WO1999014355A2 - Molecules hautement affines d'acide nucleinique, production et application - Google Patents

Molecules hautement affines d'acide nucleinique, production et application Download PDF

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Publication number
WO1999014355A2
WO1999014355A2 PCT/DE1998/002813 DE9802813W WO9914355A2 WO 1999014355 A2 WO1999014355 A2 WO 1999014355A2 DE 9802813 W DE9802813 W DE 9802813W WO 9914355 A2 WO9914355 A2 WO 9914355A2
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Prior art keywords
nucleic acid
acid molecules
molecules
functional groups
nucleotides
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PCT/DE1998/002813
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German (de)
English (en)
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WO1999014355A3 (fr
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Stefan WÖLFL
Marian Kujau
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Hans-Knöll-Institut für Naturstoff-Forschung e.V.
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Publication of WO1999014355A2 publication Critical patent/WO1999014355A2/fr
Publication of WO1999014355A3 publication Critical patent/WO1999014355A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions

Definitions

  • the invention relates to a method for inserting functional groups into combinatorial nucleic acid libraries.
  • the nucleic acid molecules modified in this way thereby obtain additional properties which facilitate the recognition of specific target molecules or complex molecular target structures.
  • This new modification enables in particular the binding to target molecules which otherwise cannot be bound or only to a limited extent by nucleic acid molecules.
  • nucleic acid aptamers In vitro selection of nucleic acid aptamers combines the possibilities that combinatorial synthesis and the enzymatic amplifiability of nucleic acid molecules offer in order to select from a large library of nucleic acid oligomer molecules that specifically bind to target molecules.
  • This method which is often referred to as SELEX (Systematic Evolution of Ligands by Exponential Enrichment, Tuerk and Gold, 1990; overview in Osborne and Ellington, 1997), offers the possibility to combine positive and negative selection steps
  • nucleic acid aptamers that bind to target molecules, such as those with low molecular weight, e.g. Cibacron blue (Ellington and Szostak, 1990), ATP (Sassanfar and Szostak, 1993), Theophillin (Jenison et al. 1994) or Arginin (Burgstaller et al. 1995; Geiger et al. 1996), or also biological macromolecules, e.g. Thrombin (Bock et al. 1992), HIV Rev Protein (Giver et al. 1993) or vasodermal endothelial growth factor (VEGF; Jeliinek et al. 1994) (see Figure 1).
  • target molecules such as those with low molecular weight, e.g. Cibacron blue (Ellington and Szostak, 1990), ATP (Sassanfar and Szostak, 1993), Theophillin (Jenison et al. 1994) or Argin
  • the possibilities for using this method are essentially limited by two factors: the stability of the nucleic acid molecules, especially in biological fluids, and the lack of functional groups, such as lipophilic side chains, in the nucleic acids, which therefore only offers limited possibilities for binding to lipophilic ones Have domains (surfaces) of other molecules.
  • the stability of the nucleic acids can be increased by various measures. A number of nucleic acid modifications have been developed in recent years, all of which are essentially based on a chemical change in the sugar-phosphate chain. Increased resistance to degradation is achieved by using phosphorothioates or 2'-amino-, 2'-fluoro- and 2'-O- Get methyl sugars (Heidenreich et al. 1993).
  • modified nucleotide triphosphates When using modified nucleotide triphosphates, it is necessary to modify all bases of one type: If a mixture of modified and unmodified nucleotide triphosphates is added to a base for enzymatic synthesis, the incorporation is random, i.e. the position of the modification in the nucleic acid sequence cannot be influenced; the search for a specific modification is hindered. Therefore, in this process, all positions of a base (e.g. uridine) are usually made with the modified base.
  • a base e.g. uridine
  • nucleic acid chain Another possibility of introducing additional groups into nucleic acids is coupling to the 3 'and 5' ends of the nucleic acid chain, which is particularly common in oligonucleotide synthesis (Jaschke et al., 1994).
  • the molecules obtained in this way lack an essential quality. Due to the coupling to the ends of the nucleic acid molecules, the additional groups are far away from the randomized area and have hardly any influence on the structure of the binding domains, which are formed by sequence motifs of the randomized area. Terminal modifications, for example for the labeling of nucleic acid molecules, are therefore preferred because these generally do not influence the structural properties. Terminal modifications also allow independent functional groups to be added. A number of combinations are possible that can be used to construct bi-functional molecules. The free availability of the ends in high-affinity nucleic acid molecules which have been produced in accordance with the invention also enables these to be used as building blocks of bi-functional molecules. Subject of the invention
  • the object of the invention is the production of high-affinity nucleic acid molecules (nucleic acid aptamers or nucleic acid ligands).
  • These high-affinity nucleic acid molecules which contain functional groups for the specific recognition of target molecules, are characterized in that they have 2'-amino sugars in the pyrimidine nucleotides and in a few internal 2'-amino sugars of these pyrimidine nucleotides Amino groups are functional groups linked, and that they have a high binding affinity for selected target molecules.
  • the functional group mediates increased lipophilicity or other additional properties in the area of the binding domain.
  • Such functional groups are, for example, a Cn chain (n greater than 4), an amino acid residue, a peptide residue or a light-activatable side chain (for example N-succinimidyl- [4-azidophenyl] -1, 3'-dithiopropionate) .
  • These high affinity nucleic acid molecules are obtained from libraries by enrichment methods. These libraries consist of nucleic acid molecules which consist of a randomized area and two flanking areas, the randomized area containing approximately 20 to 200 and the defined flanking areas containing approximately 20 nucleotides.
