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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.

No MeSH data available.


Synthesisof Target Phosphoramidite 6DMTr = 4,4′-dimethoxytrityl;Py = pyren-1-yl; Ms = methanesulfonyl; TMG = 1,1,3,3-tetramethylguanidine;PCl reagent = 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.
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sch1: Synthesisof Target Phosphoramidite 6DMTr = 4,4′-dimethoxytrityl;Py = pyren-1-yl; Ms = methanesulfonyl; TMG = 1,1,3,3-tetramethylguanidine;PCl reagent = 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.


Merging Two Strategiesfor Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes
Synthesisof Target Phosphoramidite 6DMTr = 4,4′-dimethoxytrityl;Py = pyren-1-yl; Ms = methanesulfonyl; TMG = 1,1,3,3-tetramethylguanidine;PCl reagent = 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.
© Copyright Policy
Related In: Results  -  Collection

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

sch1: Synthesisof Target Phosphoramidite 6DMTr = 4,4′-dimethoxytrityl;Py = pyren-1-yl; Ms = methanesulfonyl; TMG = 1,1,3,3-tetramethylguanidine;PCl reagent = 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.

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.