NUCLEIC ACID PROBE COUPLED TO RADIOACTIVE LABEL

Abstract: A probe comprising a nucleic acid moiety coupled to a radioactive label through an -S-S-, -Hg-S-, -CONH- or -CH₂N- linkage. This permits nucleic acid assays to be made radioactively.

Description: Nucleic acid hybridization methods are wiSeIy¯u-sea for the detection and isolation of specific genes. To date, radioactively labeled nucleic acid probes constitute the most sensitive detection system. The method of radioactive labeling includes the nick translation process, the use of kinase to phosphorylate the 5' hydroxy residue and the use of terminal transferase (TdT). For oligonucleotides, the nick translation method is not applicable and the use of TdT (for labeling purposes) can produce heterogeneous distribution of molecules. Moreover, for restriction fragments, kinase mediated reaction will work only after the removal of existing 5' - phosphate by alkaline phosphatase. All these procedures can be practiced only by skilled workers in the art. After all the above mentioned reactions, difficult purifications and separation of the labeled probe from the enzyme and unreacted radioactive starting materials are still required. It is accordingly an object of the present invention to provide a method of labeling oligonucleotide and single or double stranded DNA or RNA, via a nonenzymatic chemical method. This and other objects and advantages are realized in accordance with the present invention pursuant to which a nucleic acid to serve as a probe is modified at the 3' or 5' end to form a nucleotide with a primary alkylamine residue. The primary amine is then chemically reacted with the radioactive molecule. The reaction can be of the following types: EMI1.1 EMI2.1 The new compounds invented for this purpose include nucleic acids with terminal nucleotides with - NH2, - SH, - Hg. They are described sghematically in the drawings wherein Figures 1 to 3 are schematic flow sheets of the synthesis and labelling of modified nucleic acid probes in accordance with the present invention. Referring now more particulatey to the drawings, in Fig. 1, a single or double stranded nucleic acid probe is enzymatically coupled to a ribonucleoside triphosphate residue containing either of two types of bases. If the base is B-hexylamino A or 5-allylamino U as at the right, the product is directly ready for radioactive labelling. With other bases, as at the left in Fig. 1, the product is subjected to periodate oxidation to split the cis-diol, forming two aldehyde groups which then condense with a diamine or polyamine as in Schiff's base condensation, followed by sodium borohydride reduction of the Schiff's base function to form the corresponding saturated amine. If the aldehyd groups are condensed with the amino group of an aminoalkylthioalcohol, the result is a probe which carries an -SH group. In Fig. 2, (A) at the top, the products of Fig. 1 with free-Nez groups are reacted with oxidized z 32p-labelled ATP to produce a Schiff's base which is borohydride-reduced to give a 32p-labelled product. In Fig. 2(B), at the bottom, the products of Fig. 1 are directly reacted with Bolton Hunter reagent to form an amide carrying a radioactive 125I residue. On the other hand, the nucleoside triphosphate may carry a Hg or SH radical, as on the purine or pyrimidine ring. In Fig. 3, (A) the starting material is reacted with nucleoside triphosphate carrying an Hg or SH residue. The Hg or SH radical can be provided by employing a nucleoside triphosphate such as thio-ITP or mercurated UPT or CTP or the like, and the ultimate linkage can be through -S-Hg or -S-S-. Otherwise noted, a nucleotide can be enzymatically coupled to one end of a (NA) probe. Such nucleotide can have a modified structure on its heterocyclic ring (mod-N'), i.e. the purine or pyrimidine ring carries a SH or Hg radical. At (B), the product is reacted with a radioactive reagent such as 2-35S- mercaptoethanol to produce a radioactively labeled product. If the nucleotide enzymatically coupled to one end of a (NA) probe is a triphosphate such as etheno ATP or CTP, it will constitute a directly-readable label, being fluorescent. The invention will be further described in the following illustrative examples wherein all part are by weight unless otherwise expressed: Example 1. Preparation of modified oligonucleotide with a spermine residue at the 3' end. HB 19 A' or HB 19S (Conner et al, Proc. Natl. Acad. Sci., U.S., 80, 278 (1983)) have been prepared by a known triester method (Dembek et al., J. Am. Chem. Soc., 103, 706(1981). 