Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Abstract

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.

Figure 1.

Working principle of basic NESA. Single-stranded target DNA contains the recognition sequence of a nicking enzyme. A molecular beacon is designed with its loop sequence complementary to the target. When one target molecule hybridizes with one beacon molecule, a full recognition site forms for the nicking enzyme to cleave the beacon strand. The nicking enzyme binds to the hybrid, and makes a nick in the beacon strand. After nicking, the complex dissociates, finishing one reaction cycle. The net result of one reaction cycle is a cleaved molecular beacon. The target molecule and nicking enzyme can be re-used for next cycle of cleavage. This way, each target can go through many cycles, resulting in cleavage of many beacon molecules. In each cycle, one target causes one beacon molecule to open and fluoresce, contributing one beacon signal. After N (N is an integer) cycles, one target gives rise to N beacons signals, achieving a linear signal amplification.

Figure 2.

Time courses for basic NESA at target concentrations of 400 nM (a) and 2 nM (b), respectively. Each time course consists of three stages: background of molecular beacon, hybridization and nicking reaction. The gap between the stages corresponds to about 15 s during which the sample was taken out of the spectrofluorometer and a new component added. The final concentration of the molecular beacon is 200 nM for both time courses.

Figure 3.

Test of the detection limit of basic NESA and traditional hybridization assay. Time courses were recorded for each assay at a series of dilutions of the target. (a) Time courses for basic NESA. (b) Time courses for hybridization.

Figure 4.

Working principle of extended NESA. Basic NESA is integrated with rolling circle amplification (RCA), in order to recognize target DNA of any sequence of interest. The central element in RCA is the padlock probe, which contains, at its two ends, a target recognition sequence, and, in the middle, a sequence (green color) identical to that of the major portion (loop plus one arm) of a molecular beacon. The molecular beacon contains a nicking enzyme recognition sequence. Extended NESA includes three sequential steps: ligation, polymerization and nicking. At ligation step, a padlock probe hybridizes at two ends to target DNA and is circularized by DNA ligase. At polymerization step, a primer binds to the circularized padlock probe, and is extended by DNA polymerase, producing a long single-stranded DNA composed of tandem copies of the complementary sequence of the padlock probe, with each copy containing a complementary sequence (red color) for the molecular beacon. At nicking step, each red color sequence, like the target sequence in basic NESA, acts as a mobile catalytic site, and leads to the nicking of many molecular beacons in the presence of nicking enzymes. To be consistent with the terms used in the basic nicking assay, we designated the target DNA as the primary target, and the red color sequence in the RCA product the secondary target. In extended NESA, the primary target does not need to contain nicking enzyme recognition sequence. Extended NESA has two levels of signal amplification: each primary target induces many secondary targets through RCA, and each secondary target brings about cleavage of many beacon molecules.

Figure 5.

Time course of extended NESA at various target concentration. All time courses are divided into two stages: RCA (a) and nicking (b). RCA was conducted without the nicking enzyme, and nicking reaction started after the nicking enzyme was added into the finished RCA reaction mixture. Seven concentrations of target were examined: 1 nM, 100 pM, 10 pM, 1 pM, 0.5 pM, 0.1 pM, 0. In (a), target concentrations <10 pM are not shown since they were not detectable. In (b), target concentrations >10 pM are omitted because the majority of beacon molecules were already opened at RCA stage.

Figure 6.

Construction of standard curves using initial reaction rate. The two charts are double log plots of initial reaction rate (V₀) and target concentration (C) for basic NESA (a) and extended NESA (b), respectively. Each data point represents the average of four measurements, with the error bar standing for standard deviation. The data points were fitted with least square linear regression. Shown above each trend line are equation and coefficient of determination. In each equation, x and y stand for Log(C) and Log (V₀), respectively. The slopes of the trend lines are close to 1 in both tests, verifying the linear relationship between the initial reaction rate and target concentration.

Figure 7.

Detection of single base mutation. All mutations were made by substituting the original base by its complementary base. Initial reaction rates for perfect target (P) and various mutants were compared. (a) Basic NESA. Beacon 1 was used. The sequences of beacon 1 (gray) and the target (black) are shown at the top of the chart. N.BstNB I recognition sequence is underlined. The position of mutation is labeled alphabetically. (b) Extended NESA. At the top of the chart is part of the sequence of the hybrid between the target (black) and padlock probe (gray) near the nick. Mutation positions are numbered. The nick in the padlock probe was sealed by either T4 ligase or E. coli ligase. After rolling circle polymerization, cleavage reaction was carried out, and beacon 2 was used to monitor the cleavage reaction. Two groups of results (gray and black) are presented. T4 ligase was used to obtain the gray group results, and E. coli ligase used to obtain the black group. Each datum is the average of four measurements, and the error bar is the standard deviation.

Model 1.

Model 2.