Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

Abstract

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5' end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled ('cold') oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10(-8) M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10(-11) M solutions of PCR amplicons from M.tuberculosis in only 100 s.

Figure 1

Principle of operation of smart probes. The fluorophore is attached to the 5′ end of the oligonucleotide and quenched by guanosine residues in the complementary stem. Hybridization to the target sequence forces the stem apart and completely restores the fluorescence because efficient quenching of the fluorophore by guanosine residues requires contact formation.

Figure 2

Fluorescence intensity with time (MCS-trace) recorded from a 5 × 10⁻¹⁰ M solution of a smart probe (1 ms/bin). Fluorescence bursts are defined by a recognition threshold, i.e. a count rate of 30 kHz (black line). Origin and end of a burst were defined by a count rate of 20 kHz (dashed line). The fluorescence lifetimes of recognized bursts were calculated applying a MLE-algorithm. Using the described burst recognition definition, we identify two fluorescence bursts at ∼25 ms (223 photon counts, 3 ms burst duration, fluorescence lifetime, τ = 2.9 ns) and at ∼58 ms (138 photon counts, 4 ms burst duration, fluorescence lifetime, τ = 2.2 ns).

Figure 3

mfold structures (modeling parameters: 140 mM Na⁺, 0 mM Mg²⁺, 25°C) of the 157 bp PCR amplicon of M.tuberculosis. The binding sites for the loop sequence of the smart probe marked dark gray. The binding sites for the ‘cold’ oligonuleotides 1–6 are marked light gray (58–73: cold oligonucleotides 1; 74–98: cold oligonucleotides 2; 99–133: cold oligonucleotides 3; 134–157: cold oligonucleotides 4; 1–19: cold oligonucleotides 5; 20–40: cold oligonucleotides 6).

Figure 4

Fluorescence intensity (MCS)-traces observed from a 5 × 10⁻¹⁰ M solution of smart probe-M before and after addition of a 100-fold excess of an artificial target DNA. Measurements were carried out at room temperature in PBS (pH 7.0) containing 140 mM NaCl at an average excitation power of 0.6 mW at the sample.

Figure 5

Fluorescence intensity traces observed from 5 × 10⁻¹⁰ M solution of the smart probe-M in absence (a) and presence of (b) 2 × 10⁻⁸ M, (c) 1 × 10⁻⁹ M and (d) 5 × 10⁻¹¹ M of the matching PCR-product. To all samples the cold oligonucleotides 1–6 were added. Before measuring all samples were heated to 65°C and cooled down to 25°C over night. Measurements were carried out at room temperature in PBS (pH 7.0) containing 140 mM NaCl using an average excitation power of 0.6 mW.

Figure 6

(a) Burst size, (b) fluorescence decay time and (c) burst duration distribution of 3500 photon bursts detected from a 5 × 10⁻¹⁰ M solution of smart probe-M in the absence (gray) and presence (open) of 2 × 10⁻⁸ M matching PCR product.

Figure 7

Fluorescence lifetime of each signal measured from a 5 × 10⁻¹⁰ M solution of the smart probe-M (a) in the absence and (b) presence of 2 × 10⁻⁸ M of the matching PCR-product is plotted versus the respective burst size (photon counts/burst). Signals with a burst duration ≥4 ms are marked red.

Figure 8

Detected signals of the smart probe-M (triangles) and the smart probe-WT (squares) with a burst size of >150 photons, a fluorescence lifetime of >2.25 ns and a burst duration of ≥4 ms during a measurement time of 100 s plotted versus the concentration of PCR-product of the wild type and the mutant of M.tuberculosis, respectively. The data points obtained from the matching PCR-product are black and the respective reference sample containing the other PCR-product containing one mismatch is marked gray.

Figure 9

(a) FCS measurements of a 5 × 10⁻⁹ M solution of the smart probe before (dotted line) and after addition of the artificial target DNA (dashed line) and target PCR-product (solid line), respectively. Furthermore, 200 ms cut-outs of the MCS-traces of a 5 × 10⁻¹⁰ M smart probe sample in the absence (b) and the presence (c) of the target PCR product.