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WO1994012518A2 - Reactif pour le couplage de diverses substances a des acides nucleiques et procede pour sa preparation - Google Patents

Reactif pour le couplage de diverses substances a des acides nucleiques et procede pour sa preparation Download PDF

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Publication number
WO1994012518A2
WO1994012518A2 PCT/DE1993/001101 DE9301101W WO9412518A2 WO 1994012518 A2 WO1994012518 A2 WO 1994012518A2 DE 9301101 W DE9301101 W DE 9301101W WO 9412518 A2 WO9412518 A2 WO 9412518A2
Authority
WO
WIPO (PCT)
Prior art keywords
ribooligonucleotide
rna
reagent
nucleic acids
molecule
Prior art date
Application number
PCT/DE1993/001101
Other languages
German (de)
English (en)
Other versions
WO1994012518A3 (fr
Inventor
Gabor Igloi
Original Assignee
Gabor Igloi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gabor Igloi filed Critical Gabor Igloi
Publication of WO1994012518A2 publication Critical patent/WO1994012518A2/fr
Publication of WO1994012518A3 publication Critical patent/WO1994012518A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates to a modified ribooligonucleotide, that is to say a reagent for coupling at least one molecule to nucleic acids, in particular ribonucleic acids (RNA), a method for its production, its use and a reagent kit based on this modified ribooligonucleotide.
  • This coupling reagent is a specifically modified ribooligonucleotide, which in principle consists of any number, but at least one ribonucleotide.
  • this coupling reagent it is possible to use a variety of labels, in particular fluorescent labels, but also digoxigenin or biotin derivatives, or else other molecules, such as e.g. B. to couple proteins to nucleic acids, especially ribonucleic acids (RNA).
  • nucleic acids The coupling of a wide variety of molecules to nucleic acids is a widely used method in molecular biology. Such methods are particularly important for labeling nucleic acids. They are used for protein-nucleic acid interaction studies, nucleic acid structure studies, for DNA and RNA sequencing or, for medical diagnostics of enormous importance, in hybridization methods, especially in-situ hybridization methods. Labeling has so far been carried out mainly on a radioactive basis with the aid of isotope-substituted nucleotides and corresponding enzymes. However, since radioactively labeled substances are quite expensive, handling them is not safe and the disposal of radioactive waste is problematic for ecological reasons, efforts have been made for some time to use alternative (i.e. non-radioactive) labeling options.
  • RNA RNA molecule that is extended by one nucleotide and bears a radioactive label at the 3 "terminus. This method only allows the radioactive labeling of RNA.
  • the object of the present invention is to develop a special ribooligonucleotide, i.e. H. to provide a reagent for coupling at least one molecule, more generally considered different substances, in particular a fluorescent marker, a biotin derivative, a digoxigenin derivative or a protein to nucleic acids, in particular ribonucleic acids (RNA), which avoids the disadvantages mentioned, i.e. its use on enzymatic Implementations is based and therefore has a high specificity that is easy to carry out and that allows many different substances to be coupled to nucleic acids. Furthermore, it is the object of the present invention to provide specific possible uses for the modified ribooligonucleotide, as well as a reagent kit based on this ribooligonucleotide.
  • a special ribooligonucleotide i.e. H. to provide a reagent for coupling at least one molecule, more generally considered different substances, in particular a fluorescent marker,
  • Nucleic acid is coupled, is preferably a fluorescent marker, for example a fluorescein derivative, but can also be a digoxigenin or biotin derivative or a protein, for example. Regardless of the nature of this molecule R 3 , it is referred to below as a marker for the sake of simplicity.
  • a radioactive isotope is also possible, in contrast to the pCp conventionally used for radioactive labeling with the Coupling reagent according to the invention also "exotic" isotopes, such as 125 J can be introduced.
