39

GATA 9(2): 39-47, 1992

ORIGINAL ARTICLES

Alkaline Phosphatase Inhibitors as Labels of DNA Probes ENRICO DAVINI, CRISTINA DI LEO, ANTONIO ROSSODIVITA, and PIERGIORGIO ZAPPELLI A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competitive inhibitor of the chromogenic enzyme alkaline phosphatase. The nucleic acids labeled in such a way were able to bind themselves to the enzyme, whose activity was restored in the presence of a chromogenic substrate. Five phosphonic-acid-containing reagents were synthesized and coupled to linearized pBR322 plasmid DNA by different condensation methods. Eight probes thus obtained were assayed in a modified dot-blot detection procedure obtaining the best nucleic acid detection sensitivity of 25 pg. Finally, five of the above probes were tested in hybridization experiments, reaching sensitivity of 50 pg.

Introduction DNA or RNA probes can be used for detection of nucleic acid sequences in cells. With the aim of eliminating the health hazard for clinical laboratories caused by the radioactivity of the commercially available 32p-labeled probes, various products and procedures have been reported for the nonradioactive DNA labeling [1-6]. In particular, nonradioactive labeling of nucleic acids can be achieved by (a) incorporation of a nonradioactively labeled nucleotide into the DNA [2, 7-10]; or (b) chemical modification of DNA with a signaling reagent (hapten, biotin, or enzyme) that

From Eniricerche Spa, Monterotondo,Italy. Address correspondence to Dr. P. Zappelli, EnirichercheSpa,

Via Ercole Ramarini 32, 00016 Monterotondo,Italy. Received 31 January 1992; revised and accepted 10 April 1992. Part of this work was presented at the Conventionon Biotechnologies in Medicine, Biochemistry,Environment,Agriculture and Zoology,Turin, Italy, 10-14 October 1988 (poster communication27); and at the 18thItalianChemicalSocietyCongress, S. Margheritadi Pula, Italy, 8-13 October 1989 (abst C-16).

is then recognized by an immunoenzymatic-colorimetric system [3, 4, 11-15]. Also from our laboratory has been reported work on biotin-labeled DNA probes [5, 6, 16]. Now we wish to refer to a new approach directed to explore a labeling method [17] based on peculiar features. We argue that it would be useful to take advantage of properties of the chromogenic enzyme additional to the catalytic activity in color production. A reversible inhibitor of the enzyme could provide the specific recognition and binding of the enzyme itself. With the aid of a bridging molecule bringing a linking group in one end to DNA, and in the other a reversible enzyme inhibitor, the nucleic acid and the enzyme might be put together directly without accessory linking compounds such as in the biotinavidin system. After that, the catalytic activity of the enzyme could be restored in the presence of the substrate, providing the chromogenic response. The overall procedure is depicted in Figure 1. This new approach to nucleic acid labeling was developed by synthesizing reagents having a competitive inhibitor of alkaline phosphatase (EC 3.1.3.1) in the labeling moiety. Phosphonic acids with general formula RR'CHPO3H2 have been reported to be inhibitors of alkaline phosphatase. In particular, 2-amino-3-(4-hydroxy-3-[4-phosphonomethyl phenylazo])-phenyl propionic acid (I) and 4-aminobenzyl phosphonic acid (II) derivatives were used as ligands for affinity chromatography [18] (see Figure 2). One of the approaches reported from our and other laboratories to obtain nonradioactive DNA probes deals with the chemical labeling of nucleic acid by means of a complex reagent having the general structure DNA-linking group - - s p a c e r - reporter group According to this formula, we synthesized five labeling reagents having as reporter groups the inhibitor moieties derived from compounds I and II, and different combinations of the linker and of the spacer portions. These new reagents were coupled to DNA (linearized plasmid pBR322) and the probes were tested for the colorimetric detection of target DNA, immobilized on nitrocellulose filter, in hybridization experiments.

Materials and Methods N-Carbobenzyloxy-glycylglycyl-L-tyrosine was from Bachem; 4-fluoro-3-nitrophenyl azide was from Aid-

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40

GATA 9(2): 39-47, 1992

/

E. Davini et al.

Labeling

• ¢>-..,>

/

NH2~HCOOH CH~

--

\ j

J Hybrldizatlon

~'XN=N--@CH

zPO3H2

OH A',\\\\\\\\\\\\'~

5

' Detection

N H = / / ~ ~ CHzPO3Hz II

// ~

Chromogenl¢ re=Ponse

Figure 1. Enzyme inhibitor approach for nonradioactive DNA labeling and detection. DNA is first labeled with a bifunctional reagent, bringing, as reporter group, a reversible inhibitor of a chromogenicenzyme. The labeled probe is then hybridized with target DNA, immobilized on nitrocellulose filter. Finally, the double-stranded labeled DNA is linked to the enzyme, and the complex left to react with a chromogenic substrate for the enzyme, obtaining the DNA detection. L, linking group to DNA; 1, enzyme reversible inhibitor; E, enzyme; and S, chromogenic substrate.

