Endo IV assay
An example of the new Endo IV-based assay is shown in . A probe and an enhancer are hybridized to a complementary synthetic target with a one base gap between them.
The signal is substantially diminished in the absence of the enhancer. A rigid hydroxyprolinol linker () between the oligonucleotide and fluorophore FL1, critical for sensitivity and specificity, was used in this experiment. The results in illustrate the importance of all components used in the assay. The cleavage of the fluorophore is highly target dependent and its rate is amplified in the presence of the enhancer oligonucleotide. Very low non-target-dependent cleavage is an important feature of the Endo IV assay.
Optimization of Endo IV enzyme activity
Effects of pH and cations on E.coli Endo IV activity was determined using the probe, enhancer and target (). It was shown that Endo IV cleavage of the probe has a relatively flat pH optimum in the range between pH 8.5 and 9.5. For compatibility with post-PCR Endo IV amplification the assay buffer with pH 8.6 was chosen, similar to that of the PCR master mixture. Inclusion of the divalent magnesium cation in the buffer ranging from 0 to 20 mM, resulted in more than 2-fold increase in cleavage rate, the optimum range was ~5–8 mM. A concentration of 5 mM was chosen for the Endo IV assay. It was determined that the monovalent cations, such as Li, Na, K and Rb, inhibit Endo IV cleavage activity for the concentrations studied in the range from 0 to 100 mM. An inhibition of ~30% was observed at 20 mM monovalent cation concentration, which increased to ~60–70% at 100 mM concentration. The optimum probe concentration was determined to be ~600 nM, please see Supplementary Figures 1–3.
Optimization of fluorophore and linker structures
shows a model Endo IV assay that requires a target complementary to an enhancer and a probe labeled at the 5′ end with an Eclipse Dark Quencher and a fluorescent dye at the 3′ end. As shown, the probe (calculated Tm = 46.7°C) and enhancer (calculated Tm = 50.2°C) are separated by one base to mimic a natural abasic site. Four probes with different fluorescent dyes and linkers () were evaluated for their performance in the new assay. Fluorophores FL1, FL2 and FL3 of the probes I, II and III (with similar quantum yields) contained a hydroxyprolinol linker between the 3′-phosphate of the probe sequence and the core of the dyes. Fluorophore FL4 of probe IV was analogous to FL3 of probe III, with an additional aminocaproic spacer introduced between the hydroxylprolinol moiety and the dye; the purpose of this spacer was to distance the fluorophore from the cleavage site.
The probe specificity was evaluated by comparing the cleavage rates in the presence and absence of target. Target-dependent (specific) and target-independent (non-specific) cleavage rates of the probe were measured as the percent target cleaved per minute. The results are summarized in . Probes I–III demonstrated similar specific cleavage. A 2- to 3-fold decrease in the rate of non-specific cleavage for probes II and III versus probe I was observed. These low, non-specific cleavage rates translate into high-specific to non-specific cleavage ratios. It appears that chloro- or chloro/methyl substitutions in the fluorophores of probes II and III, respectively, are beneficial for the reduction of non-specific cleavage. One possible explanation for this reduction may be an effect of increased hydrophobicity of FL2 and FL3 on enzyme activity. In the case of probe IV, with the extended hydroxyprolinol linker, the rate of specific cleavage almost doubled. However, the corresponding increase in the rate of non-specific cleavage was ~20 times higher than that observed in probe III, substantially compromising the ratio of specific to non-specific cleavage rates. Once again, this result implies the existence of inhibiting interactions between Endo IV enzymatic activity and fluorophore which can be adjusted by moving the fluorophore away from the active center.
Effects of dyes and linkers on the rates and ratios of specific and non-specific cleavage
The rigid hydroxyprolinol linker with low non-specific cleavage rates utilized in probes I–III was identified as the linker of choice in the Endo IV assays.
The use of a flexible, straight chain linker (C6) instead of hydroxyprolinol-based linker increased the rates of non-specific cleavage to even higher degree than the extended hydroxyprolinol linker and reduced the specific to non-specific ratios to an unacceptable level (data not shown).
It appears that both the structure of the sterically constrained hydroxyprolinol linker and proximity of the bulky dye moiety to the cleavage site moderate cleavability of the 3′-terminal phosphodiester bond and practically eliminate non-template-dependent cleavage.
shows the target, probe and the enhancer aligned with the target to indicate different gaps. The Tm of the enhancer is typically chosen to be at least 5°C higher than that of the probe. The cleavage rates in the presence of enhancer are shown with 1–5 base gaps (). As expected, based on the natural abasic substrate requirement shown in , a one base gap gave the highest cleavage rate. The probe in the absence of enhancer showed a cleavage rate nearly equivalent to that of a 4 base gap in the presence of enhancer. As shown in , the maximum cleavage rate is achieved at a concentration of enhancer greater than ~80 nM. In practice, the concentration of the enhancer is typically at least equivalent or larger than that of the probe. Some cleavage is observed with no enhancer present.
Figure 4 (A) Endo IV assay investigating a gap of 0–5 bases between the probe and primer. (B) Shows the cleavage rate dependence on the gap between the probe and primer in reference to the cleavage rate when no enhancer is present. (C) Shows the cleavage (more ...)
