Detection of DNA targets with biotinylated and fluoresceinated RNA probes

ANALYTICAL

BIOCHEMISTRY

182,30%314

(1989)

Detection of DNA Targets with Biotinylated Fluoresceinated RNA Probes Effects of the Extent of Derivitization Virginia

Folsom,

Markus

J. Hunkeler,

On Detection Albert0

Molecular Biology Research and Development, Bethesda 8717 Grovemont Circle, Gaithersburg, Maryland 20877

Received

May

Hates,

Press,

Sensitivity and John

Research Laboratories,

D. Harding Life Technologies,

Inc.,

22,1989

The substituted nucleotide aminohexyl-ATP (AHATP) was used for synthesis of RNA probes from a plasmid template using the T7 phage promoter. Following synthesis, RNA probes were modified by reaction with N-hydroxysuccinimide (NHS) esters of biotin or fluorescein. Nearest-neighbor analysis was used to quantitate both the incorporation of the substituted nucleotide into RNA and the subsequent modification of the incorporated nucleotide by the NHS esters. The results indicate that AH-ATP is efficiently incorporated into RNA and that modification of the amine group is also efficient. The T7 polymerase shows a bias for ATP over AH-ATP and truncated transcripts are produced if 100% AH-ATP is used for synthesis. However, the use of 50% AH-ATP in the synthesis reaction yields fulllength RNA probes that contain on average one aminelabeled nucleotide every 12 bases. This RNA is readily modified by the respective NHS esters to obtain one biotin group per 15-18 total RNA bases or one fluorescein group per 25-35 bases. Probes modified with biotin or fluorescein were used to detect picogram levels of target DNA in a dot blot hybridization format. o 1989 ACE,demic

and

Inc.

The use of nucleic acid hybridization to detect and quantitate specific target sequences is well established. Radiolabeled probes afford high sensitivity and specificity in hybridization experiments, but are inherently unstable and pose potential health hazards as well as problems associated with the disposal of radioactive waste. Thus, there is a need to develop sensitive nonradioactive hybridization systems to circumvent these problems. Perhaps the best characterized and most widely used nonradioactive hybridization system utilizes biotin as a reporter molecule (for a review, see Ref. (1)). Biotin can 0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

be incorporated into a nucleic acid probe enzymatically (2,3), chemically (4,5), or photo chemically (6). Following hybridization, the biotinylated probe-target complex is often detected using streptavidin or avidin coupled to an enzyme such as alkaline phosphatase (7). The streptavidin moiety binds to the biotin with high affinity and the location and/or amount of the bound complex is determined from the product of the enzyme reaction. Alternative detection systems that utilize enzymes linked directly to oligonucleotide probes (8) or antibodies to recognize RNA-DNA hybrids (9) or hapten-linked probes (10) have also been described. Several considerations suggest that the sensitivity of nonradioactive detection of nucleic acid targets can be increased by the use of RNA, rather than DNA, probes. RNA probes are readily synthesized in vitro using cloning vectors that contain bacteria phage RNA polymerase promoters; a high yield of probe is achieved from a relatively small input of template DNA (11). In contrast to double-stranded DNA probes, RNA probes are strand specific. RNA/DNA or RNA/RNA hybrids are more stable than DNA/DNA hybrids, thus allowing the use of shorter hybridization times and more stringent posthybridization washes. Backgrounds on solid supports arising from nonspecifically bound probe can be further reduced by posthybridization RNase treatment. Biotinylated RNA probes have been synthesized by direct incorporation of biotinylated UTP by phage RNA polymerases. However, the yield of RNA can be low and, depending on the composition of the DNA template, truncated transcripts that are suboptimal for hybridization can be synthesized (12). These problems can be circumvented in part by the use of a two-step labeling protocol in which the polymerase first incorporates a nucleotide analog containing a primary amine attached to the base via an aliphatic linker (12). The resulting RNA is derivitized with biotin or another reporter mole309

Inc. reserved.