rich; L-tyrosine terbutylester, N,N'-dicyclohexylcarbodiimide, and Tween 20, were from Fluka; pBR322 plasmid, BAMHI restriction enzyme, and 4-nitrophenylphosphate disodium salt, were from Boehringer-Mannheim; and Escherichia coli DNA, bovine serum albumin (BSA), nitro-blue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nick translation kit, Ficoll 400, alkaline phosphatase, and 4-aminobenzylphosphonic acid were from Sigma. The other reagents were purchased as follows: biotinylated alkaline phosphatase from BRL, streptavidin from Amersham International, agarose from Pharmacia, sodium dodecylsulfate (SDS) from Carlo Erba, and polyvinylpyrrolidone (PVP) from Merck. Nitrocellulose filters were obtained from Schleicher and Schuell. IH nuclear magnetic resonance (NMR) spectra were recorded on Varian XR 400 whereas ultraviolet (UV) spectra were recorded on Perkin-Elmer Lambda 5 and LKB. Fast atom bombardment mass spectra MS (FAB) were recorded on Finnigan Mat 90. Thinlayer chromatography (TLC) was performed on silica gel plates from Merck.

Synthesis of labeling reagents Diazonium Salt of 4-Aminobenzylphosphonic Acid. NaNOz (69 mg, 1 mmol) was added in small

Fisure 2. Scheme 1. Structures of alkaline phosphatase inhibitors: (1) 2-amino-3-(4-hydroxy-3-[4-phosphonomethyl-phenylazo])-phenylpropionicacid; and (I1)3-aminobenzylphosphonic acid.

portions to an ice-cooled, stirred solution of 4-aminobenzylphosphonic acid (II, 189 mg, 1 mmol) in 1 M HC1 (2.5 ml). The solution was left under stirring for 1 h and then diluted to 10 ml with H20 (final concentration 0.1 M) and stored in an ice bath until its utilization in the following coupling reaction. UV (water): hmax287 nm.

2-(Glycvlglycyl)Amino-3-(4-Hydroxy-3-[4-Phosphonomethylphenylazo] )-Phenyl Propionic Acid (IV). N-Carbobenzyloxy-glycylglycyl-L-tyrosine (III, 200 mg, 0.47 mmol) was suspended in 6.5 ml of 0.01 M NaOH; the mixture was cooled to 0°C, and 5 ml of 0.1 M solution of the diazonium salt of II were added under stirring, keeping the pH between 9 and 9.5 by NaOH addition. The solution was left under stirring at 5°C overnight and then acidified with HC1 to pH 1. The resulting precipitate was filtered, dissolved in 1 N NaOH, precipitated with concentrated HC1, and filtered. The resulting product was washed first with EtOH and then with Et20 and dried under vacuum (147 mg, 63% yield). MS (FAB) was in agreement with the structure. By carefully controlling the acidification step of the diazo coupling product (not exceeding pH 3.5), the benzyloxycarbonyl intermediate was isolated by extraction with 2-butanol. 1H NMR (D6-DMSO): 8 11.10 (broad s, IH, COOH), 8.22 (d, 1H, NH-tyr), 8.05 (t, IH, NHgly), 7.9-7.0 (13H, aromatics, Ph-carbobenzyloxy, OCONH and Ph-tyr), 5.03 (s, 2H, OCHzPh), 4.45 (m, 1H, CH tyr.), 3.8-3.6 (4H, CHz-gly), and 3 . 1 5 2.85 (4H, CH2P and C H 2 - t y r ) .

N-Carbobenzyloxy-6-Aminohexanoic Acid (VI). To a cooled ( - 10°C) solution of 6-aminohexanoic acid

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41

GATA 9(2): 39-47, 1992

Enzyme Inhibitors as D N A Labels

Table 1. CouplingProcedures of the SynthesizedAlkaline Phosphatase Inhibitors to DNA for the Preparation of the Labeled Probes Compound IV X XIII IV X XIII XV XVII

Procedure

Probe

One-step condensation One-step condensation One-step condensation Two-step condensation Two-step condensation Two-step condensation Photoactivation Alkylation

PP-35 PP-40 PP-34 PP-42 PP-43 PP-41 PP-44 PP-45

Table 2. Direct Dot-Blot Detection SensitivityResults of pBR322 DNA Probes PP-34 and PP-35 Using Different Developing Procedures DNA detected conc. (ng) Probe

Procedure 1

Procedure 2

Procedure 3

Procedure 4

PP-34 PP-35

>500 >500

50 500

50 500

0.5 0.5

DNA detection system by BRL--procedure 1: alkaline phosphatase (AP) from Sigma; procedure 2: AP from BRL; procedure 3: BRL biotinylated AP, streptavidin, and biotinylated AP; and procedure 4: Blugene from BRL.

