A single molecular beacon probe is sufficient for the analysis of multiple nucleic acid sequences

Abstract

Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping.

Figure 1

Principal Scheme of indirect binding of MB probe to the analyte by using a binary approach.[15b] The probe consists of an MB probe and the two synthetic oligodeoxyribonucleotides m and f, which co-exist in dissociated state in the absence of a DNA analyte (left). Addition of the specific nucleic acid analyte results in the formation of a quadripartite associate, in which MB probe adopts the open conformation (right). The complex is unstable if there is a mismatch base-pairing in the hybrid of analyte and strand m.

Figure 2

Design and performance of the universal MB probe (UMB). A) Structures of UMB and complementary oligonucleotides. B) Fluorescent response of UMB in the presence of different concentrations of complementary DNA: UMB complement C24 (●), UMB complement C22 (○) or UMB complement C18 (■). C) Signal-to-background ratio for the complexes of the MB probe in the presence of low concentrations of C24, C22 or C18. The S/B threshold of 2 is shown by the horizontal dashed line. Assay conditions: 100 nm MB, 50 mm Tris–HCl, pH 7.4, 50 mm MgCl₂, 20°C.

Figure 3

Structures of BDPs in the complex with cognate analytes. Triethylene glycol linkers are represented as dashed lines. BHQ1 is black hole quencher 1; FAM represents fluorescein residue. Only the 3′-end sequences of each analyte are shown (see Table 1 in the Experimental Section for full sequences). SNP sites are underlined. Analyte-binding arms of strands m and f are in italic.

Figure 4

Two alternative conformations of binary probe quadripartite complex. The left conformation contains a MB probe in an elongated conformation thus allowing a high fluorescent signal. The right conformation contains fluorophore close to the quencher, which prevents significant increase in fluorescence of the quadripartite complex.

Figure 5

Analysis of BDP rs14-A in 12% native polyacrylamide gel by using UMB′ (FAM-5′-CGC GTT AAC ATA CAA TAG ATC GCG). UMB′ (100 nm) was incubated either alone (lane 1) or with different combinations of 300 nm strands m and f and 75 nm analytes rs14-A (matched) or rs14-G (mismatched). Lane 2: UMB′, m and f; lane 3: UMB′, m, f and rs14-G; lane 4: UMB′, m, f, rs14-A; lane 5: UMB′ and f; lane 6: UMB′, f and rs14-G; lane 7: UMB′, f and rs14-A; lanes 8–10 contained pure oligonucleotides m, f or rs14-A, respectively. A) Gel without staining (only UMB′-containing bands are detected). B) The same gel after SYBR Gold staining (all oligonucleotides in the gel are detected). Note, strands m and f were used in fourfold excess over analytes; thus no analyte was observed in its free form in the samples containing strands m and f.

Figure 6

A) Fluorescence enhancement (S/B) for BDP rs14-G (300 nm strands m and f and 100 nm UMB) in the presence of perfectly matched analyte rs14-G (●) or mismatched analyte rs14-A (○) as functions of the analyte concentrations. Fluorescence emission of the BDP rs14-G at 517 nm in the absence of the analyte is taken as a background. B) Fluorescent enhancement for all BDPs at low analyte concentrations. The S/B threshold of 2 is shown by a horizontal dashed line. Assay conditions: 100 nm UMB; strands: 300 nm for BDP rs14-G (●) or BDP rs14-A (○), 120 nm for BDP rs71-G (□) or BDP rs71-A (■), 600 nm for BDP rs87-C (▲), 1000 nm for BDP rs87-T (△).

Figure 7

Specificity and selectivity of binary DNA probes. A) Fluorescence enhancement of BDPs in the presence of the correspondent matched (white bars) or mismatched (gray bars) analytes. B) Fluorescence enhancement of BDPs in the presence of all six DNA analytes (white bars) or all DNA analytes except for the correspondent matched analyte (gray bars). The dashed line indicates the signal-threshold level. All experiments were carried out in triplicate. The error bars represent three standard deviations. Assay conditions: 100 nm UMB, 25 nm analytes, 300 nm BDP rs14-G and BDP rs14A, 120 nm BDP rs71-A and BDP rs71-G, 1000 nm BDP rs87-T, 600 nm BDP rs87-C, 50 mm Tris–HCl, pH 7.4, 50 mm MgCl₂, 20°C.