  • Target molecules can be, for example, low molecular weight organic substances or biological macromolecules.
  • the specific recognition of target molecules is achieved by incorporating the functional groups on internal amino groups in 2'-amino-pyrimidine nucleic acids with the aid of activated molecules (for example succinimide esters) (see FIG. 2).
  • the modification takes place only after the enzymatic synthesis. It is influenced by the secondary structure and can be controlled by the reaction conditions.
  • the reaction conditions buffer, temperature and time
  • the nucleic acids thus produced then contain an (possibly several) additional functional group which is bound to an internal sugar, ie a sugar which is located in the nucleic acid chain and not at one of the ends.
  • the coupling at an internal position facilitates the direct influence on the formation of the specific binding domain by the newly inserted group.
  • the libraries of combinatorially modified nucleic acid molecules produced in this way are then selected via the specific target binding and the enriched binding molecules are then amplified (see FIG. 3).
  • 2'-amino-modified pyrimidine nucleotide triphosphates are used, which are already used for the production of nuclease-resistant nucleic acid molecules. This is a major advantage compared to other methods for producing functionalized, such as lipophilic, nucleic acid molecules for in vitro selection.
  • the nucleic acid molecules obtained in this way have both increased stability against nucleases and also that for the secondary modification required reactive amino groups.
  • the secondary modification then incorporates the additional functional groups which, for example, increase the lipophilicity of the nucleic acid molecule.
  • the method presented here differs significantly from the known methods in which bases which are lipophilic substituted are already used. Only the primary modification, which is necessary both for stability against nucleases and for the secondary modification, is found at the 2'-position of all sugars of pyrimidine nucleotides.
  • the functional secondary modification takes place selectively only at a few positions on the molecule.
  • Another major advantage of the method presented here is the internal modification of the nucleic acid.
  • the formation of a binding domain is essentially determined by the spatial proximity of the groups involved in the construction of the domain.
  • the probability of the formation of a uniform domain is increased in the case of a polymer, such as the nucleic acid here, in that the groups involved are arranged in direct proximity on the polymer.
  • the method according to the invention therefore offers a significant advantage over the known possibilities of inserting functional groups at the ends of the nucleic acid polymer.
  • An unmodified nucleic acid essentially shows two structural features. Double-stranded helix elements are formed when there are reversely complementary sequences.
  • Single-stranded areas that connect the ends of helix elements are called loops, and direct transitions between helix elements are called junctions.
  • the surface features of these elements which can form a specific binding domain, are: the sugar-phosphate chain, the small and the large groove (minor, major groove) and the stacking of the bases (base stacking); and for the loop: the sugar-phosphate chain and 'freely' accessible bases. Modifications with additional side chains make it possible to insert new, selectable chemical properties into this system. With internal modifications, an influence on the structure of the nucleic acids, ie on the formation of helix and loop domains, can also be expected.
  • the high-affinity nucleic acid molecules found can be used for a number of applications which are based on the specific interaction with the respective target molecule. In this way, they can be used to set up analytical methods for the target molecule, such as organic molecules or biological molecules (see above). Of particular interest is the use for the detection of biomolecules. High binding affinity and binding specificity, while at the same time being stable against nucleases, make it possible to find the molecules found both for diagnostic methods, in vitro, ex vivo and in vivo, and for therapy, for example for activating or inhibiting physiological processes or for drug targeting by the specific one Interaction with the respective target molecule. Examples:
  • the template contained two defined, flanking sequence elements that match the sequence of the primers and a central region with a randomized sequence with a length of 75 nucleotides (N75).
  • N75 75 nucleotides
  • a double-stranded DNA pool was made from 100 nmol template (> 1 ⁇ 15 molecules) and 500 nmol primer each with Taq DNA polymerase and dNTPs. This dsDNA pool was overwritten in 2'-amino-pyrimidine-RNA by T7-RNA polymerase in the presence of 2'-amino-dUTP, 2'-amino-dCTP, GTP and ATP.
  • the 2'-amino RNA pool which contains a 2'-amino group at each pyrimidine position, was incubated with succinimide-activated fluorescein for 2 hours at room temperature. The reaction was stopped by adding hydroxylamine. The nucleic acid molecules were purified by ethanol precipitation. The library with the modified nucleic acid molecules (> 10 ⁇ molecules) was taken up in the binding buffer, heated to 95 ° C. and cooled on ice. This molecule library was incubated with VEGF receptors immobilized on a plastic dish. After 2 hours of incubation, the supernatant was removed and the bound molecules were washed briefly three times.
  • the nucleic acid molecules (bound fraction) which were also bound after washing were denatured by heating and released from the binding.
  • the bound fraction was then amplified by means of reverse transcription and subsequent PCR.
  • the dsDNA pool thus obtained was used again as a starting material for a further affinity purification.
  • the nucleic acid molecules of the binding fraction were ligated into a plasmid vector and transformed into E. coli. Single colonies plasmids were isolated and the inserts sequenced.
  • the bound nucleic acids were detached both by denaturation and by cleavage of disulfide bridges.
  • Faseb J 7 90-6 Jäschke, A., Harrison, J.P., Nordhoff, E., Hillenkamp, F., Cech, D., and Erdmann,