CHCH2), 2.10 (m, 2H, CHzCO), and 1.43 (9H, s, terbutyl). (V) (750 mg, 5.7 mmol) in 4.5 ml of 4 N NaOH were added, dropwise and under stirring, 1.2 ml (9.05 mmol) of benzyl chloroformate. The mixture was allowed to warm up to 0°C under stirring (1 h), then extracted with Et20 to eliminate unreacted benzylchloroformate, and, after acidification with 6 N HCI, extracted with EtOAc. The acetate extracts, washed with H20, dried (Na2SO4), and evaporated in vacuo, gave 1.2 g (80% yield) ofVI. TLC (Silica F): nBuOHAcOH-H20 6:2:2. IH NMR (D6-DMSO): B 7.35 (5H, aromatic), 7.25 (t, 1H, NH), 5.0 (s, 2H, CH2Ph), 2.95 (q, 2H, CH2N), 2.20 (m, 2H, CH20), and 1.51.4 (m, 6H, CH2).

N-( N-C arbobenzyloxy-6-Aminohexanoyl )-L- Tyrosine Terbutyl Ester (VIII). To a cooled (0°C) and stirred solution of VI (150 mg, 0.57 mmol) and of u-tyrosine terbutyl ester VII (133 mg, 0.57 mmol) in dimethoxyethane (2 ml) was added N,N'-dicyclohexylcarbodiimide (116 mg, 0.57 mmol). The solution was left under stirring at 4°C overnight. The resulting mixture was filtered and the precipitate was washed several times with dimethoxyethane. The combined solutions and washings were concentrated in vacuo and the residue dissolved in EtOAc. The solution was washed successively with 0.1 M citric acid (to eliminate unreacted VII), then with 0.5 M NaHCO3 (to eliminate unreacted VI), and finally with water. The organic extract dried (Na2SO4) and evaporated in vacuo gave a residue that was chromatographed on Lobar Lichroprep Si 60 column, eluted with AcOEt-petrol ether 40-70 (3:2). Fractions with positive UV(h=280nm) and Folin's reagent response were collected and evaporated in vacuo to give 90 mg (33% yield) of VIII. IH NMR (CDC13): ~ 7.80 (s, IH, OH phenolic), 7.45 (m, 5H, aromatic), 7.40 (t, 1H, OCONH), 7.00-6.70 (d, d, 4H, aromatic), 6.10 (d, 1H, NHCH), 5.05 (s, 2H, OCH2), 4.70 (q, 1H, CH), 3.10 (q, 2H, NHCH2), 2.85 (dd, 2H,

2-( N-Carbobenzyloxy-6-Aminohexanoyl) Amino-3 (4 -Hydroxy- 3-[4 -P hosphonomethylphenylazo ]) -P henyl Propionic Acid (IX). VIII (90 mg, 0.18 mmol) was dissolved in 1 ml of MeOH, and 1 M NaOH was added until pH 10. To the cooled (5°C) solution, 1.8 ml of solution of the diazonium salt of II (prepared as above) was added dropwise, maintaining the pH in the range 9-9.5 by addition of 0.5 N NaOH; some drops of MeOH were also added to keep all products in solution. After 18 h at 5°C, 6 N HC1 was added until pH 1; the resulting precipitate was filtered, dissolved in 0.1 N NaOH, precipitated with 6 N HC1, and filtered. The product was dried in vacuo to give 30 mg of IX, conmining minor amounts of X, as determined by IH NMR (D6-DMSO). 2 - (6- Aminohexano yl )Amino- 3 -( 4 -Hydr oxy- 3 - [4Phosphonomethylphenylazo])-Phenyl Propionic Acid (X). Under stirring, 0.3 ml of 33% HBr in AcOH were added to IX (30 mg). The solution was left 1 h at r.t.; Et20 was added and the red precipitate was filtered, obtaining 19 mg (14% yield) of X as hydrobromide. MS (FAB) was in agreement with the structure. (N-C arbobenzyloxy )-6-Aminohexanoyl-N- Hydroxysuccinimidester (XI). N-hydroxysuccinimide (436 mg, 3.79 mmol) and N,N'-dicyclohexylcarbodiimide (858 mg, 4.16 mmol) were added under stirring to a cooled (0°C) solution of VI (1 g, 3.7 mmol) in dimethoxyethane (1.5 ml). The mixture was left under stirring for 20 h. The precipitate was filtered off and washed with anhydrous Et20. The organic solution evaporated in vacuo left a residue that chromatographed on silica gel with EtOAc-petrol ether 40-70 (7:3), gave XI (793 mg, 58% yield). IH NMR (D6-DMSO): B 7.35 (m, 5H, aromatic), 7.22 (t, 1H, NH), 5.01 (s, 2H, CH2Ph), 2.98 (q, 2H, CH2NH),

