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WO1999063111A2 - Methode de marquage d'acide nucleique - Google Patents

Methode de marquage d'acide nucleique Download PDF

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Publication number
WO1999063111A2
WO1999063111A2 PCT/IB1999/001116 IB9901116W WO9963111A2 WO 1999063111 A2 WO1999063111 A2 WO 1999063111A2 IB 9901116 W IB9901116 W IB 9901116W WO 9963111 A2 WO9963111 A2 WO 9963111A2
Authority
WO
WIPO (PCT)
Prior art keywords
group
peptide
oligonucleotide
tert
mercaptoethyl
Prior art date
Application number
PCT/IB1999/001116
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English (en)
Other versions
WO1999063111A3 (fr
Inventor
Ramon Eritja
Dirk Gottschling
Hartmut Seliger
Original Assignee
European Molecular Biology Laboratory
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
Priority claimed from GBGB9811771.6A external-priority patent/GB9811771D0/en
Priority claimed from GBGB9813765.6A external-priority patent/GB9813765D0/en
Application filed by European Molecular Biology Laboratory filed Critical European Molecular Biology Laboratory
Priority to AU39525/99A priority Critical patent/AU3952599A/en
Publication of WO1999063111A2 publication Critical patent/WO1999063111A2/fr
Publication of WO1999063111A3 publication Critical patent/WO1999063111A3/fr

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Classifications

    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B27/00—Layered products comprising a layer of synthetic resin
    • B32B27/06—Layered products comprising a layer of synthetic resin as the main or only constituent of a layer, which is next to another layer of the same or of a different material