310

FOLSOM

cule by reaction with an appropriate ester, such as E-caproylamidobiotin-N-hydroxysuccinimide ester (CABNHS ester).’ An important, relatively uncharacterized, issue in regard to synthesis and use of nonradioactive RNA probes is the actual extent of derivitization with the reporter molecule and the effects of this parameter on sensitivity of detection. In this paper, we examine these questions for biotinylated and fluoresceinated RNA probes synthesized by enzymatic incorporation of the nucleotide analog, N6-aminohexyl-ATP, followed by reaction with CAB-NHS or 5-carboxyflourescein-NHS ester. We show that classical nearest-neighbor analysis (13,14) affords a sensitive assay for the extent of incorporation of AH-ATP and derivitization with the reporter molecule. We further examine the sensitivity of detection of filter-bound targets using RNA probes containing known amounts of biotin or fluorescein. MATERIALS

AND

METHODS

For synthesis of AH-ATP, 6-chloropurine monophosphate triethylammonium salt was reacted with tributylammonium pyrophosphate using carbonyldiimidazole as a coupling agent. The resulting triphosphate was treated with 1,6-diaminohexane, under basic conditions, to afford the desired AH-ATP in a 50% yield. Plasmid pT7-18s contains a 7-kb BamHl fragment from human papillomavirus type 18 cloned into the pT7 plasmid. RNA was synthesized from linearized pT7-18s template using T7 RNA polymerase (11). Reactions contained 40 mM Tris-HCl, pH 8.0, 8 mM MgC&, 75 InM NaCl, 2 mM spermidine, 5 IrIM DTT, and 0.67 mM each of CTP, GTP, and UTP. Reactions also contained ATP and/or AH-ATP at a total concentration of 0.67 mM; the relative concentrations of AH-ATP and ATP varied, but total concentration was maintained at 0.67 mM. A 20-~1 reaction contained 0.2 pg of template, 140 units of T7 polymerase, and 200 &!i of [a-32P]CTP at 800 Ci/mmol (Amersham). Some reactions contained [3H]UTP (20 &i at 40 Ci/mmol, DuPont) in place of [32P]CTP. Reactions were incubated at 37°C for 15 min and terminated by the addition of 200 ~1 of 10 mM Tris-HCl, pH 7.6,20 mM EDTA. Aliquots from each reaction were diluted and assayed by trichloroacetic acid precipitation to determine the percentage incorporation of the trace label. The calculated yield from 0.2 pg of template was 3-7 pg of RNA. RNA samples were adjusted to 0.5 M Tris-HCI, pH 7.6, precipitated with 2.5 vol of ethanol, dried, and dissolved in water. Aliquots of the RNA samples were ’ Abbreviations used: AH-ATP, N’-aminohexyl-ATP; CAB-NHS ester, c-caproylamidobiotin-N-hydroxysuccinimide ester; HPV 18, human papillomavirus type 18; DTT, dithiothreitoh SDS, sodium dodecyl sulfate; PEI, polyethyleneimine; BSA, bovine serum albumin; NBT, nitroblue tetrazolium; BCIP, &bromo-4-chloro-3-indolylphosphate; FITC, fluorescein isothiocyanate.

ET

AL.

I;(“b&‘JH,

OH

OH

R =Et,NH’ FIG.

1.

Structure

of aminohexyl-adenosine

triphosphate

(AH-

ATP).

lyophilized, dissolved in gel-loading buffer, and analyzed by electrophoresis in formaldehyde agarose gels. Gels were dried and autoradiographed. The CAB-NHS ester was obtained from BRL and the 5-carboxyfluorescein-NHS ester was from Research Organics. For modification reactions, l-2 pg of RNA in 25 ~1 of water was added to 25 ~1 of freshly prepared 0.4 M sodium bicarbonate (about pH 8.2). Each ester was dissolved at 10 mg/ml in dimethylformamide immediately before use. Ten microliters of ester was added per reaction (1.7 mg/ml final concentration) and incubation was at room temperature for 2 h. Reactions were quenched with 50 ~1 of 1 M Tris-HCl, pH 7.6. One hundred microliters of 1X SSC (0.015 M sodium citrate, 0.15 M NaCl), 0.05% SDS was added and the RNA was separated from unincorporated ester on a 6-ml Sephadex G50 column in 1X SSC, 0.05% SDS. RNA was collected in the excluded volume and used directly for nearest-neighbor analysis or hybridization. For nearest-neighbor analysis, 2-3 ng of RNA was incubated with 30 units of T2 ribonuclease (BRL) in 40 ~1 of 25 mM sodium citrate, pH 5.0, for 2 h at 37°C (13,14). Polyethyleneimine (PEI) cellulose chromatography plates (Sigma) were predeveloped in 1 M NaCl followed by rinsing in distilled water. Samples (5-10 ~1) were applied to the plate in small aliquots with a pipetman, drying between applications. Plates were rinsed with 100% methanol to remove salt from spotted samples and then air-dried. Plates were developed in isobutyric acidwater: 28% ammonium hydroxide, 66:33.5:0.5 (15,16). Development time was about 4 h. Plates were autoradiographed for 24-48 h at -80°C with an intensifying screen. After autoradiography, spots corresponding to radioactive nucleotides were cut from the chromatography plate and quantitated by scintillation counting. For identification of biotin-AMP (see Fig. 5A), T2-digested samples were incubated with streptavidin-agarose (BRL); the streptavidin-agarose was removed by centrifugation and the supernatant was applied to the PEI plate. For dot blot analysis, an EcoRl restriction fragment containing the entire human papillomavirus type 18 genome (about 8 kb) was isolated from a recombinant plas-