  • the coupling reagent according to the invention consists of a ribooligonucleotide of any length in which a reactive group, in particular an amino group, is bonded directly or via a spacer group to one or more 3 ′′ phosphate groups.
  • a sulfhydryl group can also be used as a reactive group "However, it is more sensitive and does not react with as many substituents as an amino group.
  • Ri is a phosphate group
  • R 2 is a nucleobase, especially cytidine
  • R 3 is the molecule to be coupled
  • a coupling reagent which also functions according to the invention is likewise obtained, but the conversion rates of the coupling reaction are up to 50% lower than when using cytidine.
  • the ribooligonucleotide can be extended on its 3 "phosphate group with further nucleotides, which can also be substituted.
  • the alkyl chain used as spacer contains no phosphate group and therefore also no R 4 radical.
  • a hydrogen atom is then instead bonded to the carbon atom of the alkyl chain to which a phosphate group is bonded according to claims 1 and 2.
  • An extension of the coupling reagent in the manner described above is then not possible and the coupling reagent has only a single marker molecule R 3 .
  • RNA ligase especially the T4 RNA ligase.
  • the minimum requirements of this enzyme must be Substrate is a 3 ", 5" ribonucleoside bisphosphate. Ribooligonucleotides which contain such a group at their 5 "end are bound by the RNA ligase to the 3" - OH group of any ribonucleic acid.
  • the reagent can also be coupled to a DNA; all that is required is to attach a ribonucleotide to the 3 "end of the DNA before coupling.
  • the reactive group which binds the marker is bonded to the 3 "phosphate group of the coupling reagent via a spacer containing phosphate.
  • the spacer is an alkyl chain with a length of between 1 and 30 carbon atoms, to which a phosphate group can be bound.
  • the coupling reagent can be extended further at this phosphate group of the spacer: Either by attaching another nucleoside to which a marker can in turn be bound (see formula I), or by directly introducing further marker molecules with a spacer (see formula II) .
  • the length of the ribooligonucleotide can be selected as required and there is in principle the possibility of binding markers to any or all of the phosphate groups with the exception of the 5 "terminal phosphate group, a reagent is available with the coupling reagent according to the invention with which nucleic acids can be used in one step can be coupled with several markers, which, for example, gives a stronger signal, particularly in connection with in situ hybridization methods, which is a major advantage of the coupling reagent according to the invention, since it is very complex to obtain a signal amplification in conventional labeling methods.
  • the coupling reagent is synthesized analogously to the automatic DNA synthesis. It is essential to the invention that the coupling reagent can be synthesized automatically with the aid of conventional DNA synthesizers, the synthesis cycle being suitably modified to the requirements of RNA synthesis.
  • ribonucleotides are used as building blocks instead of deoxyribonucleotides and uracil is used instead of thymidine.
  • the 2 "-OH group is protected by a protective group, for example a silyl group, during the reaction.
  • the reaction time in RNA synthesis is approximately 10 minutes compared to approximately 3 minutes in DNA synthesis.
  • ribooligonucleotide is synthesized according to the phosphoramidite method, which is described in detail by T. Atkinson and M. Smith (in: Oligonucleotide Synthesis; A Practical approach; MJ Gait (ed.) IRL Press, Oxford, Washington DC, 1984) on the solid phase from 3 "to 5".
  • a reactive amino group is preferably introduced with the aid of a special amination reagent of the form (l-dimethoxytrityloxy-3-fluorenylmethoxycarbonylamino-propan-2-yl) - (2-cyanoethyl) - (N, N-diisopropyl) - phosphoramidite (for example from the company Glen Research [Sterling, Vancouver, Canada] available as 5 "-branched modifier C3).
  • amination reagent results in a coupling reagent with a 3" -terminal nucleoside.
  • This can be modified further, for example by oxidizing the 2 "- and 3" -OH groups to aldehyde groups.