Probe Tm optimization
shows how the cleavage rate of probes with different Tms changes with temperature. Probe lengths ranging from a 6mer to a 14mer were investigated and are shown in with calculated Tms of between 14 and 60°C. All the probes showed a bell-shaped relationship for cleavage rate with temperature. As expected, the cleavage rate for all probes increased with increased temperature. A relatively sharp cleavage rate optimum was observed for the probes a, b and c with calculated Tms of 60, 48 and 42, respectively. It appears that the best probe cleavage occurs close to the calculated Tm or slightly above it. Performing the assay at a temperature slightly higher than the Tm () showed not only optimum activity but also appears to allow probe cleavage cycling, an important feature of this assay.
Figure 5 The effect of temperature on the cleavage rate of probes with different length and calculated Tm. The numbers in parentheses are the determined Tms. ‘nd’ is not determined. The target and enhancers sequences are, respectively, 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′ (more ...)
Determination of assay sensitivity
The ability of the Endo IV assay to detect different target concentrations is shown in . It was observed that in the case of 125 nM target concentration, the probe-based fluorescence plateaued in ~1 h, while in the presence of 25 nM target a fluorescence plateau was reached in ~3 h. The limit of detection of this assay is 0.04 nM, clearly distinguishable from the stable background (). This stable background, a characteristic feature of the assay, is maintained over a period of 15 h. This property is also observed in the assay with other nucleic acid targets.
Figure 6 Dependence of rates of fluorescence generation on target concentration. Probe, enhancer and target sequences are shown in . The concentration of reagents were 150 nM probe, 150 nM enhancer, 0.2 U/μl enzyme, 5 mM MgCl2 in 20 mM Tris–HCl (more ...)
The ability of the Endo IV assay to discriminate mismatches at different positions of a 14mer probe is illustrated in . Mismatches were introduced in the probe one at a time, from positions 1 to 8. The Endo IV assay shows excellent specificity for the different mismatches from base 1 to base 6 with large match/mismatch signal ratios. As shown in , the mismatch signal is often about the same as that of the NTC or slightly higher. The exceptions are the difficult 4-G/T- and 8-T/G-mismatches (24
) where match/mismatch ratios of ~16 and 7 were observed, respectively. These ratios are quite adequate to differentiate matched and mismatched sequences. It appears (data not shown) that the discrimination in positions 1 and 2 is largely determined by substrate requirements while discrimination in other positions is determined by thermodynamic considerations. Experiments with a large number of assays have also indicated that, with 10mer to 12mer probes, satisfactory discrimination is observed with the mismatch position in all except the last three bases at the 5′ end.
Figure 7 The comparison of the change in relative signal fluorescence of match and different mismatches at different positions in a 14mer probe in an Endo IV assay run at 60°C. The probe sequence of the matched probe and target sequence are, respectively, (more ...)
The exquisite specificity of the Endo IV assay appears to be the result of at least two factors. The first is the strict intrinsic substrate specificity requirement of the Endo IV enzyme which appears to require the rigid prolinol linker (, n
= 0) used in the probes I–III. Second, the use of short probes (10mer to 12mer) gives the Endo IV a distinct advantage over most other assays where probes are typically much longer (1
Post-PCR Endo IV genotyping
The application of the Endo IV cleavage as a post-PCR detection system is shown in and . Following an asymmetric PCR using primers to produce predominantly single-stranded amplicon, two allele-specific probes, an enhancer and Endo IV enzyme are added to the amplification mixture and incubated isothermally for about an hour. The cleaved fluorescence from the two allele-specific probes, each labeled with a different fluorescent dye, is then plotted in a scatter diagram as shown in and for the genotyping analysis of two single nucleotide polymorphisms (SNPs) which are of immediate importance to in cancer (http://snp500cancer.nci.nih.gov
). The scatter diagrams of two polymorphisms, namely agouti signal molecular epidemiology studies protein (ASIP-01) and adenomatous polyposis coli (APC-03) (http://snp500cancer.nci.nih.gov
) are shown in and , respectively. In both cases, a probe specific to the wild-type allele is labeled with FL1 and a probe specific for the mutant allele labeled with FL3 is used.
Figure 8 A scatter plot of the genotyping of an agouti signal protein (ASIP-01) mutation. The probe sequences for the wild-type and mutant alleles are, respectively, 5′-Q-ACCTTCATTCCC-FL1-3′ and 5′-Q-GACCTTCATTCTC-FL3-3′. The mismatch (more ...)
Figure 9 A scatter plot of the genotyping of an adenomatous polyposis coli (APC-03) mutation. The probe sequences for the wild-type and mutant alleles are, respectively, 5′-Q-TACTTTCTGTGGC-FL1-3′ and 5′-Q-CTTTTCCGTGGC-FL3-3′. The (more ...)
The probe sets, designed to analyze ASIP-01 and APC-03 polymorphisms, were used to assay 102 unrelated human DNA samples obtained from the Coriell Institute. In the case of the ASIP-01 target the mismatch was situated two bases from the 3′ end of the probe, while in the case of APC-03, the mismatch was six bases from the 3′ end of the probe.
From our experience, the spacing of the allele clusters in and are comparable or better than those scatter plots observed in other methods (2
). The exquisite specificity achieved with the Endo IV enzyme, combined with the shorter probes used and the high signal accumulation, gives this assay an advantage over other SNP assays. In comparison with other signal accumulating assays (2
), the Endo IV assay has similar levels of high signal accumulation, but uses shorter probes with higher specificity than those assays. In the case of hybridization-based assays (5
), the Endo IV assay has not only a signal accumulation advantage, but also uses shorter probes with a specificity advantage. In addition, the shorter probes allow much more flexibility in assay design compared to other methods with longer probes.