NONRADIOACTIVE TABLE

DETECTION

1

Incorporation of [32P]CTP Trace Label by T7 RNA Polymerase in the Presence of Increasing Concentrations of AHATP Relative AH-ATP (% of total 10% 25% 50% 75% 100%

A) b

AH-ATP AH-ATP AH-ATP AH-ATP AH-ATP

Expt

1

incorporation

of trace

Expt

95

91

48

41

label

2

a Incorporation of label using 100% ATP is defined as 100%. * AH-ATP concentration is reported as percentage of the total concentration (on a molar basis) in the synthesis reaction.

(%)(I Expt

3

102 100 93 77

ATP

mid. The DNA samples were diluted in TE buffer and denatured by boiling. After cooling on ice, an equal volume of 2 M ammonium acetate was added and samples were dot blotted onto nitrocellulose paper that had been preequilibrated in 1 M ammonium acetate. The filters were washed briefly in 20x SSC, air-dried, and baked at 80°C under vacuum. Prehybridization (2 h) and hybridization (16 h) reactions were performed in 50% formamide, 5X SSC, 1X Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.2 mg/ml salmon sperm DNA, 1 mM EDTA, and 0.1% SDS at 42°C. Probes were used at 100 rig/ml. After hybridization, filters were washed once with 2X SSC, once with 2X SSC containing 1 pg/ml RNase A, and twice with 0.2~ SSC, 0.1% SDS. Filters were then washed twice with 0.16X SSC, 0.1% SDS at 50°C. Filters were rinsed briefly in 2X SSC, rinsed briefly in Buffer 1 (0.1 M TrisHCl, pH 7.5,0.15 M NaCl), and then blocked with Buffer 2 (3% BSA in Buffer 1) for 1 h at 65°C. Alkaline phosphatase conjugate was diluted in Buffer 1 and incubated with filters for 30 min at room temperature. After binding of conjugate, filters were washed three times in Buffer 1 and rinsed briefly in Buffer 3 (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM MgC1.J. For color development, filters were incubated for 1 h at room temperature with nitroblue tetrazolium (NBT) at 0.33 mg/ml and 5bromo-4-chloro-3-indolylphosphate (BCIP) at 0.17 mg/ ml in Buffer 3. All washes were performed for 15 min at room temperature unless otherwise specified. Biotinylated probes were detected with streptavidin-alkaline phosphatase (BRL). Fluorescein-modified probes were detected with an alkaline phosphatase conjugate of an anti-fluorescein isothiocyanate mouse monoclonal antibody (EIA Co., San Francisco). RESULTS

AND

DISCUSSION

The structure of AH-ATP is shown in Fig. 1. The molecule contains a primary amino group attached to the

USING

RNA

311

PROBES

N6 position of the adenine ring via a six carbon linker arm. After incorporation of AH-ATP into RNA the amino group can be derivitized with a reporter group such as biotin or fluorescein. We have used incorporation of an [w~‘P]CTP trace label to assess the efficiency of RNA synthesis from the pT7-18s template using AHATP (or mixtures of AH-ATP and ATP) and T7 RNA polymerase (Table 1). In reactions containing 50% or less of AH-ATP, there is little effect on the yield of RNA. However, an increase in the relative concentration of AH-ATP to 75% results in a somewhat decreased yield of RNA; further increase in the AH-ATP concentration to 100% results in yields of RNA at 45% of that of control reactions (100% ATP, no AH-ATP). Thus T7 polymerase shows a bias for ATP over AH-ATP.