  • These aldehyde groups can react with amino groups to form a Schiff base. If the aldehyde groups are reacted with corresponding amino groups of enzymes, the enzyme is bound to the oligonucleotide.
  • the terminal nucleoside is not modified, there is a risk that the 3 "OH group of this nucleoside reacts with the 5" phosphate group of the same type of molecule.
  • the nucleoside can be split off at the 3 "end by means of ⁇ -elimination and the self-ligation can thereby be prevented.
  • the reactive amino group can also be introduced with an amination reagent of the form (l-dimethoxytrityloxy-3-fluorenylmethoxycarbonylamino-propane-2-succinoyl) - aminoalkane (available, for example, from Glen Research as a 3 "amino-modifier-CPG), where directly a 3 "phosphate group according to formula (I) or (II) results.
  • an amination reagent of the form (l-dimethoxytrityloxy-3-fluorenylmethoxycarbonylamino-propane-2-succinoyl) - aminoalkane (available, for example, from Glen Research as a 3 "amino-modifier-CPG), where directly a 3 "phosphate group according to formula (I) or (II) results.
  • the ⁇ -elimination is saved if you do not want a terminal nucleoside.
  • R 3 Various substances (R 3 ) which easily react with an amino group, such as fluorescent markers, for example a fluorescein derivative, or digoxigenin or biotin derivatives or else proteins, in particular nucleases, are then linked to the introduced amino group.
  • Activated substituents can exist as isothiocyanates, isocyanates, as N-hydroxysuccinimide esters, p-nitrophenyl esters, sulfonic acid chlorides, triazine chlorides or as acid azides.
  • the following substances are preferably used as markers: fluorescein succinimide ester, NBD-F (4-fluoro-7-nitrobenz-2-oxa-1,3-diazole), tetramethylrhodamine 5 (6) isothiocyanate, Texas red (sulphorhodamine-101-acid chloride) , Biotin succinimide ester, digoxygenin succinimide ester.
  • proteins in particular nucleases
  • this "crosslinking" of an enzyme to an oligonucleotide can in principle be carried out in the following ways: on the one hand, a free sulfhydryl group can be incorporated into the enzyme by incorporating a cysteine residue (see R. Zuckermann et al., Journal "J. Am. Chem. Soc.
  • hetero cross-linking reagent eg N-succinimidyl 3- (2-pyridyldithio) propionate
  • the succinimidyl residue of the hetero crosslinking reagent reacts with the amino group of the coupling reagent, causing the enzyme to cross the
  • Crosslinking reagent is bound to the coupling reagent.
  • Another way of introducing a free SH group into the corresponding enzyme is to chemically convert an amino group into an SH group (see Carlsson et al., Journal "Biochem. J”. 173, 723-737 (1978)) .
  • the actual cross-linking takes place as described above.
  • a third possibility of binding an enzyme to the coupling reagent is to use a homo cross-linking reagent which, in contrast to the hetero cross-linking reagent mentioned above, has two succinimidyl residues. It reacts both with the amino group of the coupling reagent and with an amino group of the enzyme.
  • a phosphate group at the 5 "terminus of the ribooligonucleotide which is necessary for the reaction of the ribooligonucleotide with the nucleic acid to be labeled, is preferably carried out by means of a phosphorylation reagent of the form: 2- [2- (4,4" -dimethoxotrityloxy) ethylsulfonyl ] ethyl- (2-cyanoethyl) - (N, N-diisopropyl) - phosphoramidite (available from Glen Research as a chemical phosphorylation reagent).
  • a 5 "phosphorylation is also possible with other methods, for example using ATP and the enzyme polynucleotide kinase. However, this method is extremely cumbersome and lengthy, especially on a larger scale.
  • the coupling reagent according to the invention can be used with the aid of conventional DNA synthesizers are automatically synthesized.
  • the synthesis of a coupling reagent of the form 5 "pCpCp fluorescein is explained in more detail below as an exemplary embodiment.
  • RNA triplet was first produced from the following building blocks (direction: 5 "- 3") using the phosphoramidite method:
  • the reagents were obtained from Glen Research, but are also available from other companies.
  • RiboA-CPG are filled into a column and this is then sealed.
