Acridinium ester probes

Fig. 7.3. Principle of acridinium-ester probe hybridization in solution and detection. Acridiniura-NHS is linked to allyl nucleotides (Section 7.3.1.2) in the probe. When the probe hybridizes with its target in solution, the acridinium moiety intercalates in the duplex. Unhybridized acridinium ester probes can be hydrolyzed selectively (Table 7.7, step 2), whereas protected acridinium ester remains intact (ID. After nonspecific chemiluminescence is sufficiently eliminated, specific chemiluminescence is measured (in). The whole procedure takes less than 30 min.

Hybridization protection assay with acridinium ester probes... 

Hybridize methyl acridinium ester probe with target nucleic acid (single-stranded or heat-denatured) for 10 min at 60°C in 0.1 M lithium succinate buffer (pH 5.2) containing 10% (w/v) lithium lauryl sulfate, 2 mM EDTA and 2 mM EGTA (total volume 100 xl with 0.2 pmol of probe in a 12x75 mm tube). 

Acridinium esters have also been utilized for chemiluminescent detection of cDNA probes (Fig. 5) [9-11], The hydrolysis rate is much faster when the ester is conjugated to single-stranded DNA, rather than to double-stranded DNA. This means that the chemiluminescence from unhybridized acridinium ester-labeled probe is rapidly lost, whereas the chemiluminescence from the hybridized probe is minimally affected. This permits discrimination between hybridized and unhybridized acridinium ester-labeled DNA probes without separation steps. 

Therefore, chemiluminescent methods using an acridinium ester-labeled cDNA probe allow the discrimination of a mismatched DNA sequence in a homogeneous assay. 

CHEMILUMINESCENT DETECTION OF ACRIDINIUM ESTER-LABELED PROBE... 

The acridinium ester (AE) in an AE-labeled cDNA probe hybridized to target DNA is less likely to be hydrolyzed than in the unhybridized conformation (Fig. 10) [9-11]. Single-base mismatches in the duplex adjacent to the site of AE attachment disrupt this protection, resulting in rapid AE hydrolysis [11]. Hydrolysis by a weak base renders AE permanently nonchemiluminescent. After hydrolysis, it is possible to use the remaining chemiluminescence as a direct measure of the amount of hybrid present. This selective degradation process is a highly specific chemical hydrolysis reaction, which is sensitive to the local environment of the acridinium ester. The matched duplex can be detected and quantified readily, whereas the mismatched duplex produces a minimal signal. 

Figure 10 Mismatch detection by using a chemiluminescent AE-labeled cDNA probe. Procedure [9, 11] Acridinium ester-labeled probes specific for either wild-type or mutant sequence corresponding to a target DNA are hybridized with the sample DNA for 1.0 h at 60°C in a hybridization buffer (pH 5.2). Hybridized and nonhybridized probes are discriminated by the hydrolysis reaction for 12 min at 62.5°C in the presence of Na2B407 (pH 8.5) and Triton X-100. The chemiluminescence of each sample is then measured in a luminometer.

Figure 2 illustrates the reaction mechanisms of acridinium ester label probes and alkaline phosphatase probes using dioxetane chemiluminescent detection. Table 2 summarizes approaches for labeling DNA. 

Label amino function in DNA probe with N-hydroxy succinimide derivatives of acridinium esters. 

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

A3. Arnold, L. J., Jr., Hammond, P. W., Wiese, W. A., and Nelson, N. C., Assay formats involving acridinium-ester-labeled DNA probes. Clin. Chem. (Winston-Salem, N.C.) 35, 1588-1594(1989). 

N4. Nelson, N. C., and Kacian, D. L., Chemiluminescent DNA probes A comparison of the acridinium ester and dioxetane detection systems and their use in clinical diagnostic assays. Clin. Chim. Acta 194, 73-90 (1990). 

Chemiluminescence assays are ultrasensitive (attomole to zeptomole detection limits) and have wide dynamic ranges. They are now widely used in automated immunoassay and DNA probe assay systems, (e.g., acridinium ester and acri-dinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labels and the enhanced-luminol reaction for horseradish peroxidase labels [see Chapter 9]). 