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42

GATA 9(2): 39-47, 1992

2.80 (s, 4H, COCH2CH2CO), 2.65 (t, 2H, CHzCOO), and 1.60-1.30 (m, 6H, CH2).

N- (N- Carbobenzyloxy)- 6- Aminohexanoyl-4-Aminobenzylphosphonic Acid (XII). To 228 mg (0.63 mmol) of XI, dissolved in 10 ml of anhydrous dimethoxyethane, a solution of I! (117 rag, 0.63 mmol) in 6 ml of 0.25 M NaHCO3 was added at r.t. under stirring (final pH, 7.7). Stirring was continued for 7.5 h and then the solution was evaporated in vacuo. The residue was dissolved in water (10 ml) and extracted with Et20 to eliminate unreacted XI. The aqueous solution, cooled in an ice bath, was acidified to pH 1 with 6 N HCI. The resulting precipitate was filtered, washed with water, and dried in vacuo at 35°C, giving XII (98 mg, 36% yield). IH NMR (D6DMSO): 8 9.80 (s, IH, NH-Ph), 7.30 (m, 5H, aromatic), 7.20 (t, 1H, NHCH2), 7.50-7.10 (d, d, 4H, aromatic), 5.00 (s, 2H, CH2Ph), 2.95 (q, 2H, CH2NH), 2.87 (d, 2H, CHzP), 2.25 (t, 2H, CHzCO), and 1.601.20 (m, 6H, CH2).

N-(6-Aminohexanoyl)-4-Aminobenzylphosphonic Acid (Xlll). Under stirring, 0.3 ml of 33% HBr in AcOH were added to XII (33 mg). The solution was left 1 h at r.t.; Et20 (10 ml) was added and the precipitate, washed with 3 x 5 ml of Et20, was filtered and dried in vacuo. The residue was dissolved in water and lyophilized to give XIII (29 mg, 90% yield as hydrobromide). 1H NMR (D6-DMSO): 8 9.90 (s, IH, NH-Ph), 7.80 (broad s, 3H, NH3+), 7.50-7.10 (d, d, 4H, aromatic), 2.92 (d, 2H, CHAP), 2.80 (q, 2H, CH2NH), 2.28 (t, 2H, CH2CO), and 1.60-1.20 (m, 6H, CH2).

(N-[4-Azido-2-Nitrophenylamino-6-Hexanoyl])-4Aminobenzylphosphonic Acid (XV). This preparation was made in dark glassware. 4-Fluoro-3-nitrophenyl azide XIV (22 mg, 0.12 mmol), dissolved in 0.8 ml of EtOH, was added to a solution of XIII (38.6 mg, 0.1 mmol) in 2 ml of 0.3 M NaHCO3. The solution was left overnight at r.t. under stirring and then extracted with EtzO (3 x 5 ml) to eliminate unreacted XIV. The resulting water solution was acidified with 6 N HC1 to pH 1 and extracted with EtOAc (3 x 10 ml). The collected extracts, dried over NazSO4 and concentrated in vacuo, afforded 6.7 mg (14.5% yield) ofXV. 1H NMR (D6-DMSO): 8 9.79 (s, 1H, NH-Ph), 7.70 (1H, aromatic),7.45 (d, 2H, aromatic), 7.31 (m, 1H, aromatic), 7.12 (m, 3H, aromatic), 2.80 (q, 2H, CH2NH), 2.90 (d, 2H, CH2P), 2.90 (2H, CH2NH, partly overlapped by the solvent), 2.30 (t, 2H, CH2CO), 1.65 (broad s, 4H, CH2), and 1.40 (m, 2H, CH2).