  • the RNA synthesis cycle is started.
  • the setting on the DNA synthesizer is: Tr off, auto. This means that the protective groups are split off and the product is split off from the carrier.
  • the column after the reaction is rinsed with ethanol: NH 3/1: 3.
  • the product thus obtained is lyophilized and then taken up again in 0.2 ml of 1 M tetraethylammonium fluoride in tetrahydrofuran. It is then incubated for 6 h at room temperature, as a result of which the protective groups on the ribose are split off. 0.3 ml of 1 M ammonium acetate solution, pH 5.3, is then added to this solution and diluted with 5 ml of water.
  • the substance can be stored at -20 ° C or lyophilized for a long time without loss.
  • the incubation medium contains:
  • the incubation medium contains: - 50 ⁇ ⁇ labeled ribooligonucleotide (from b)
  • the coupling reagent is used for coupling to nucleic acids.
  • An enzyme namely an RNA ligase, preferably the T4 RNA ligase, catalyzes the bond between the 5 "phosphate end of the coupling reagent and the 3" OH end of the nucleic acid to be modified.
  • the reaction is carried out in a special RNA ligase buffer which contains magnesium chloride, an organic solvent, ATP, coupling reagent, the nucleic acid to be modified and the RNA ligase at a slightly alkaline pH. This mixture is incubated at temperatures between 0 and 10 ° C. for 4 to 24 h and the modified nucleic acid is then extracted with phenol and precipitated with ethanol.
  • RNA with fluorescein is carried out below using the coupling reagent, the synthesis of which was described in the example above.
  • the total volume of the incubation medium contains 30 ⁇ :
  • RNA ligase buffer 100 mM Hepes / KOH, pH 7.8; 40 mM magnesium chloride; 7 mM dithiothreitol; 20 ⁇ g / ml bovine serum albumin; 20% dimethyl sulfoxide
  • a main application of the coupling reagent according to the invention is the coupling of labeling substances (R 3 ), in particular fluorescent markers, to nucleic acids.
  • labeling substances R 3
  • the straightforwardness of the method of binding marker substances to the coupling reagent and the coupling reagent to a nucleic acid makes it possible, for example in the form of a kit, to provide the user with the still unmodified coupling reagent and separately various marker substances. The user can then easily produce the coupling reagent as required and in the required modification as required. Since the coupling of the modified coupling reagent to the nucleic acid to be labeled is an enzymatic method, the user has a simple possibility of coupling any nucleic acids with markers of his choice very gently and with high specificity.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
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  • Engineering & Computer Science (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un ribo-oligonucléotide de formule (I) ou (II) dans lesquelles R1 désigne un groupe hydroxyle ou un groupe phosphate ou au moins un 3', 5'nucléotide-bisphosphate, R2 désigne une nucléobase, R3 est la molécule à coupler et R4 représente un atome d'hydrogène ou un nucléoside. Dans lesdites formules, m désigne un chiffre compris entre 1 et 100, et en particulier entre 2 et 5, n1 désigne 0 ou un chiffre compris entre 1 et 30, et en particulier 1, et n2 désigne 0 ou un chiffre compris entre 1 et 30, et en particulier 1. Le ribo-oligonucléotide convient pour le couplage de substances de marquage, en particulier des marqueurs fluorescents, mais également des protéines, en particulier des nucléases, avec catalyse d'une ligase ARN à des acides nucléiques. Ce ribo-oligonucléotide modifié peut être employé a) pour des procédés d'hybridation ou b) pour des études de la structure de l'ARN ou c) pour des études des interactions protéine-ARN ou d) pour des opérations particulières sur des acides nucléiques.
PCT/DE1993/001101 1992-11-25 1993-11-19 Reactif pour le couplage de diverses substances a des acides nucleiques et procede pour sa preparation WO1994012518A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4239531.3 1992-11-25
DE19924239531 DE4239531A1 (de) 1992-11-25 1992-11-25 Reagenz zur Kupplung verschiedener Substanzen an Nukleinsäuren sowie Verfahren zu dessen Herstellung

