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.


Recognition of dsDNAmodel target DH8 using differentInvader probes. (a) Illustration of recognition process; (b) representativeelectrophoretograms for recognition of DH8 using 1- to500-fold excess of X5:X6, DY5:DY6 DSX1:DSX2, or DSX3:DSX4; (c) dose–response curves (average of at leastthree independent experiments, error bars represent standard deviation).The sequence of DNA hairpin DH8 is shown in Figure 4. For experimentalconditions, see Figure 2.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Recognition of dsDNAmodel target DH8 using differentInvader probes. (a) Illustration of recognition process; (b) representativeelectrophoretograms for recognition of DH8 using 1- to500-fold excess of X5:X6, DY5:DY6 DSX1:DSX2, or DSX3:DSX4; (c) dose–response curves (average of at leastthree independent experiments, error bars represent standard deviation).The sequence of DNA hairpin DH8 is shown in Figure 4. For experimentalconditions, see Figure 2.

Mentions: On the basis of the observed ΔGrec293 values, we decided to evaluate the dsDNA-targetingefficiency of DSX1:DSX2, DSX3:DSX4, X5:X6, and DY5:DY6 using a similar electrophoretic mobility shiftassay as used in the 9-mer series (Figure 3a). Thus, a DIG-labeled DNA hairpin (DH8)—comprised of a 13-mer double-stranded mixed-sequencestem that is linked by a T10 loop—was used as amodel dsDNA target. Incubation of DH8 with the various13-mer Invader probes results in dose-dependent formation of a slowermoving band on nondenaturing PAGE gels (Figure 3b). As expected from the initial 9-mer studies,the parent Invader X5:X6 recognizes DH8 more effectively at low probe concentrations than DY5:DY6, which has a pseudocomplementary energetichotspot (Figure 3c).Gratifyingly, Invader probes with separated pseudocomplementary basepairs and energetic hotspots display improved recognition efficiency(see curves for DSX1:DSX2 and DSX3:DSX4, respectively, Figure 3c), which follows the observed trend in ΔGrec293 values. It is particularly noteworthy thatthe use of as little as 1.0 mol equiv of DSX3:DSX4 results in ∼20% recognition of DH8, especiallywhen considering that optimal Invader design normally calls for incorporationof multiple energetic hotspots.33 Thissuggests that spatial separation of pseudocomplementary base pairsand energetic hotspots is a promising principle for the design ofefficient dsDNA-targeting Invader probes.


Merging Two Strategiesfor Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes
Recognition of dsDNAmodel target DH8 using differentInvader probes. (a) Illustration of recognition process; (b) representativeelectrophoretograms for recognition of DH8 using 1- to500-fold excess of X5:X6, DY5:DY6 DSX1:DSX2, or DSX3:DSX4; (c) dose–response curves (average of at leastthree independent experiments, error bars represent standard deviation).The sequence of DNA hairpin DH8 is shown in Figure 4. For experimentalconditions, see Figure 2.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Recognition of dsDNAmodel target DH8 using differentInvader probes. (a) Illustration of recognition process; (b) representativeelectrophoretograms for recognition of DH8 using 1- to500-fold excess of X5:X6, DY5:DY6 DSX1:DSX2, or DSX3:DSX4; (c) dose–response curves (average of at leastthree independent experiments, error bars represent standard deviation).The sequence of DNA hairpin DH8 is shown in Figure 4. For experimentalconditions, see Figure 2.
Mentions: On the basis of the observed ΔGrec293 values, we decided to evaluate the dsDNA-targetingefficiency of DSX1:DSX2, DSX3:DSX4, X5:X6, and DY5:DY6 using a similar electrophoretic mobility shiftassay as used in the 9-mer series (Figure 3a). Thus, a DIG-labeled DNA hairpin (DH8)—comprised of a 13-mer double-stranded mixed-sequencestem that is linked by a T10 loop—was used as amodel dsDNA target. Incubation of DH8 with the various13-mer Invader probes results in dose-dependent formation of a slowermoving band on nondenaturing PAGE gels (Figure 3b). As expected from the initial 9-mer studies,the parent Invader X5:X6 recognizes DH8 more effectively at low probe concentrations than DY5:DY6, which has a pseudocomplementary energetichotspot (Figure 3c).Gratifyingly, Invader probes with separated pseudocomplementary basepairs and energetic hotspots display improved recognition efficiency(see curves for DSX1:DSX2 and DSX3:DSX4, respectively, Figure 3c), which follows the observed trend in ΔGrec293 values. It is particularly noteworthy thatthe use of as little as 1.0 mol equiv of DSX3:DSX4 results in ∼20% recognition of DH8, especiallywhen considering that optimal Invader design normally calls for incorporationof multiple energetic hotspots.33 Thissuggests that spatial separation of pseudocomplementary base pairsand energetic hotspots is a promising principle for the design ofefficient dsDNA-targeting 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.