An acridinium ester-labeling method has been developed by Gen-Probe, Inc.. Because of high quantum yield and flash reaction kinetics in the presence of base and H2O2, chemiluminescent acridinium esters provide the possibility of designing sensitive nonradioactive probes. The detectability of these systems is 5 x 10 mol and acridinium-labeled probes are fully compatible with hybridization. Arnold et al. (1989) discriminated hybridized from unhybridized acridinium ester-labeled DNA probes without prior separation (Fig. 7.3). In a typical experiment (Table 7.7), the ss probe is hybridized to... 

The fo5 of the acridinium ester in the hybridized probe decreases if mismatches are present. In such experiments, hydrolysis is omitted and RNA/DNA-probe hybrids are adsorbed to hydroxyapatite. Although about 80% may be hybridized, half-lives could be reduced, for some mismatches, to close to that of unhybridized probes (Arnold et al., 1989). Probes with mismatches cannot be used in solution hybridization but can be used in hybridization on solid phase where... 

A luminescent probe detection system called the hybridization protection assay, or HP A, makes use of an acridinium ester-derivatized oligonucleotide that is hybridized to the amplified DNA (02). Unhybridized probe is preferentially... 

The ability to synthesize short oligonucleotide DNA probes with an acridinium ester attached at any defined position within the sequence (A16, N5) has allowed for the use of such probes in hybridization-based assays for RNA and DNA target sequences. Two techniques have recently emerged, together with a combination of the two (for enhanced sensitivity). 

The second technique uses a homogeneous assay format with no physical separation between hybridized and nonhybridized probes. The method is the result of a discovery that for a suitably positioned acridinium ester, within an oligonucleotide sequence, hybridization prevents hydrolytic attack on the ester by hydroxide ions. Such hydrolysis would lead to the inactivation of the chemiluminescent label (see Section 3.2.1). This differential susceptibility to hydrolysis forms the basis for a so-called hybridization-protection assay (HPA), which is schematically illustrated in Fig. 30. 

Fig. 30. Schematic illustration of the hybridization protection assay (HPA). The acridinium ester is covalently attached to a DNA probe. Upon hydrolysis of the phenyl ester group, there is no possibility of forming a dioxetaneone intermediate and hence the reaction is dark i.e., there is no light emission. By contrast, chemiluminescence of the hybridized probe (shown in the lower part of the figure) is minimally affected. Adapted from Nelson et at. (N6), with permission.

G9. Goto, M., Shinichi, O., Okuzumi, K., Kimura, S., and Shimada, K., Evaluation of acridinium ester-labeled DNA probes for the identification of Mycobacterium tuberculosis and Mycobacterium avium-Mycobacterium intracellulare complex in culture. J. Clin. Microbiol. 29, 2473-2476 (1991). 

Septak, M., Acridinium ester-labeled oligonucleotide probes. J. Biolumin. Chemilumin. 4, 351-356 (1989). 

Although acridinium esters can be used to determine hydrogen peroxide, they are more commonly used as labels for immunoassay or DNA probes. As no catalyst is required, acridinium ester chemiluminescence has a lower chemical blank than the oxidation of luminol and therefore has superior detection limits. A strongly alkaline solution (pH 12-13) is required, but under these conditions acridinium esters undergo reversible conversion to the non-chemiluminescent pseudobase form (Scheme 1), which slowly decomposes. As a consequence, acidic hydrogen peroxide is added to reconvert the pseudobase to the acridinium ester before the chemiluminescence reaction is initiated with a sodium hydroxide solution. 

Becker, M., Lerum, V., Dickson, S., Nelson, N.C., Matsuda, E. The double hehx is dehydrated evidence from the hydrolysis of acridinium ester-labeled probes. Biochemistry 1999, 38(17), 5603—5611. 

Acridinium-NHS (AT-hydroxysuccinimide) ester labeling reagent is synthesized as described by Weeks et al. (1983) and probes containing an allylamine linker arm (Fig. 7.2.1) are prepared either as described for nucleotides and for enzymatically labeled probes (Section 7.6.2.2), by transamination (Section 7.8.1) or during oligomer synthesis (Section 6.4). The acridinium-NHS ester is then reacted with the linker arm by standard methods (Section 7.4.1). 

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