Limits...
Merging Two Strategiesfor Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes

View Article: PubMed Central - PubMed

ABSTRACT

The development of molecular strategiesthat enable recognitionof specific double-stranded DNA (dsDNA) regions has been a longstandinggoal as evidenced by the emergence of triplex-forming oligonucleotides,peptide nucleic acids (PNAs), minor groove binding polyamides, and—morerecently—engineered proteins such as CRISPR/Cas9. Despite thisprogress, an unmet need remains for simple hybridization-based probesthat recognize specific mixed-sequence dsDNA regions under physiologicalconditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probesare chimeras between pseudocomplementary DNA (pcDNA) and Invader probes,which are activated for mixed-sequence dsDNA-recognition through theintroduction of pseudocomplementary base pairs comprised of 2-thiothymineand 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalizednucleotides, respectively. We demonstrate that certain pseudocomplementaryInvader probe designs result in very efficient and specific recognitionof model dsDNA targets in buffers of high ionic strength. These chimericprobes, therefore, present themselves as a promising strategy formixed-sequence recognition of dsDNA targets for applications in molecularbiology and nucleic acid diagnostics.

No MeSH data available.


Related in: MedlinePlus

Recognition of DNA hairpins using Invader probes. (a) Illustrationof recognition process; (b) sequences and thermal denaturation temperaturesof DNA hairpins with isosequential (DH1) or nonisosequentialstems (DH2–DH7); underlined nucleotidesindicate sequence deviations relative to probes; (c) representativeelectrophoretograms of recognition of DH1 using 1- to500-fold molar excess of Y1:Y3 or DY1:DY4; (d) dose–response curves (averageof at least three independent experiments; error bars represent standarddeviation); (e) electrophoretograms illustrating incubation of DH1–DH7 with 200-fold molar excess of X1:X3, Y1:Y3, or DY1:DY4. Experimental conditions for electrophoreticmobility shift assay: separately preannealed targets (34.4 nM) andprobes (variable concentrations) were incubated for 12–16 hat ambient temperature in 1X HEPES buffer (50 mM HEPES, 100 mM NaCl,5 mM MgCl2, 10% sucrose, 1.4 mM spermine tetrahydrochloride,pH 7.2) and then resolved on 16% nondenaturing PAGE (70 V, 2.5 h,∼4 °C) using 0.5× TBE as a running buffer (45 mMTris, 45 mM boric acid, 1 mM EDTA); DIG: digoxigenin.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836393&req=5

fig2: Recognition of DNA hairpins using Invader probes. (a) Illustrationof recognition process; (b) sequences and thermal denaturation temperaturesof DNA hairpins with isosequential (DH1) or nonisosequentialstems (DH2–DH7); underlined nucleotidesindicate sequence deviations relative to probes; (c) representativeelectrophoretograms of recognition of DH1 using 1- to500-fold molar excess of Y1:Y3 or DY1:DY4; (d) dose–response curves (averageof at least three independent experiments; error bars represent standarddeviation); (e) electrophoretograms illustrating incubation of DH1–DH7 with 200-fold molar excess of X1:X3, Y1:Y3, or DY1:DY4. Experimental conditions for electrophoreticmobility shift assay: separately preannealed targets (34.4 nM) andprobes (variable concentrations) were incubated for 12–16 hat ambient temperature in 1X HEPES buffer (50 mM HEPES, 100 mM NaCl,5 mM MgCl2, 10% sucrose, 1.4 mM spermine tetrahydrochloride,pH 7.2) and then resolved on 16% nondenaturing PAGE (70 V, 2.5 h,∼4 °C) using 0.5× TBE as a running buffer (45 mMTris, 45 mM boric acid, 1 mM EDTA); DIG: digoxigenin.

Mentions: On thebasis of the observed ΔGrec293 values,we decided to determine the dsDNA-recognition properties of Y1:Y3 and DY1:DY4 relativeto benchmark Invader X1:X3 using an electrophoreticmobility shift assay from our earlier studies.22 Thus, a digoxigenin (DIG) labeled DNA hairpin (DH)—comprisedof a 9-mer double-stranded mixed-sequence stem, which is linked bya T10 loop—was used as a model dsDNA target (Figure 2a and 2b). Room temperature incubation of DH1 with Y1:Y3, DY1:DY4, or X1:X3 in a HEPES buffer of considerable ionicstrength, results in dose-dependent formation of a ternary recognitioncomplex as evidenced by the emergence of a slower migrating band onnondenaturing PAGE gels (Figure 2c). Nonlinear regression fits of dose–responsecurves reveal that X1:X3, Y1:Y3, and DY1:DY4 have C50 values of ∼0.8 μM, ∼2.8μM, and ∼1.5 μM, respectively (Figure 2d), which mirrors the trendin ΔGrec293 values (Table 2).


