relates to a process for producing L-nucleic acids interacting with a target molecule having natural configuration, said process comprising the following steps:
(a) producing a heterogeneous population of D-nucleic acids;
(b) bringing the population mentioned in step (a) into contact with the optical antipode of the target molecule;
(c) separating the D-nucleic acids interacting with the optical antipode of the target molecule;
(d) sequencing the D-nucleic acids interacting with the optical antipode of the target molecule;
(e) synthesizing L-nucleic acids, th
Bald Rolf
Erdmann Volker A.
Fürste Jens Peter
Epps-Ford Janet L.
McGarry Sean
Pillsbury & Winthrop LLP
Wetherell, Jr. John R.
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