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Abstract

La présente invention porte sur la production de molécules hautement affines d'acide nucléinique (aptamères-acide nucléinique ou ligands-acide nucléinique). Ces molécules hautement affines d'acide nucléinique contiennent des groupes fonctionnels de reconnaissance spécifique de molécules-cibles et sont en outre caractérisées en ce qu'elles disposent de 2'-aminosucre dans les nucléotides-pyrimidine et que des groupes fonctionnels sont liés par l'intermédiaire du groupe aminé aux 2'-aminosucres les moins internes de ces nucléotides-pyrimidine. Le groupe fonctionnel permet de mettre en évidence une lipophilie accrue ou une autre propriété supplémentaire du domaine de liaison: Les molécules hautement affines de l'acide nucléinique peuvent s'utiliser pour des applications reposant sur l'interaction spécifique avec la molécule-cible concernée. Une grande affinité de liaison, une grande spécificité de liaison et, en parallèle, une stabilité assurée contre les nucléases permettent d'utiliser ces molécules d'acide nucléinique aussi bien à des fins diagnostiques - in vitro, ex vivo et in vivo - que thérapeutiques, par exemple pour stimuler ou inhiber des processus physiologiques ou pour le ciblage de médicament.
PCT/DE1998/002813 1997-09-18 1998-09-16 Molecules hautement affines d'acide nucleinique, production et application WO1999014355A2 (fr)