Merging Two Strategiesfor Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes
Recognition of DNA hairpins using Invader probes. (a) Illustrationof recognition process; (b) sequences and thermal denaturation temperaturesof DNA hairpins with isosequential (DH1) or nonisosequentialstems (DH2–DH7); underlined nucleotidesindicate sequence deviations relative to probes; (c) representativeelectrophoretograms of recognition of DH1 using 1- to500-fold molar excess of Y1:Y3 or DY1:DY4; (d) dose–response curves (averageof at least three independent experiments; error bars represent standarddeviation); (e) electrophoretograms illustrating incubation of DH1–DH7 with 200-fold molar excess of X1:X3, Y1:Y3, or DY1:DY4. Experimental conditions for electrophoreticmobility shift assay: separately preannealed targets (34.4 nM) andprobes (variable concentrations) were incubated for 12–16 hat ambient temperature in 1X HEPES buffer (50 mM HEPES, 100 mM NaCl,5 mM MgCl2, 10% sucrose, 1.4 mM spermine tetrahydrochloride,pH 7.2) and then resolved on 16% nondenaturing PAGE (70 V, 2.5 h,∼4 °C) using 0.5× TBE as a running buffer (45 mMTris, 45 mM boric acid, 1 mM EDTA); DIG: digoxigenin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836393&req=5

fig2: Recognition of DNA hairpins using Invader probes. (a) Illustrationof recognition process; (b) sequences and thermal denaturation temperaturesof DNA hairpins with isosequential (DH1) or nonisosequentialstems (DH2–DH7); underlined nucleotidesindicate sequence deviations relative to probes; (c) representativeelectrophoretograms of recognition of DH1 using 1- to500-fold molar excess of Y1:Y3 or DY1:DY4; (d) dose–response curves (averageof at least three independent experiments; error bars represent standarddeviation); (e) electrophoretograms illustrating incubation of DH1–DH7 with 200-fold molar excess of X1:X3, Y1:Y3, or DY1:DY4. Experimental conditions for electrophoreticmobility shift assay: separately preannealed targets (34.4 nM) andprobes (variable concentrations) were incubated for 12–16 hat ambient temperature in 1X HEPES buffer (50 mM HEPES, 100 mM NaCl,5 mM MgCl2, 10% sucrose, 1.4 mM spermine tetrahydrochloride,pH 7.2) and then resolved on 16% nondenaturing PAGE (70 V, 2.5 h,∼4 °C) using 0.5× TBE as a running buffer (45 mMTris, 45 mM boric acid, 1 mM EDTA); DIG: digoxigenin.
Mentions: On thebasis of the observed ΔGrec293 values,we decided to determine the dsDNA-recognition properties of Y1:Y3 and DY1:DY4 relativeto benchmark Invader X1:X3 using an electrophoreticmobility shift assay from our earlier studies.22 Thus, a digoxigenin (DIG) labeled DNA hairpin (DH)—comprisedof a 9-mer double-stranded mixed-sequence stem, which is linked bya T10 loop—was used as a model dsDNA target (Figure 2a and 2b). Room temperature incubation of DH1 with Y1:Y3, DY1:DY4, or X1:X3 in a HEPES buffer of considerable ionicstrength, results in dose-dependent formation of a ternary recognitioncomplex as evidenced by the emergence of a slower migrating band onnondenaturing PAGE gels (Figure 2c). Nonlinear regression fits of dose–responsecurves reveal that X1:X3, Y1:Y3, and DY1:DY4 have C50 values of ∼0.8 μM, ∼2.8μM, and ∼1.5 μM, respectively (Figure 2d), which mirrors the trendin ΔGrec293 values (Table 2).

View Article: PubMed Central - PubMed

ABSTRACT

The development of molecular strategiesthat enable recognitionof specific double-stranded DNA (dsDNA) regions has been a longstandinggoal as evidenced by the emergence of triplex-forming oligonucleotides,peptide nucleic acids (PNAs), minor groove binding polyamides, and—morerecently—engineered proteins such as CRISPR/Cas9. Despite thisprogress, an unmet need remains for simple hybridization-based probesthat recognize specific mixed-sequence dsDNA regions under physiologicalconditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probesare chimeras between pseudocomplementary DNA (pcDNA) and Invader probes,which are activated for mixed-sequence dsDNA-recognition through theintroduction of pseudocomplementary base pairs comprised of 2-thiothymineand 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalizednucleotides, respectively. We demonstrate that certain pseudocomplementaryInvader probe designs result in very efficient and specific recognitionof model dsDNA targets in buffers of high ionic strength. These chimericprobes, therefore, present themselves as a promising strategy formixed-sequence recognition of dsDNA targets for applications in molecularbiology and nucleic acid diagnostics.

No MeSH data available.


Related in: MedlinePlus