40 g of the oligonucleotide is dissolved in 1QOpS TdT buffer (potassium cacodylate 200 mM (pH 7.2); 5 m@i magnesium chloride; 0.2 mM dithiothreitol). To the solution, 10 l lmM ATP is added. The mixture is incubated at 370C for 5 minutes. Then 400 units of terminal deoxynucleotidyl transferase (TdT) is added. (TdT was purchased from P.L. Biochemicals). The reaction mixture is incubated at 370C for 45-60 minutes, then quenched by adding EDTA (final concentration 10mM) and warming the solution to 600C. The pH of the solution is then raised to 12-13 by adding 1M sodium hydroxide. The whole solution is then heated at 600C for'bne hour. This procedure removes all but one terminal ribonucleotide residue. The solution is then brought to pH 9 with hydrochloric acid and 10 units of bacterial alkaline phosphatase is added. The mixture is incubated at 370C for 10 hours and then neutralized with HC1 to pH 7. The solution is then diluted to about lml (10 times dilution) with water. This reduces the ionic strength of the solution. The mixture is then loaded onto a DEAE cellulose column (5cm length, 0.5 cm diameter) which has been preequilibriated with TE (lOmM Tris, lmM EDTA; pH 7.2, adjusted with hydrochloric acid). The column is then washed with TE buffer containing lOOmM NaCl. Oligonucleotide with the terminal ribonucleotide residue is eluted with TE containing 600 mM NaCl. The nucleic acid sample is then partially desalted on a Sephadex G10 column. The final volume of solution containing the nucleic acid is about 300 pl. To this solution, acetic acid (1M) is added to adjust the pH to 5 and then a freshly prepared solution of sodium periodate (pH 5, Na-acetate buffer) is added to a final concentration of lmM. The oxidation is allowed to proceed for 30 min at 250C. Then the pH of the solution is increased to 8.5 with sodium hydroxide and one tenth volume of lOmM hexamethylene diamine or spermine solution (pH adjusted to 7-8 for solubilization)in water is added. The reaction mixture is incubated at 250C for 60 minutes and the Schiff's base formed is reduced with sodium borohydride by adding 10mug borohydride solid in five steps. A 15 minute time interval is allowed between each step. A similar procedure is followed for DNA. The exception is in the use of phenol extraction and ethanol precipitation for deproteinization. No DEAE column has been used when DNA is modified. Summarizing the steps used: EMI6.1 <tb> TdT <tb> Oligonucleotides <SEP> A <SEP> Oligonuc-A-A-A <tb> <SEP> 1. <SEP> Alkali <SEP> digestion <tb> <SEP> 2. <SEP> Alkaline <SEP> Phosphatase <tb> <SEP> treatment <tb> <SEP> 3. <SEP> DEAE <SEP> column <tb> <SEP> 4. <SEP> Desalting <SEP> on <SEP> G10 <tb> <SEP> [Ol <tb> <SEP> Oligonuc <SEP> - <SEP> CHOs <SEP> Oligonuc-A <tb> B2N¯XIBz <SEP> 1 <tb> <SEP> Oligonuc <SEP> - <SEP> CR <SEP> = <SEP> N¯¯v¯¯aNH2 <tb> <SEP> Reduction <tb> <SEP> Reduction <tb> <SEP> Oligonuc <SEP> - <SEP> CH2-NH <SEP> 2 <tb> If DNA is used, in place of steps 3 and 4, phenol extraction and ethanol precipitation are used. Example 2 Coupling of hexylamino ATP to DNA or oligonucleotide at the 3' end. Nucleic acid sample as in example 1 is taken in TdT buffer. To the solution, 10p1 1 mM hexylamino ATP is added. Then the TdT reaction is carried out as in example 1. Following the reaction, the sample is treated with 1M sodium hydroxide at 600C for 30 minutes. Then it is neutralized with HC1 to pH 7.2. For DNA, phenol extraction and ethanol precipitation is done by shaking the mixture with phenol, removing the phenol phase, adding ethanol and cooling the mixture on dry ice. For oligonucleotide, a DEAE column is used as in example 1. The same procedure can be followed to couple any -NH2 containing nucleotide to DNA or oligonucleotide provided the residue is covalently linked to the nucleotide base in proper position. Example 3 Reaction of the product of Example 1 with BOLTON HUNTER REAGENT for radio iodination. Since DNA is not structurally affected by benzene, the aqueous solution of the product of example 1 (10 100 ug in 100 ul of 0.1M borate buffer, pH 8) is mixed with lmCi - Bolton Hunter reagent in the benzene solution supplied and the mixture is shaken for 15 minutes at OOC. The reaction works at any temperature between 0 and 500C and at pH between 7 and 9.5. At higher pH and higher temperature, the life time of the reagent is short and it reacts faster with water; hence the yield can go down to 10 - 15%. After the reaction, the whole mixture is passed through a Sephadex G25 column and the excluded radioactive fractions are pooled for hybridization purposes. Example 4 32p labeling without the use of an enzyme. 