Europalsches Patentamt J European Patent Office @ Publication number: 0 212 546 A2 Office europeen des brevets @ EUROPEAN PATENT APPLICATION @ Application number: 86111137.5 © Int. CI": C07H 21/00 // C12Q1/68 <g> Date of filing : 12.08.86 Priority: 13.08.85 US 765288 © Applicant : ENZO BIOCHEM, INC., 325 Hudson Street, New York, N.Y. 10013 (US) Inventor: Stavrianopoulos, Jannls, 1380 Riverside Drive Date of publication of application: 04.03.87 Apartment 8E, New York New York 10033 (US) Bulletin 87/10 Inventor: Rabbanl, Elazar, 69 Fifth Avenue, New York New York 10003 (US) Representative: Vossius & Partner, @ Designated Contracting States : AT BE CH DE FR GB IT LI Slebertstrasse 4 P.O. Box 86 07 67, LUNLSE D-8000M(inchen86(DE) <§) Method for labeling polynucleotide sequences. @ A method for labeling a polynucleotide sequence to form a polynucleotide probe is disclosed. The method comprises forming a modified polynucleotide sequence wherein the mod- ification comprises the cleavage of preselected bases to gen- erate sugar aldehydes. The preselected bases are either qua- ternized nitrogen purines or uracil. A signalling moiety is att- ached to the modified polynucleotide sequence by means of the sugar aldehydes. CM < to <* 10 CM 01 o & HI *CTORUM AG Ourref.: U 796 EP V08sius*pARTn__ 0912546 Case: ENZ-22 ao*ISNTANW*'--5 \ 2 AUQ Enzo Biochem, Inc. iV-^JS^- New York, USA 47407V 4 METHOD FOR LABELING POLYMOCLEOTIDE SEQUENCES BACKGROUND OF THE INVENTION 5 Recent advances in molecular biology have allowed the development of improved methods for detecting infectious agents in clinical specimens. One promising method is that of hybridizing a 10 polynucleotide probe to a polynucleotide analyte of an infectious agent. The hybridization can be carried out in intact cells or in sample extracts. This technique has the advantage over immunoassays/ e.g. ELISA, in that the detection of the infectious 15 agent can be achieved even when epitopes of the particular infectious agent are not being expressed. This permits the detection of an infectious agent that might otherwise escape detection. 20 Polynucleotide probes consist essentially of two components: a polynucleotide sequence and a signalling moiety. The polynucleotide sequence is capable of hybridizing to a segment of the target genome. The sequence can be comprised of 25 deoxyr ibonucleotides or ribonucleotides and can range in length from about fifteen to several thousand nucleotide's? The signalling moiety is a fragment or molecule that provides a signal or the means for generating a signal to indicate that hybridization 30 has taken place. 32 First generation signalling moieties comprised P atoms and provided a radioactive signal. The use of 32 these signalling moieties has drawbacks in that P 3? has a 14-day half -life, poses health risks, and requires special disposal facilities. This lead to 3212546 -2- :he development of the current second-generation lon-isotopic signalling moieties that permit one to jenerate a signal by means of fluorescence, Luminescence or chromogen. These second generation probes have a drawback in that the signalling moiety is attached to the base portions of the polynucleotide; the signalling moiety can thus sterically interfere with proper base pairing. Recently, a polynucleotide probe was modified in which a fluorescent signalling moiety was attached to the sugar portions of the polynucleotide. The method comprised depurinating the polynucleotide at a high temperature and acidic pH to generate sugar aldehydes. Acriflavin dyes were attached to the sugars via a Schiff base at the 1 position of the sugar. See "Fluorescent Labeling of DNA by Covalent Binding of Acriflavin", J.W. Levinson, A. deSostoa, L.F. Kiebes, and J.J. McCormick, Biophysical Journal, 16: 91a (1976) . This method has a drawback, in that the acidic conditions required for depurination catalyzes the formation of cross-links between the sugar aldehydes and amino bases. The cross-links interfere with proper base hybridization to the target genome. A second drawback is that one dye per two hundred sugars was the highest ratio that could be achieved that would still permit the DNA to hybridize to itself at 85% of the value of the control DNA. The ability to selectively remove pyrimidines and N-quaternized purines from polynucleotides is known. been with However , the removal in the past has not the intent of labeling a polynucleotide sequence. 0212546 -3- Rather , it was to break the polynucleotide chain to determine its base sequence. See Allan M. Maxam and Walter Gilbert, Methods In Enzymology, 6j>, 449-560, 1980. OBJECTS OF THE INVENTION 10 It is an object of this invention to provide a method for labeling a polynucleotide sequence by attaching a signalling moiety to the sugar portion of the polynucleotide sequence. 15 It is another object of this invention to provide a method for generating many sugar aldehydes under conditions wherein cross-linking between the aldehyde and amino bases is severely minimized. 