A

B

C

2.41

0 i24-

FIG. 2. Formaldehyde agarose gel of “P-labeled RNA samples synthesized with AH-ATP using T7 RNA polymerase. The synthesis reaction contained 100% ATP (A), 50% AH-ATP + 50% ATP (B), or 100% AH-ATP (C). Approximately 1 X lo5 cpm was loaded per lane. Exposure to Kodak XAR-5 film was for 2 h at room temperature. The numbers to the left of the autoradiogram indicate position of ethidium bromide-visualized RNA marker fragments (BRL 0.24-0.95 kb RNA ladder).

312

FOLSOM

AH-AMP

CMP UMP, GMP

origin

A

B

C

FIG. 3. Nearest-neighbor analysis of RNA samples. The T2 ribonuclease digestion products of RNA samples were analyzed by thinlayer chromatography on a PEI cellulose plate. RNA was synthesized with 100% ATP (A), 50% AH-ATP + 50% ATP (B), or 100% ATP (C). Approximately 5 X lo4 cpm was loaded per lane. Exposure to film was for 2 h at room temperature.

RNA synthesized with AH-ATP was analyzed on denaturing agarose gels. As shown in Fig. 2, the size distribution of the product synthesized with 50% AH-ATP (Lane B) is similar to that of the product synthesized with 100% ATP (Lane A). The reactions produce a similar number of full-length transcripts (7.5 kb) relative to shorter fragments. When the concentration of AH-ATP is increased to lOO%, many fewer full-length transcripts are synthesized (Lane C). A number of discrete bands of shorter length are also detected. The short fragments may result from the failure of the polymerase to incorporate stretches of AH-ATP. A similar observation has been made for biotin-11-UTP; use of this nucleotide with AT-rich templates leads to low yields of RNA and the generation of truncated transcripts (12). Nearest-neighbor analysis was used to analyze the incorporation of AH-ATP into RNA. RNA was synthesized with a-labeled [32P]CTP followed by digestion to completion with ribonuclease T2. Ribonuclease T2 cleaves 5’ phosphate linkages and thus transfers the 32Plabeled phosphate that was introduced with the [32P]CTP to its 5’ neighbor. The method utilizes a commercially available radiolabeled compound and allows radioactive tagging of the molecule of interest (i.e., AHATP) after its incorporation into RNA. Use of a radiolabeled AH-ATP is therefore not necessary. For analysis of incorporation of AH-ATP, labeled CTP, UTP, or GTP could be used. The products of T2 digestion (3’-monophosphates) were analyzed by thin-layer chromatography and auto-

ET

AL.

radiography. As shown in Fig. 3, 3’-AMP and 3’-AHAMP are well separated in the chromatography system we have utilized, the relative incorporation of AH-ATP and ATP can thus be quantitated by cutting the TLC plate and scintillation counting. There is no evidence of 3’-AMP in the T2 digest of RNA synthesized with 100% AH-ATP, indicating that the AH-ATP reagent contains no contaminating ATP. We have analyzed the relative amounts of AMP and AH-AMP in RNA synthesized at several relative concentrations of AH-ATP, as shown in Fig. 4. If AH-ATP were incorporated with the same efficiency as ATP, the experimental points would lie on the dashed line in the figure. The experimental points are all below the dashed line, indicating that the RNA polymerase preferentially incorporates ATP over AH-ATP. The bias of the enzyme for ATP is most pronounced at low concentrations of AH-ATP. For example, the ratio of the percentage of AH-AMP incorporated to the percentage of AH-ATP in the reaction is 0.56 for a reaction containing 10% AHATP and 0.75 for a reaction containing 50% AH-ATP. Due to the bias of the enzyme for ATP over AH-ATP and because a large fraction of the input triphosphates is utilized during synthesis, we expect the ratio of AMP/ AH-AMP incorporated to be high at the beginning of the

0

20

40 AH-ATP

(%

60 of

ATP

80

100

m Reactm)

FIG. 4. Incorporation of AH-ATP into RNA. RNA was synthesized with increasing concentrations of AH-ATP (from 10 to 75% AHATP). The efficiency of incorporation of AH-ATP was determined by T2 ribonuclease digestion and chromatography on a PEI cellulose plate. Radiolabeled nucleotides were cut from the PEI plate and quantitated by scintillation counting. The dotted line indicates the theoretical case in which the polymerase does not discriminate between ATP and AH-ATP (the percentage AH-ATP in the reaction is identical to the percentage AH-AMP incorporated). The autoradiogram of the PEI plate is shown in the inset. RNA samples were synthesized with 100% ATP (A), 10% AH-ATP (B), 25% AH-ATP (C), 50% AH-ATP (D), and 75% AH-ATP (E). Approximately 6 X lo3 cpm was loaded per lane; exposure to EAR-5 film was for 40 h at -80°C.