E. Daviniet al.

lodoacetyI-N-Hydroxysuccinimidester (XVI). This preparation was made in dark glassware. To a stirred and cooled (0°C) solution of iodoacetic acid (1 g, 5.38 mmol) andN-hydroxysuccinimide (0.62 g, 5.38 mmol) in 10 ml of dimethoxyethane, N,N'-dicyclohexyl-carbodiimide (1.11 g, 5.38 mmol) was added. After overnight stirring at 4°C, the mixture was filtered off and the solution was evaporated in vacuo at r.t., giving 1.33 g (87% yield) of XVI. (N-[lodoacetylamino-6-hexanoyl] )-4-Aminobenzylphosphonic Acid (XVII). This preparation was made in dark glassware. To a solution of XIII (35 mg, 0.092 mmol) in concentrated aqueous NaOH (2 ml, final pH 9.5), a solution of XVI (26 mg, 0.092 mmol) in 1 ml of dimethoxyethane was added. The mixture was stirred for 6 h, the pH adjusted to 9 with NaOH, and evaporated in vacuo. The residue was extracted with dimethoxyethane (3 x 15 ml) to eliminate unreacted XVI. The residue was dissolved in 2 ml of water and the solution, acidified to pH 1 with 6 N HCI, was extracted with EtOAc (3 x 20 ml). The combined extracts, dried over Na2SO4 and concentrated in vacuo, afforded 14.5 mg (33.7% yield) of XVII. IH NMR (D6DMSO): 8 9.85 (s, 1H, NH-Ph), 8.25 (t, 1H, NHCHz), 7.50 (d, 2H, aromatic), 7.10 (d, 2H, aromatic), 4.5-4.0 (2H, CH2I), 3.08 (q, 2H, CH2NH), 2.90 (d, 2H, CH2P), 2.30 (t, 2H, CH2CO), and 1.60-1.20 (m, 6H, CH2).

Inhibition Kinetics Inhibition kinetics of alkaline phosphatase by the synthesized phosphonic acids were determined at pH 8.0, in 0. l M Tris-HC1 buffer. 4-Nitrophenyl phosphate at concentrations of 1, 0.35, 0.2, 0.15, and 0.1 mM was used as substrate. Assays were performed at r.t. in an UV spectrometer at 405-nm wavelength, using 1-ml cuvettes. Changes in optical density were recorded for 3-4 rain at 15-s intervals. Kinetic data were elaborated with a linear regression program on an IBM computer and Ki values were determined from differences in slope. The Km for the substrate, 4-nitrophenyl phosphate, determined under the same conditions was 3.63 x 10-5 M. The inhibition data for the phosphonic acids tested were as follows: I (Ki = 1.67 mM), II (Ki = 5.11 mM), IV (Ki = 0.07 mM), X (Ki = 0.19 mM), and XIII (Ki = 3.14 raM). To exclude the influence of one of the spacer arms used, we also tested compound III (Ki = 160 mM).

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43 Enzyme Inhibitors as DNA Labels

GATA 9(2): 39-47, 1992

Labeling o f DNA Probes

with Et20, as previously described in the preparation of probe PP-34. The probe was precipitated by cold ( - 20°C) ethanol addition, dried in vacuo, dissolved in 0.1 mM EDTA, and stored at -20°C.

Probe PP-34. Reagent XIII was linked to DNA via the condensation reaction. To 3 mg of the reagent in 50 ixl of 50 mM sodium phosphate buffer, pH 6.5, were added 10 ixl of a solution of 1 txg/ixl of DNA in 0.1 mM EDTA and 60 p.l of a solution of 0.2 M EDC ( 1-ethyl-3-[N,N-dimethylamino]-isopropylcarbodiimide hydrochloride) and 0.2 M N-methylimidazole. The reaction was carried out overnight at room temperature. The solution was alkalinized to pH 9 with 1 M NaOH, extracted first with 2butanol (2 x 100 I~1 and 1 x 50 ~xl) and then with Et20 (2 x 300 ixl), and DNA precipitated with cold EtOH. The pellet was dissolved in 500 I~1 of 50 mM phosphate buffer, pH 6.8, and purified on a Sephadex G-25 column, eluted with the same buffer. DNAcontaining fractions were concentrated with 2-butanol and later purified by Sephadex chromatography as above. Probe PP-35. The product was obtained as described for probe PP-34, but by substituting reagent IV for XIII. Probe PP-40. The product was obtained as described for probe PP-34, but by substituting reagent X for XIII. Probes PP-41, PP-42, and PP-43. Reagents XIII, IV, and X were also linked to DNA with a different procedure via a two-step condensation method: 20 Ixl of a solution of 0.5 txg/ixl of denatured DNA in 0.1 mM EDTA were added to 100 p~l of a solution of 0.12 M of N-methyl imidazole and 0.12 M of EDC, pH 6. After a 60-rain reaction at r.t., DNA was cold ethanol precipitated and dissolved in 100 ixl of an aqueous solution containing - 3 mg of the labeling reagent (pH of the mixture, 8). After a 1-h reaction at 50°C, DNA was purified as previously described for the one-step procedure. Probe PP-44. Reagent XV was coupled to DNA via the photoactivation reaction (all manipulations of reagent XV were performed in subdued light). A solution 1 txg/txl of reagent XV in water was added to an equal volume of a solution 1 Ixg/l~l of DNA in 0.1 mM EDTA (generally 10-15 ~g of DNA were labeled) and sealed in a glass capillary tube. The last, placed in an ice-water bath, was irradiated 15 min by a visible-light lamp (OSRAM MB/U 400 W). The reaction mixture was diluted to 100 txl with 0.1 M Tris-HC1, pH 9, and extracted with 2-butanol and