20 SUMMARY OF THE INVENTION This invention relates to a method for labeling a polynucleotide sequence comprising generating a sugar aldehyde on that sequence and then attaching a c 5 signalling moiety to the sequence by means of the sugar aldehyde. The sugar aldehyde may be generated by removing- an N-alkylated adenine or guanine base from a modified polynucleotide sequence, or a uracil base from a polynucleotide sequence. Removal of the N-alkylated adenine or guanine base (depurination) is carried out by hydrolysis which generates the sugar aldehyde. Removal of the uracil base is carried out by reacting the polynucleotide sequence with hydroxylamine . The hydroxylamine cleaves the uracil base while forming a sugar oxime. Contacting the sugar oxime with a ketone-containing compound 0212546 -4- generates the sugar aldehyde. ^ The depurination is carried out from about pH 5.0 to about pH 7.5, preferably from about pH 6.0 to about 7.5 at a temperature from about 40° to about 60°C. A signalling moiety or bridging moiety is present during the depurination step. 10 DETAILED DESCRIPTION OF THE INVENTION A method is provided for modifying a polynucleotide sequence for use as a probe. The signalling moiety is attached to the sugar portion of the sequence. The method comprises removing a preselected purine or uracil base from the sequence to generate apurinic or apyrimidinic sites. The sugars at those sites contain either an aldehyde, i.e. a sugar aldehyde, or Schiff base at the 1-position depending on if the base removed is a quaternized purine or uracil respectively. Sugars containing a Schiff base as a result of uracil cleavage are hydrolyzed to the aldehyde, i.e. a sugar aldehyde is formed. A signalling moiety is then attached to the polynucleotide sequence via the sugar aldehyde. The preselected purine bases adaptable to this invention must contain at least one quaternized nitrogen; i.e. N-l, N-3, and N-7 position. Quaternizing the nitrogen destabilizes the glycosidic bond permitting the cleavage of the purine from the polynucleotide at neutral conditions. The carrying out of this reaction at neutral conditions minimizes the formation of cross-links between the aldehyde group of the sugar and the amino bases. Release of 0212546 -5- the purine generates a sugar aldehyde at the site of cleavage . Purine nucleotide as used in this invention means either an individual nucleotide or a nucleotide that is integral to a polynucleotide. Quaterni zat ion of a purine nucleotide is generally carried out by reacting a purine nucleotide with an alkylating agent. When the alkylation is carried out at the nucleotide level, the quaternized nitrogen purine nucleotide can be chemically or enzymatically incorporated into the polynucleotide sequence. The alkylations should be carried out at a pH ranging from about 6.0 to about 8.0. At a pH below 6.0, the acidity will adversely affect the product. When alkylation is carried out at the nucleotide level, the acidity will promote cleavage of the quaternized nitrogen purine from the sugar. This cleavage will decrease the yield of product that can be utilized as a substrate by the polymerase enzyme (polymerase enzymes require a substrate comprising a base, sugar, and phosphate group for enzymatic incorporation) . When alkylation is carried out at the polynucleotide level, the* acidity will promote cross-linking of the bases to the sugar distorting the polynucleotide tertiary structure, and precluding the use of the polynucleotide as a probe. If the alkylation is done at a pH greater than about 8.0, the polynucleotide sequence can be cut at the site of the alkylated base, generating small ineffective pieces of probe. The polynucleotide sequence comprising a quaternized nitrogen purine nucleotide is referred to herein as a alkylated polynucleotide sequence. 0212546 -6- Alkylating agents that are suitable for use with this 5 invention include, for example, alkyl halides, dialkyl sulfates, dialkylsulfonates , and diazomethane . Preferred alkylating agents are the alkyl iodides, alkyl halides, dialkyl sulfates and diazomethane. Particularly preferred are methyl iodide, dimethyl sulfate, and diazomethane. The purine nitrogen quaternized when alkylation is carried out at the nucleotide level should be the N-7. This is because alkylation at N-l or at N-3 2-5 will distort the template and interfere with the base pairing of the purine to its complementary pyrimidine base. This base pairing is a requirement for incorporation by nick translation. The purine nitrogen that is quaternized when alkylation is carried out at the polynucleotide level can be N-l, N-3, or N-7 since the purine is already incorporated into the polynucleotide. It is preferred to purify the alkylated nucleotide 2- prior to incorporation into the polynucleotide sequence, and to purify the alkylated polynucleotide prior to dsguri nation. The alkylated nucleotide can be purified by chromatography, for example, ion exchange chromatography, and Sephadex chromatography.