Applications Claiming Priority (2)

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DE19741084.7 1997-09-18
DE19741084A DE19741084A1 (de) 1997-09-18 1997-09-18 Hochaffine Nukleinsäuremoleküle mit funktionellen Gruppen zur spezifischen Erkennung von Zielmolekülen, ihre Herstellung und Verwendung

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WO1999014355A2 true WO1999014355A2 (fr) 1999-03-25
WO1999014355A3 WO1999014355A3 (fr) 1999-08-19

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000201A1 (fr) * 1986-06-24 1988-01-14 California Institute Of Technology, Inc. Nouveaux phosphoramidites de desoxyribonucleosides et leur utilisation pour la preparation d'oligonucleotides
WO1995031572A1 (fr) * 1994-05-18 1995-11-23 Pharmgenics, Inc. Oligonucleotides contenant une fraction phosphonate d'aminohydrocarbure et utilisation desdits oligonucleotides

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US5641629A (en) * 1990-06-11 1997-06-24 Nexstar Pharmacueticals Inc Spectroscopically detectable nucleic acid ligands
US5853984A (en) * 1990-06-11 1998-12-29 Nexstar Pharmaceuticals, Inc. Use of nucleic acid ligands in flow cytometry
US5705337A (en) * 1990-06-11 1998-01-06 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: chemi-SELEX
US5686592A (en) * 1990-06-11 1997-11-11 Nexstar Pharmaceuticals, Inc. High-affinity oligonucleotide ligands to immunoglobulin E (IgE)
US5789163A (en) * 1990-06-11 1998-08-04 Nexstar Pharmaceuticals, Inc. Enzyme linked oligonucleotide assays (ELONAS)
YU187991A (sh) * 1990-12-11 1994-09-09 Hoechst Aktiengesellschaft 3-(2)-amino-ali tiol-modifikovani, s fluorescentnom bojom vezani nukleozidi, nukleotidi i oligonukleotidi, postupak za njihovo dobijanje i njihova upotreba
IL114235A0 (en) * 1994-07-14 1995-10-31 Schering Ag Oligonucleotide conjugates and processes for noninvasive diagnosis utilizing the same
DE4424923A1 (de) * 1994-07-14 1996-01-18 Schering Ag Konjugate aus Metallkomplexen und Oligonucleotiden, die spezifisch an bestimmte Zielstrukturen binden für MRI
DE4424922A1 (de) * 1994-07-14 1996-01-18 Schering Ag Konjugate aus Metallkomplexen und Oligonucleotiden, die spezifisch an bestimmte Zielstrukturen binden
WO1996027604A1 (fr) * 1995-03-06 1996-09-12 Nexstar Pharmaceuticals, Inc. Ligands oligonucleotidiques de haute affinite se liant a la phospholipase secretoire a2 (spla2)
DE19543232A1 (de) * 1995-11-07 1997-05-15 Knoell Hans Forschung Ev Herstellung einer Matrix-gebundenen miniaturisierten kombinatorischen Poly- und Oligomerbibliothek
DE19619373A1 (de) * 1996-05-14 1997-11-20 Hoechst Ag Neue Substanzbibliothek und damit hergestellte supramolekulare Komplexe

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000201A1 (fr) * 1986-06-24 1988-01-14 California Institute Of Technology, Inc. Nouveaux phosphoramidites de desoxyribonucleosides et leur utilisation pour la preparation d'oligonucleotides
WO1995031572A1 (fr) * 1994-05-18 1995-11-23 Pharmgenics, Inc. Oligonucleotides contenant une fraction phosphonate d'aminohydrocarbure et utilisation desdits oligonucleotides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OSBORNE S E ET AL: "NUCLEIC ACID SELECTION AND THE CHALLENGE OF COMBINATORIAL CHEMISTRY" CHEMICAL REVIEWS, Bd. 97, Nr. 2, 1. M{rz 1997, Seiten 349-370, XP000683273 in der Anmeldung erw{hnt *

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WO1999014355A3 (fr) 1999-08-19

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