Probe PP-45. Reagent XVII was coupled to DNA by direct alkylation. To 10 ixl of a solution of 1 txg/~zl of DNA in 0.1 mM EDTA were added 200 txl of a solution of 2.5 mg of XVII in 125 mM sodium phosphate buffer, pH 6.5. The pH was adjusted to 6.8 by addition of 12 txl of 1 M NaOH. After 6 days' reaction at r.t., the solution was extracted with 2butanol, and DNA recovered by ethanol precipitation. General DNA Techniques Dot Blots. pBR322 plasmid DNA was linearized with BAMHI; Escherichia coli DNA was sonicated to obtain fragments of 100-150 base pairs. DNA samples were serially diluted, heat denatured in boiling water for 7 min, quickly cooled on ice, and spotted onto nitrocellulose filters (presoaked 20 min in 20 x SSC [20 x SSC = 3 M sodium chloride, 0.3 M sodium citrate, pH 7]). Filters were air dried and baked at 80°C for 2 h under vacuum. Hybridization. Filters were incubated in sealed plastic bags with prehybridization buffer at 42°C for 2 h. Prehybridization buffer was 50% deionized formamide, 50 mM sodium phosphate, pH 6.5, 5 x Denhardt (1 x Denhardt solution = 0.002% BSA, PVP, and Ficoll 400), 2 x SSC, 0.1 mg/ml denatured DNA carrier (herring sperm DNA), and H20. After prehybridization, the buffer was squeezed out, hybridization buffer was added, bags were sealed again, and filters were incubated at 42°C overnight. The hybridization buffer was the same as the prehybridization one, but added with the probe (200 ng/ml) denatured by boiling before use. After hybridization, the filters were washed first with 2 x SSC, 0.1% SDS (2 x 5 min), followed by 0.2 x SSC, 0.1% SDS (2 x 5 min), and finally with 0.16 x SSC, 0.1% SDS (2 x 15 min) at 50°C. Detection of Probes. Filters were subjected to the following sequence of operations: (a) gentle shaking in blocking buffer (100 mM NaCI, 100 mM TrisHC1, 3 mM MgClz, and 0.5% Tween 20, pH 7.5) for 90 min at r.t.; (b) incubation with gentle shaking in incubation buffer (similar to the blocking buffer plus 0.005% Tween 20) containing biotinylated alkaline phosphatase (1 txg/ixl) for 10 min; (c) washing

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44 E. Davini et al.

GATA9(2):39-47,1992

o\ /

CH,POfi

CH,OCONHCHrCONHCHICONH~HCOOH CH,

III OH

n* H ,NCH,CONH

CH,CONH

HCOOH E Hz

IV N=N

CH,PO,H,

OH

SCHEME '2

-

CH,OCONH(CH,)JOOH H,N$ZiCOOC(CH,),

VI

VII

-o‘_’

CH,POl%

CH,OCONH(CH,),CONH~~COOC[CH,),

IX

N=N

’ u-

-



CH,PO,H*

OH

H,N-(cH,),CONH AcOH-HB

r

OH

SCHEME3 Figure 3. Schemes 2-4. Synthetic procedures for the preparation of the bifunctional reagents used in DNA-labeling experiments. In scheme 2 is reported the synthesis of reagent IV. Reagent X was obtained as illustrated in scheme 3. Finally, m scheme 4, the syntheses of reagents XII and XVII are described.

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45 GATA9(2): 39-47, 1992

Enzyme Inhibitors as DNA Labels

HzN--(CHz)sCOO" BzOCOC/~_CH,OCONH(CHz) ' COOH V

NHS- DCC

VI

HzN---~CHzPO3H=

o

II =

Xl



o

CHzOCONH(CH=)sCONH~ C H 2 P O 3 H

2

AcOH-HBr J.