Definitions

  • the present invention relates to a method of labelling nucleic acid using oligonucleotide-peptide conjugates to hybridise to the nucleic acid.
  • the invention also relates to methods of production of oligonucleotide-peptide conjugates, to nucleotide intermediates used in the generation of these conjugates and to the use of these conjugates to label nucleic acid.
  • the nucleotide intermediates used in the conjugation process possess mercaptoethyl groups protected by tert-butylthio groups that can form oligonucleotide-peptide conjugates through the reaction of the thiol-oligonucleotides with a maleimido-peptide.
  • nucleic acid labelling There are numerous applications in which it is desirable or necessary to label a nucleic acid, ranging from initial qualitative detection to quantitative measurement of nucleic acid concentration. Historically, methods of labelling nucleic acid have utilised radioactive isotopes. However, over the last decade, there has been a concerted effort to find alternative ways of nucleic acid labelling, for reasons both of safety and of environmental necessity.
  • oligonucleotides can be labelled with non-radioactive reporter groups such as fluorescent compounds, biotin, digoxigenin, 2,4-nitrophenyl groups and enzymes. Fluorescent derivatives are detected by their fluorescent properties and by the use of antibodies. Biotin can be detected with streptavidin and its derivatives. All of these techniques have respective advantages and disadvantages and there remains a great need for alternative methods of non-radioactive nucleic acid labelling to suit particular applications.
  • non-radioactive reporter groups such as fluorescent compounds, biotin, digoxigenin, 2,4-nitrophenyl groups and enzymes. Fluorescent derivatives are detected by their fluorescent properties and by the use of antibodies. Biotin can be detected with streptavidin and its derivatives. All of these techniques have respective advantages and disadvantages and there remains a great need for alternative methods of non-radioactive nucleic acid labelling to suit particular applications.
  • a method of labelling nucleic acid comprising hybridising an oligonucleotide-peptide conjugate to the nucleic acid.
  • the peptide portion of the oligonucleotide-peptide conjugate is antigenic and may comprise between 2 and 50 amino acids, preferably between 8 and 20 amino acids.
  • Suitable epitopes are well known to those of skill in the art and include the myc-TAG sequence (Evan et al, 1985), the FLAG peptide (Hopp et al., 1988), po ⁇ yhistidine (Smith et al, 1988) and anti-VSV-G (Kreis et al., 1986).
  • a method of conjugation of the oligonucleotide-peptide conjugate involving the steps of a) modifying a nucleotide base so as to contain a mercaptoethyl group protected with a tert-butylthio group and then b) reacting the modified nucleotide with a maleimido peptide.
  • this method is used in a method of labelling nucleic acid by hybridising an oligonucleotide-peptide conjugate to the nucleic acid.
  • an oligonucleotide-peptide conjugate obtainable according to any one of the above- described methods.
  • Oligonucleotides carrying thiol groups at certain positions have been previously used in various applications including a) introduction of biotin, fluorescent compounds and other non-radioactive labels (Beaucage and Iyer 1993, Connolly 1985); b) preparation of conformationally-restricted oligonucleotides (Ferentz et al 1993, Glick 1991, Osborne 1997); c) immobilisation of oligonucleotides to solid supports such as affinity columns (Blanks and McLaughlin 1988) and gold particles (Elghanian, R. et al 1997, Mirkin et al. 1996); and d) preparation of oligonucleotide-peptide conjugates (Corey 1995, Eritja 1991, Zuckermann and Schultz 1988).
  • thiol groups have conventionally been introduced on the 5' and 3' ends of the oligonucleotides in order to maintain the hybridisation properties of the oligonucleotides (Beaucage and Iyer 1993, Connolly 1985). Thiol groups have additionally been introduced at the phosphate bond (Fidanza and McLaughlin 1992), at the 2 '-position (Gundlach et al. 1997) and on the nucleobases (Goodwin and Glick 1993, Osborne 1997).
  • the invention also provides novel nucleotide intermediates modified so as to possess a mercaptoethyl group protected by a tertbutyl-thio group.
  • the mercaptoethyl group may be introduced at positions 2, 6 or 8 (adenine), 2 or 8 (guanine), 4 or 5 (cytosine) or position 5 (thymine and uracil) in the structure of the base portion of the nucleotide.
  • Rl is selected from the group consisting of H, protecting group
  • R2 is selected from the group consisting of H, phosphoramidite, H-phosphonate or phosphate diester;
  • R3 is selected from H, OH, protecting group
  • B is selected from adenine (with a mercaptoethyl group protected by a tert-butylthio group at positions 2, 6, or 8); guanine (with a mercaptoethyl group protected by a tert- butylthio group at positions 2 or 8); cytosine (with a mercaptoethyl group protected by a tert-butylthio group at positions 4 or 5); thymine (with a mercaptoethyl group protected by a tert-butylthio group at position 5), or uracil (with a mercaptoethyl group protected by a tert-butylthio group at position 5).
  • Oligonucleotides carrying these nucleotides can be purified and characterised.
  • the tert-butylthio group can be removed during or after standard ammonia deprotection yielding oligonucleotides carrying free thiol functions for subsequent reaction with maleimido- peptide.
  • novel intermediate nucleotides may be modified in the sugar portion of the nucleotide with a protecting group at the 5' hydroxy position.
  • protecting group is meant any chemical group that is effective to prevent the molecule from reacting during chemical treatment in which the molecule is not intended to participate. Suitable protecting groups may be removed highly specifically under mild conditions and
  • ET RULE 26 include groups such as dimethoxytrityl (DMT). Other suitable protecting groups will be known to those of skill in the art. A protecting group may also be included at position C3 of the sugar portion of the nucleotide molecule.
  • DMT dimethoxytrityl
  • the nucleotide may also be phosphitylated at position 4 to give a phosphoramidite derivative according to standard protocols that will be known to those of skill in the art.
  • oligonucleotide-peptide conjugates suitable for use in labelling nucleic acid, using the novel nucleotide intermediates described above.
  • An oligonucleotide containing one or more nucleotide intermediates is reacted with one or more maleimido- peptides, to yield oligonucleotide-peptide conjugates with a defined structure.
  • Conjugates formed by this method may for example comprise an oligonucleotide sequence that is complementary to a target gene sequence and a peptide sequence that is recognised by a specific antibody. These oligonucleotide-peptide conjugates maintain the hybridisation capacity of the unmodified oligonucleotide sequence.
  • peptide epitopes may be used for coding different oligonucleotide sequences in multiple hybridisation experiments. Furthermore, this technology allows the use of oligonucleotides carrying multiple labels. Peptide epitopes may also be used to introduce a peptide code for each of the four natural bases; such a system would have application in the field of DNA sequencing.
  • oligonucleotide-peptide conjugates so prepared to label nucleic acid.
  • Figure 1 Synthesis of the N 4 -(tert-2-mercaptoethyl)-dC phosphoramidite derivative.
  • A) Hexadimethyldisilazane followed by mesitylensulfonyl chloride, Et3N, DMAP and p- nitrophenol.
  • B) 2aminoethyl-t-butyldisulphide.
  • oligonucleotide-peptide conjugate For the preparation of the oligonucleotide-peptide conjugate, the protocol based on the reactivity of the thiol group was used (Eritja et al 1991). In this method a thiol group is introduced on the oligonucleotide that will react with a maleimido-peptide carrying the c-myc TAG epitope.
  • oligonucleotides carrying mercaptoethylcytosine residues using standard deprotection conditions.
  • These oligonucleotides are used for the preparation of an olgonucleotide- peptide conjugate carrying the c-myc epitope, that has been shown herein to be specifically recognised by a monoclonal antibody directed against the peptide epitope.
  • the monoclonal antibody used is 9E10 (Evan et al., 1985).
  • the oligonucleotide-peptide conjugate was specifically bound with a similar sensitivity to that seen in other non-radioactive labelling systems. Synthesis of monomers for oligonucleotide synthesis
  • transient protection of alcohol functions was performed with hexamethyldisilazane
  • activation of position 4 of DU was done with mesytilnesulfonyl chloride
  • DMAP mesytilnesulfonyl chloride
  • triethylamine instead of phosphoryl chloride
  • 1,2,4-triazole 1,2,4-triazole and smaller excess of p-nitrophenol was used.
  • the desired product (1) was isolated pure without silica gel purification in a 47% yield. Displacement of the p-nitrophenyl group with protected 2-mercaptoethylamine gave protected N4-mercaptoethyl-dC (compound 2).
  • the S-tert-butylthio group Goodwin and Glick 1993, Gundlach et al.
  • the S-tert-butylthio group is stable to oligonucleotide synthesis conditions and easily removed during or after standard synthesis conditions (Goodwin and Glick 1993, Gundlach et al. 1997, Osborne 1997) and for these reasons was selected.
  • Pentadecamer sequences 01 : 5'-d(TAGAGG5TCCATTGC)-3', 02: 5'- d(TAGAGG5TC5ATTGC)-3' and 03: 5'-d(TAG AGG CT55AT TGQ-3' were prepared using phosphoramidite 5 and the phosphoramidites of the natural bases protected with the more labile tert-butylphenoxzacetyl groups. Coupling efficiencies of the modified phosphoramidite were of the same order as commercially available phosporamidites (98-99%).
  • Standard ammonia deprotection conditions concentration ammonia for six hours at 55°C gave t-butylthio protected oligonucleotides that were purified using standard protocols. Purified oligonucleotides were characterised by mass spectrometry and by enzymatic digestion followed by HPLC analysis of the nucleosides.
  • DTT dithiothreitol
  • oligonucleotides containing protected mercaptoethyl groups can be purified, characterised and stored without inter and intramolecular disulfide formation.
  • t-butylthio protected oligonucleotide were deprotected with DTT and, after, the excess of DTT is eliminated by a Sephadex column (NAPTM 10) yielding a solution of the oligonucleotide the free thiol group.
  • Table 1 Melting temperatures (°C) of duplexes containing N 4 -(tert-2-mercaptoethyl)- dC (C*) and peptide-oligonucleotide conjugate (C # ) in a solution comprising 0.15 M NaCl, 0.05 N tris-HCl buffer pH 7.5.
  • Tm 48.9 °C 5' TAGAGGCTC*C*ATTGC 3' 3 ' ATCTCCGAG G TAACG 5 '
  • Non-radioactive detection of oligonucleotide-peptide conjugate was assayed on a dot- blot format (data not shown). Small amounts (from 5 ⁇ g to 0.3ng) of oligonucleotide- peptide conjugate were fixed in a nitrocellulose membrane with UV light. The membrane was incubated with anti-c-myc monoclonal antibody 9E10 followed by incubation with a secondary antibody against mouse antibodies conjugated with horseradish peroxidase and finally, the membrane was treated with luminol (chemiluminescence substrate for peroxidase, ECLTM system, Amersham) and the emitted light was collected for 15 minutes.
  • luminol chemiluminescence substrate for peroxidase
  • Oligonucleotide carrying c-myc sequence was found to be specifically recognised by the monoclonal antibody giving positive spots.
  • the detection limit was approx. 50ng (8 pmol). No light was observed when an oligonucleotide without peptide was used as negative control.
  • Nylon membranes were also used and gave similar positive results but they had a higher background.
  • oligonucleotides carrying mercapteothyl-C residues were easily prepared using phosphoramidite 5. These oligonucleotides are useful intermediates to obtain oligonucleotide-peptide conjugates such as oligonucleotides carrying epitope peptide sequences. Oligonucleotide-peptide conjugates carrying the c- myc epitope sequence are capable to hybridise with their complementary sequences and they are specifically detected by monoclonal antibodies. For these reasons oligonucleotides carrying epitope peptide sequences should be useful as non-radioactive labelling system for nucleic acids opening the possibility of using the large diversity of available antibodies as a coding system in multiple hybridisation experiments.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

