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

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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.

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Recognitionof mismatched DNA hairpins using various types of Invaderprobes. (a) Sequences and thermal denaturation temperatures of DNAhairpins with isosequential (DH8) or nonisosequentialstems (DH9–DH14); underlined nucleotidesdenote sequence deviations relative to Invader probes. (b) Representativegel electrophoretograms illustrating incubation of DH8–DH14 with 200-fold molar excess of X5:X6, DY5:DY6, DSX1:DSX2, or DSX3:DSX4. For experimentalconditions, see Figure 2.
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fig4: Recognitionof mismatched DNA hairpins using various types of Invaderprobes. (a) Sequences and thermal denaturation temperatures of DNAhairpins with isosequential (DH8) or nonisosequentialstems (DH9–DH14); underlined nucleotidesdenote sequence deviations relative to Invader probes. (b) Representativegel electrophoretograms illustrating incubation of DH8–DH14 with 200-fold molar excess of X5:X6, DY5:DY6, DSX1:DSX2, or DSX3:DSX4. For experimentalconditions, see Figure 2.

Mentions: 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.


Merging Two Strategiesfor Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes
Recognitionof mismatched DNA hairpins using various types of Invaderprobes. (a) Sequences and thermal denaturation temperatures of DNAhairpins with isosequential (DH8) or nonisosequentialstems (DH9–DH14); underlined nucleotidesdenote sequence deviations relative to Invader probes. (b) Representativegel electrophoretograms illustrating incubation of DH8–DH14 with 200-fold molar excess of X5:X6, DY5:DY6, DSX1:DSX2, or DSX3:DSX4. For experimentalconditions, see Figure 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836393&req=5

fig4: Recognitionof mismatched DNA hairpins using various types of Invaderprobes. (a) Sequences and thermal denaturation temperatures of DNAhairpins with isosequential (DH8) or nonisosequentialstems (DH9–DH14); underlined nucleotidesdenote sequence deviations relative to Invader probes. (b) Representativegel electrophoretograms illustrating incubation of DH8–DH14 with 200-fold molar excess of X5:X6, DY5:DY6, DSX1:DSX2, or DSX3:DSX4. For experimentalconditions, see Figure 2.
Mentions: 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.

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.