XII

HzN--(CHz)sCONH~CH zPO3Hz XIII

0 ~COON? XIV

.o,

XVI

N , ' - ~ N H (CHz),CONH~ C H , P NO= XV

O,Hz

ICH=CONH(CHz)sCONH- - ~

CH=PO=Hz

SCHEME 4 Figure 3. Continued

with blocking buffer (3 × 5 min); (d) incubation with streptavidin (2 i~g/txl) in incubation buffer for 10 min; (e) washing in blocking buffer (3 x 5 rain); (f) incubation with biotinylated alkaline phosphatase (1 Ixg/txl) in incubation buffer for 10 min; (g) washing in blocking buffer (3 × 5 min) and then in developing buffer (50 mM NaC1, 50 mM Tris-HCl, 5 mM MgC12, pH 9.5) (2 × 5 min); and (h) development in substrate solution (4.4 txl stock NBT and 3.3 txl stock BCIP in 1 ml of developing solution) from 4 h to overnight. Substrate solution was freshly pre-

pared from stock solutions of NBT (75 mg/ml in 70% dimethylformamide) and BCIP (50 rng/ml in 100% dimethylformamide).

Results and Discussion Labeling reagents were synthesized by utilizing chemical procedures widely used in the chemistry of amino acids, like protection of the amino group, activation of the carboxy group, coupling, and deprotection. For compounds IV and XI, the inhibitor group

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46 GATA 9(2): 3 9 - 4 7 , 1992

E. Davini et al.

Table 3. DetectionSensitivityResults of pBR322 DNA Probes Labeled with Alkaline Phosphatase lnhibitors Dot-blot DNA detected conc. (ng) Probe

Direct

Hybridized

PP-34 PP-35 PP-40 PP-41 PP-42 PP-43 PP-44 PP-45 PP-NT a

0.5 0.5 0.05 0.025 0.05 0.5 0.025 -0.05

--0.5 0.5 5 -0.05 50 0.025

DNA detection system: Blugene by BRL. aPP-NT: biotin-labeled probe with nick-translation technique using BRL nick-translation reagent system.

was introduced by a diazotization reaction (schemes 2 and 3 in Figure 3). Compound XIII, containing a NHz as DNA-linking group, was obtained according to scheme 4 (Figure 3). The last reagent was further modified respectively with an aromatic azide, obtaining compound XV, and with an iodoacetyl group, obtaining compound XVII (scheme 4). We determined the inhibition constants of alkaline phosphatase (AP) for compounds I (Ki = 1.67 ×

PP~4]

I

5

0g

500

pg

50

pg

25

pg

PP-44

I

PP-NT

I

NEG

I

pit;

Figure 4. Colorimetric detection of D N A labeled with alkaline phosphatase inhibitors and bound to nitrocellulose. Single-stranded pBR322 D N A samples were labeled as follows: PP-41 was labeled with reagent XIII, with a two-step condensation procedure, PP-44 was labeled with reagent XV, with a photoactivated procedure; PP-NT (positive control) was labeled with the nick-translation technique; and N E G (negative control) was pBR322 D N A , as such. The labeled probes and controls were spotted onto nitrocellulose and baked, and finally detected with the Detection System Blugene (BRL) under the conditions reported in Materials and Methods.

10-3 M) and II (Ki = 5.11 x 10 -3 M), in accordance with the literature [18]; we tested also the inhibition activity of III in order to exclude any interference from one of the spacers (Ki = 0.16 M), and finally of three of the synthesized labeling reagents: IV (K~ = 7 × 10-5 M), X (Ki -- !.9 x 10-4 M), and XIII (Ki = 3.1 x 10-3M). Plasmid pBR322, linearized with BAMHI restriction enzyme, was the DNA source selected for the preliminary labeling studies because of its definite molecular weight and commercial availability, Moreover, we reported [6] on our work on biotin-labeled probes using the same plasmid pBR322 as DNA source, thus enabling a direct comparison between these different labeling methods. Reagents were coupled to DNA in different ways, depending on the linking group: compound XV was coupled via photoactivation reaction; reagents IV, X, and XIII were coupled via condensation reaction with a one-step method using EDC and N-methyl imidazole, or a two-step method with preactivation of DNA; and reagent XVII was coupled by direct alkylation. The probes obtained from the above reagents are listed in Table 1. Great effort was made to improve the detection sensitivity, in particular by using probes PP-34 and PP-35 (see Table 2): in fact, preliminary experiments for the direct detection of blotted labeled probes, according to a reported detection protocol (DNA Detection System by BRL) and using AP from Sigma, gave a poor detection sensitivity. Better results (50 ng for probe PP-35) were obtained by utilization of a crosslinked form of AP from BRL. With the aim of increasing DNA detection sensitivity, we tested biotinylated AP. By using a two-step procedure (BRL biotinylated AP, followed by the addition of streptavidin and of biotinylated AP), the lower DNA concentration detected was 50 ng for probe PP-35 and dropped to 5 ng for probe PP-44 (the one obtained from the aromatic azide-containing reagent, as DNAbinding group). Better results were obtained with the detection kit Blugene commercially available from BRL: we reached 50 pg detection sensitivity for PP-40 probe and 25 pg for probe PP-44. Further sensitivity improvement was achieved by a modification of the amino-containing reagent-linking procedure by using a preactivation DNA step with EDC and methyl imidazole, before the coupling step. The above improvement is presumably ascribable to an increase in the yield of labeled fragments at their 5'-terminal phosphate function. Two of the three probes obtained in this way enabled an increase in