32p labeled ATP (at the phosphate residue) is dissolved in 100 mM sodium acetate (pH 5) buffer (1 mM concentration) and oxidized with sodium periodate in conventional manner by adding sodium periodate (final concentration 2mM). After the reaction, the pH of the solution is adjusted to 8 with sodium hydroxide solution. Then the product is mixed with the product of Example 1 or 2 in the same buffer in equimolar concentration with respect to primary amine containing ends and the reaction is carried out for 60 minutes at 200C. The product is reduced with sodium borohydride solution as in Example 1 and purified on a Sephadex G10 column, collecting only the fraction corresponding to void volume. Example 5 Hybridization of the labeled probe The detection probe of EP 841 07 248.1 is labeled with either 1251 or 32P as in Examples 3 and 4, then hybridized with DNA from blood samples and assayed as described in the foregoing application. Example 6 Hybridization with labeled modified oligonucleotides Modified oligonucleotides prepared from Hss l9A' and H 19S (Example 1) are labeled with 32p as in example 4. The labeled modified oligonucleotides have been hybridized with blood DNA following the method of Conner et al (Proc. Natl. Acad. Sci., US. 80, 278-282 (1983). The improvement in using the modified oligonucleotides is the labeling procedure. The present labeling method does not require an enzyme and the efficiency is greatly increased {30-90%}. It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

Claims: WHAT IS CLAIMED IS: 1. A probe comprising a nucleic acid moiety coupled to a radioactive label through an -S-S-,-Hg-S-, -CONHor -CH2Nlinkage. 2. A probe according to claim 1, wherein the linkage is a -CO-NH- or -CH2-N C linkage and is carried by a heterocyclic ring of the nucleic acid. 3. A probe according to claim 1, wherein the linkage is carried by the terminal ribose residue of the nucleic acid. 4. A probe according to any of claims 1 to 3, wherein the radioactive label is present on a nucleoside phosphate. 5. A probe according to any of claims 1 to 3, wherein the radioactive label is present on the radical of a Bolton Hunter reagent. 6. A nucleic acid material comprising a nucleic acid moiety carrying an aliphatic primary amino group. 7. A nucleic acid material according to claim 6, wherein the primary amino group is carried by a heterocyclic ring of the nucleic acid. 8. A nucleic acid material according to claim 6, wherein the primary amino group is carried by the terminal ribose residue of the nucleic acid. 9. A process for the production of a probe comprising a nucleic acid moiety coupled to a radioactive label through an -S-S-, -Hg-S-, -CQNH- or -CH2Nv C linkage, carried by a heterocyclic ring wherein a nucleoside triphosphate which carries an -Hg or -SH group or an aminoalkyl moiety is enzymatically coupled to a nucleic acid and this product is coupled to radioactive material. 10. A process for the production of a probe comprising a nucleic acid moiety coupled to a radioactive label through an -S-S- ,-Hg-S-, -CONH- or -CH2N # linkage, carried by the terminal ribose residue wherein a nucleic acid which carries a ribose ring at the end is subjected to a periodate oxidation to split the cis-diol, forming two aldehyd groups, followed by a condensation with a diamin, a polyamin or an aminoalkylthioalcohol, optionally followed by a sodium borohydrid reduction of the Schiff's base fuction, the product of said condensation is a probe which carries an -NH2 or -SH moiety which is coupled to radioactive material.

Inventors: DATTAGUPTA, NANIBHUSHAN
Applicants: MOLECULAR DIAGNOSTICS, INC.
Application year: 1985
Main classification: C12Q1
IPC codes: G01N33, C12N15, C07H21, C12Q1