NONRADIOACTIVE

A

DETECTION

Bio.AMP AH,AMP FI,AMP

A

B

C

D

E

F

G

H

fluorescein

A

B

c

(A) Modification of amine-labeled RNA by CAB-NHS ester. RNA samples (6 X lo3 cpm) were digested to completion with T2 ribonuclease. Half of each TZ-digested sample was incubated with streptavidin-agarose to remove the biotinylated nucleotide monophosphate and samples were analyzed by chromatography on a PEI cellulose plate. Unmodified RNA samples were synthesized with 100% ATP (A and H) or 50% AH-ATP (B and G). Samples modified by FIG.

RNA

313

PROBES

ties corresponding to biotin-AMP is unequivocally identified by absorption on streptavidin-agarose (Fig. 5A). On the basis of counting the radioactivity in the TLC spots, the CAB-NHS ester reproducibly modifies 65-80% of the AH-AMP residues. Labeling with the

B

AH.AMP

USING

5.

CAB-NHS ester were synthesized with 50% AH-ATP (C and F) or 100% AH-ATP (D and E). Samples were analyzed after T2 digestion (A-D) or after T2 digestion and treatment with streptavidin-agarose (E-H). (B) Modification of amine-labeled RNA by 5-carboxyfluorescein-NHS ester. TLC analysis of RNA synthesized with 50% AHATP (A) and RNA synthesized with 50% AH-ATP followed by modification with the fluorescein ester (B, C).

synthesis reaction. As synthesis proceeds the relative concentration of AH-ATP increases, and the enzyme should incorporate a higher relative number of AH-ATP molecules. Therefore labeling of the probe population may not be uniform when a mixture of AH-ATP and

ATP is used. However, we have no evidence that this affects the sensitivity of detection of specific targets. On the basis of the results shown in Table 1 and Figs. 2-4, the optimal relative concentration of AH-ATP for synthesis of RNA with T7 polymerase appears to be 50%. The yield of RNA with 50% AH-ATP is similar to the yield with 100% ATP; gel analysis indicates a large proportion of full-length transcripts and nearest-neighbor analysis consistently indicates that AH-AMP represents about 37% of the total AMP incorporated when the synthesis reaction contains 50% AH-ATP. If we assume random distribution of sequence in RNA, then one AH-AMP is present about every 12 bases in transcripts synthesized from a reaction containing 50% AH-ATP. This represents a level of modification that should be sufficient for most labeling requirements. After synthesis, RNA probes were modified with CAB-NHS ester or 5-carboxyfluorescein-NHS ester. The extent of modification was quantitated by nearest,neighbor analysis and thin-layer chromatography. As seen in Fig. 5, biotin-AMP and fluorescein-AMP are well resolved from both AH-AMP and AMP. The sne-

ester

is somewhat

less efficient;

30-35%

of

the AH-AMP residues are modified. We found that increasing the concentration of ester in the modification reaction (from 1.7 to 8.5 mg/ml) improved the efficiency of modification with the fluorescein reagent by about 35%. However, increasing the duration of the modification reaction had no effect. The efficiency of the modification reaction was found to be independent of the number of incorporated amino groups. When 10, 25, 50, or 75% AH-AMP was used in the synthesis reaction, the same efficiency of modification of the incorporated amine groups was observed. This was true for both the biotin and the fluorescein esters (data not shown). The lower efficiency of labeling observed with the fluorescein reagent may in part be due to steric hindrance associated with the larger ring structure of the fluorescein moiety. The short length of the linker arm contained

within

the fluorescein

labeling

reagent

may also

increase steric hindrance. The fluorescein group is linked to the N-hydroxysuccinimide group through a single carboxy carbon; in contrast, the biotin group in the CAB-NHS ester is linked to the NHS group through a 6 carbon linker arm. We find that an increase in the temperature of the modification reaction from room temperature to 50°C improves the efficiency of modification with the fluorescein reagent, from 32 to 65% (data not shown). This result is consistent with steric interference to modification: a decrease in secondary structure

A

B

C

D

A

;

FIG. 6. Detection of HPV 18 target DNA by dot blot hybridization. The amount of target DNA in picograms in each dot is indicated at the left of the figure. Strips in A were probed with biotin-modified RNA and detected with streptavidin-alkaline phosphatase. Biotinmodified probes were synthesized with 25% AH-ATP (A), 50% AHATP (B), 75% AH-ATP (C), or 100% ATP (D). Strips in B were probed with fluorescein-modified RNA and detected with anti-FITC antibody-alkaline phosphatase. Fluorascein-modified probes were synthesized with 50% AH-ATP (A) or 100% ATP (B).