L'invention concerne une méthode de marquage d'acide nucléique au moyen de conjugués oligonucléotido-peptidiques à hybrider à l'acide nucléique. Elle porte aussi sur des méthodes de production de conjugués oligonucléotido-peptidiques, et sur des intermédiaires nucléotidiques de ces conjugués qui possèdent des groupes mercaptoéthyles protégés par des groupes butylthio. Ces intermédiaires peuvent former des conjugués oligonucléotido-peptidiques par une réaction avec un maléimido-peptide.
PCT/IB1999/001116 1998-06-01 1999-05-28 Methode de marquage d'acide nucleique WO1999063111A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39525/99A AU3952599A (en) 1998-06-01 1999-05-28 Method of labelling nucleic acid

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9811771.6 1998-06-01
GBGB9811771.6A GB9811771D0 (fr) 1998-06-01 1998-06-01
GB9813765.6 1998-06-25
GBGB9813765.6A GB9813765D0 (fr) 1998-06-25 1998-06-25

Publications (2)

Publication Number Publication Date
WO1999063111A2 true WO1999063111A2 (fr) 1999-12-09
WO1999063111A3 WO1999063111A3 (fr) 2000-02-24

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015012912A3 (fr) * 2013-04-22 2015-04-16 Northwestern University Phosphoramidites d'alcyne et préparation de constructions d'acides nucléiques sphériques

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992000989A1 (fr) * 1990-07-10 1992-01-23 Imperial Chemical Industries Plc Procede de marquage non-isotopique d'acide nucleique

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015012912A3 (fr) * 2013-04-22 2015-04-16 Northwestern University Phosphoramidites d'alcyne et préparation de constructions d'acides nucléiques sphériques

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WO1999063111A3 (fr) 2000-02-24
AU3952599A (en) 1999-12-20

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