Towards MPD enabled direct detection of DNA
Abstract: We developed a new approach to detection of DNA without the need of target amplification. It couples the MPD technique with a use of a plurality of DNA probes, including peptide nucleic acid (PNA) probes. This approach is based on the ability of short oligomers of peptide nucleic acids (PNAs) to form looped structures, P-loops, during hybridization with dsDNA. If two PNAs (so called "openers") bind specifically to neighbouring DNA sites separated by several base pairs, an extended open region emerges within the DNA duplex. This region can serve as a target for probes carrying a reporter group e.g., 125I. MPD and PNA based techniques are applied synergistically.
Towards direct detection of a low number of DNA copies: The direct detection of DNA using an appropriate hybridization technique with signal amplification may have considerable advantage for biomedical and food quality testing. The main challenges are the need for a large "signal amplification" and reduction of non-specific biological background. The sensitivity of MPD can be further enhanced by SuperTracers. Thus, dqDNA/MPD is currently not signal but "background" limited. To overcome non-specific biological background [NSBB], we implemented a series of innovative procedures which diminish the background by a few orders of magnitude when compared to prior-art techniques.
How to improve the selectivity and diminish the background: The challenge is hybridization specificity. We are developing PNA-based DNA diagnostics. It is a synergistic combination of superselective methods of probe hybridization with double-stranded DNA (dsDNA) via PNA interaction with DNA duplex (P-loop formation) using MPD reporting. This technique is referred to as PNA enabled dqDNA/MPD [dqDNA/(MPD,PNA)]. We are using: PNA openers, biotinylated DNA probe used for capture (fishing hook) and DNA probe coupled to radioiodinated molecule, e.g., SuperTracer. The considerable gain of specificity is due to the use of PNA. The use of SuperTracers and MPD instrumentation provide the needed sensitivity. Thus MPD and PNA based techniques were applied synergistically to permit a user friendly, high throughput operation.
Hybridization of a PNA to dsDNA via PD-loop is a more sequence-specific process than usual hybridization of a PNA probe to ssDNA or RNA. First, a duplex DNA flanked by two specific PNA openers have the only sites exposed and accessible for complexing with the complementary PNA or DNA probes. Most of DNA retains its duplex structure and is inaccessible for binding of an PNA with mixed purine-pyrimidine composition. Note that P-loops formed as a result of binding of separate openers are too small to form stable complexes with a PNA. The requirement for a simultaneous binding of two openers along with a probe to the dsDNA target additionally provides high specificity as it was demonstrated for the hybridization of tethered PNAs and separate PNAs having two different targeting segments complementary to target DNA or RNA. Second, the PNA probe normally consists of about 15 nucleotides (nt), so its recognition site is virtually unique in the whole genome. The entire P-loop may be somewhat larger, embracing about 20 bp or more. Because of a small length of the PNA probes, even one mismatch due to the incorrect opening of the site by the same pair of PNA openers, will result in an unstable PNA/DNA complex.
Our preliminary studies demonstrated that PNA-based DNA detection/quantitation is improved by the use of MPD Instrumentation. We have performed pilot experiments in which the P-loop technology was combined with MPD detection to increase the sensitivity. We performed pre-gel hybridization of PNA probe to plasmid dsDNA via P-loop. The P-loop technology significantly increases specificity of hybridization while the MPD technology strongly increases the assay sensitivity. We performed the initial studies on HIV-1 target.
The development of SuperTracers: In all super-sensitive detection methods, some form of signal enhancement is used: large turnover of an enzymatic reaction, amplification of the number of target molecules, etc. These methods are very efficient but often lead to a sample-to-sample variability of amplification. For large macromolecules of biological importance, the more reliable method is multiple labeling, i.e., a large but well defined number of labels are conjugated to the same target. Labeling efficiency depends on the type of label used and is often limited by steric hindrance. Also, there is an important difference between fluorescent and isotopic labels. Multiple fluorophores tend to saturate the signal due to significant "quenching" of the fluorescence caused by neighboring groups. Thus the signal amplification is not proportional to the number of tags. In the case of radiolabels, the signal is strictly proportional to the number of radiolabels, the dynamic range is very large, and the quantitation is possible over a broad range of signal levels.
For dqDNA/MPD, SuperTracers consist of DNA or PNA probe linked to a large, heavily iodinated dendrimer (see Figure 1).
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A promising class of SuperTracers is based on the dendrimer, which is a spherical polymer where polymerization is controlled to achieve step-wise propagation, resulting in a preselected number of reporter groups (preferably amino) on the surface. Products of this step-growth are called generations, and the number of amino groups is doubled in each subsequent generation. The SuperTracer can boost assay sensitivity hundred fold. The level of NSBB is crucial when using hybridization with poly-iodinated DNA probes. We demonstrated that spherical, closed shell dendrimers show lower NSBB than much larger open tree branched DNA. |
We performed chemical synthesis of phosphate-based dendrimers that contain one linker group and a strictly controlled number of reporter groups. The chemistry is based on a phosphoramidite method of the chemical synthesis of oligonucleotides. All chemical reactions are performed on solid support using commercially available reagents. It has been shown that coupling yield of an individual cycle is above 99.7%. Synthesis of the oligonucleotide hybridization probe was performed on the solid support, e.g., Controlled Pore Glass (CPG) in an automatic DNA synthesizer. In conclusion, we successfully developed the 7th generation dendrimer coupled to a DNA probe by an appropriate linker. The synthesis method is being extended to 9th and hopefully the 10th generation SuperTracer.
MPD enhanced detection using PNA/DNA constructs: We have performed pilot experiments in which the P-loop hybridization technology was combined with MPD detection using 125I-labeled probes to increase the sensitivity of hybrid detection. The P-loop complex was purified from non-bound PNAs, blotted onto nitrocellulose membrane and immobilized by UV-crosslinking. Activity of the incorporated 125I was measured using ssMPD or MPD-Imager. The results presented in Figure 2 demonstrate linearity of the signal in the whole range of complex concentrations. We showed good reproducibility and sensitivity of the dqDNA/(MPD,PNA). We detected a few attomoles of the target DNA without any amplification of the signal. The estimated limit of quantitation is about 0.2 attomole. That is about 100 times better than conventional chemiluminescent detection. These results are very encouraging and by implementing signal amplification techniques we expect to reach zeptomole sensitivity.
The limits to sensitivity of dqDNA/(MPD,PNA) is NSBB and super-stringent washing may be necessary to reach zeptomole limits of quantitation. Our collaborators, a group of Prof. M. Frank-Kamienetskii from BU, studied the use of "padlock probes" which are compatible with such super-stringent wash. Signal amplification of the probe bound to duplex DNA via P-loop using rolling circle amplification (RCA) has been documented. Efficient RCA generated single-stranded product in a primer-dependent fashion has been demonstrated.
These results show that the synergistic combination of P-loop technology and MPD detection allowed direct detection of DNA at the sub-attomole level without any signal amplification, which is a substantial increase in sensitivity as compared with chemiluminescent methods. However, the level of detection still is not sufficient to detect low levels of microbial DNA and needs to be improved. To further increase the sensitivity of MPD/PD detection, we developed a DNA polymerase-driven signal amplification meditated by the P-loop in situ hybridization.