314

FOLSOM

and/or a greater flexibility in the structure of the RNA at higher temperatures could increase the accessibility of amine groups to modification. Given the efficiency of incorporation of AH-ATP into RNA and the efficiency of modification of the incorporated amine group, a probe synthesized using 50% AHATP contains one biotin every 15-18 bases or one fluorescein every 25-35 bases. Modified RNA samples have also been analyzed by gel electrophoresis. Although the mobility of the modified RNA is slightly reduced relative to unmodified RNA (due to the presence of biotin and fluorescein groups on the RNA), the modification reaction does not significantly alter the size distribution of the RNA (data not shown). We have also synthesized RNA probes using a tritiated nucleotide as the trace label. Although the tritiumlabeled probes cannot be analyzed by nearest-neighbor analysis, we have compared the 3H-labeled probes with 32P-labeled probes (that have been subjected to TLC analysis) by dot blotting the RNA onto nitrocellulose and detecting with the appropriate conjugate. Using this criterion, the 3H-labeled probes appear to be similar to the 32P-labeled probes in regard to the amount of incorporated biotin or fluorescein. They also demonstrate the same sensitivity of detection of DNA target in filter hybridization assays (as described below). Given the observed consistency (by TLC analysis) in the efficiency of modification of AH-AMP-labeled probes with ligand, probes could be generated with no trace radiolabel and assayed for modification by dot blotting the probe and applying the relevant detection system. Biotinylated probes and fluorescein-modified probes were used to detect HPV 18 target DNA in a dot blot filter hybridization format (Fig. 6). Biotinylated probes were detected with streptavidin-alkaline phosphatase and the NBT-BCIP color development system (see Materials and Methods). There was no difference in the sensitivity of detection using biotinylated probes that were synthesized with 25,50, or 75% AH-ATP. In each case, 2 pg of target was detected. A control probe synthesized with 0% AH-ATP (i.e., 100% ATP) gave a low level signal in this system. This could be due to covalent modification by the CAB-NHS ester at sites other than the primary amine group introduced with AH-ATP. Fluorescein-modified probes were detected with an alkaline phosphatase conjugate of an anti-FITC mouse monoclonal antibody. Although 2 pg of target was detected with the fluorescein-based system, sensitivity of detection was about twofold lower than that with the biotinylated probes. This does not necessarily imply that the biotin-based system is intrinsically more sensitive than the fluorescein system, since different conjugates were used in the two experiments. Detection sensitivity in the filter hybridization assay was similar for fluorescein-modified probes synthesized with 25, 50, or 75% AH-ATP. We have shown, however, that sensitivity of

ET

AL.

detection of fluorescein-modified probes increases linearly with AH-ATP concentration (up to 75% AH-ATP) in a solution hybridization format (data not shown). Therefore, choice of the most sensitive probe may depend on the format used for hybridization. In summary, we have demonstrated the use of AHATP for generation of amine-labeled RNA probes and subsequent modification by two different NHS esters. Our results suggest that AH-ATP provides a useful reagent for attachment of ligands to RNA probes. We have further demonstrated the use of such probes for detection of target DNA in a filter hybridization format. The efficiency of incorporation of AH-ATP and the efficiency of modification of incorporated AH-AMP have been quantitated using nearest-neighbor analysis. To our knowledge, this is the first reported application of this technique for determining the extent of ligand labeling of a nucleic acid probe. The technique allows precise determination of probe composition, and the extent of ligand labeling can be correlated with the sensitivity of the probe. ACKNOWLEDGMENTS We thank Leonard Klevan and Dietmar Rabussay for support and encouragement, Mindy Goldsborough for providing HPV clones, Robert Bebee for advice on nearest-neighbor analysis, and Tillie Schneidmill for typing the manuscript.

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