© 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

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Enzyme Inhibitors as DNA Labels

detection sensitivity of 1 order of magnitude: 25 pg and 50 pg for probes PP-41 and PP-42 (compared with 0.5 ng for PP-34 and PP-35, respectively; see Table 3 and Figure 4). Many parameters involved in the complex detection procedure were examined: concentration of blocking, incubation, and detection buffers (dilution to 1:10 of the standard protocol conditions); and time contact and pH in the first step of detection protocol (1 h at pH 9.5 instead of 10 min at pH 7.5). However, the above efforts did not improve the detection sensitivity. Finally we tested probes PP-40, PP-41, PP-42, PP-44, and PP-45 in hybridization experiments using the best developing conditions found in direct dotblot sensitivity determinations. The results reported in Table 3 show the best sensitivity for probe PP-44, with a DNA detectable concentration limit of 50 pg. The detection sensitivity results achieved with DNA probes labeled with the present approach roughly parallel those obtainable by the biotin-labeling procedures reported by our [5, 6, 16] and other laboratories [12, 19, 20]. The aim of this phase of the work has been to confirm the practical realization of our new approach to nonradioactive DNA labeling. This approach, which also needs an ad hoc detection procedure, will be subjected to more research efforts to improve the detection protocol further.

The authors appreciate the work of Dr. Francesco Norelli in performing inhibition experiments.

References 1. Lebacq P: Technoscope Biofutur 11: 12-18, 1987 2. Chollet A, Kawashima EH: Nucleic Acids Res 13:15291539, 1985 3. Enrlich HA, Sheldon EL, Horn G: Bio/technology4:975981, 1986 4. Renz M, Kurz C: Nucleic Acids Res 12:3435-3444, 1984 5. Zappelli P, Di Leo C, Rossodivita A: Italian Patent Appl 22785A/87, 1987 6. Zappelli P, Di Leo C, Rossodivita A: Italian Patent Appl 23033A/87, 1987 7. Haralambidis J, Chai M, Tregear GW: Nucleic Acids Res 15:4857-4876, 1987 8. Gebeyehu G, Rao PY, SooChan P, Simms DA, Klevan L: Nucleic Acids Res 15:4513-4534, 1987 9. Langer PR, Waldrop AA, Ward DC: Proc Natl Acad Sci USA 78:6633-6637, 1981 10. Zwadyk P Jr, Cooksey RC, Thornsberry C: Curt Microbiol 14:95-100, 1986 11. Herzberg M, Reinhartz A, Ritterband M, Twizer S, Smorodinski NI, Fish F: Chimicaoggi 69-71, November 1987 12. ReisfeldA, RothenbergJM, Bayer EA, Wilchek M: Biochem Biophys Res Commun 142:519-526, 1987 13. Reines AS, Schulman LH: Methods Enzymol 59:146-156, 1979 14. SchulmanLH, PelkaH, Reines SA: NucleicAcidsRes 9:12031217, 1981 15. Draper DE: Nucleic Acids Res 12:989-1002, 1984 16. Zappelli P, Di Leo C, Rossodivita A: Biotech RIA 88 Molecular Probes: Technologyand Medical Applications, Florence, Italy, 11-13 April 1988 [poster communication], p 171 17. Zappelli P, Davini E, Rossodivita A, Di Leo C, Norelli F: Italian Patent Appl 2101 IA/88, 1988 18. Landt M, Boltz SC, Butler LG: Biochemistry 17:915-919, 1978 19. Leary JJ, Brigati DJ, Ward DC: Proc Natl Acad Sci USA 80:4045-4049, 1983 20. Forster AC, Mclnnes JL, Skingle DC, SymonsRH: Nucleic Acids Res 13:745-761, 1985

© 1992 Elsevier Science Publishing Co., Inc., 655 Avenueof the Americas, New York, NY 10010

Alkaline phosphatase inhibitors as labels of DNA probes.

A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competiti...
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