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WO1994012518A2 true WO1994012518A2 (fr) 1994-06-09
WO1994012518A3 WO1994012518A3 (fr) 1994-07-21

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2424479A (en) * 2005-01-31 2006-09-27 Agilent Technologies Inc RNA labelling using RNA ligating enzymes
US7544471B2 (en) 2005-07-30 2009-06-09 Agilent Technologies, Inc. Preparing RNA from a wax-embedded tissue specimen
US7700289B2 (en) 2006-04-03 2010-04-20 Agilent Technologies, Inc. Method for labeling RNA
US7718365B2 (en) 2005-07-09 2010-05-18 Agilent Technologies, Inc. Microarray analysis of RNA
US8076064B2 (en) 2005-07-09 2011-12-13 Agilent Technologies, Inc. Method of treatment of RNA sample

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19954934A1 (de) * 1999-11-16 2001-05-17 Otto S Wolfbeis Verfahren zur Solubilisierung von optischen Markern
EP1136569A3 (fr) 2000-03-24 2004-01-28 Bayer Corporation Sondes d'acides nucléiques qui ont des marqueurs non-nucléosidiques à haute hydrophilicité comprenant plusieurs autres marqueurs et leur utilisation
US7572585B2 (en) 2006-07-31 2009-08-11 Agilent Technologies, Inc. Enzymatic labeling of RNA

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2565996B1 (fr) * 1984-06-15 1988-01-22 Inst Nat Sante Rech Med Acides nucleiques marques chimiquement, leur utilisation et necessaire pour sa mise en oeuvre
US4757141A (en) * 1985-08-26 1988-07-12 Applied Biosystems, Incorporated Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof
US5352578A (en) * 1989-02-15 1994-10-04 Worcester Foundation For Experimental Biology Method of separating oligonucleotides from a mixture
DE3916871A1 (de) * 1989-05-24 1990-11-29 Boehringer Mannheim Gmbh Modifiziertes phosphoramidit-verfahren zur herstellung von modifizierten nukleinsaeuren
GB9009980D0 (en) * 1990-05-03 1990-06-27 Amersham Int Plc Phosphoramidite derivatives,their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
US5210015A (en) * 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
IL99068A (en) * 1990-08-09 1998-01-04 Genta Inc Non-nucleotide binding reagents for oligomers
WO1992003464A1 (fr) * 1990-08-28 1992-03-05 Microprobe Corporation Synthese sur support solide d'oligonucleotides places en queue en position 3' par l'intermediaire d'une molecule de liaison
DE4123540A1 (de) * 1991-07-16 1993-01-21 Boehringer Mannheim Gmbh Immobilisierung von nukleinsaeuren

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2424479A (en) * 2005-01-31 2006-09-27 Agilent Technologies Inc RNA labelling using RNA ligating enzymes
GB2424479B (en) * 2005-01-31 2009-02-25 Agilent Technologies Inc RNA labelling by ligation in the presence of DMSO
US7541144B2 (en) 2005-01-31 2009-06-02 Agilent Technologies, Inc. RNA labeling method
US7718365B2 (en) 2005-07-09 2010-05-18 Agilent Technologies, Inc. Microarray analysis of RNA
US8076064B2 (en) 2005-07-09 2011-12-13 Agilent Technologies, Inc. Method of treatment of RNA sample
US7544471B2 (en) 2005-07-30 2009-06-09 Agilent Technologies, Inc. Preparing RNA from a wax-embedded tissue specimen
US7700289B2 (en) 2006-04-03 2010-04-20 Agilent Technologies, Inc. Method for labeling RNA

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WO1994012518A3 (fr) 1994-07-21
DE4239531A1 (de) 1994-05-26

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