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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.


(a) Structures of monomers used herein. (b) Representationof energylevels of different dsDNA-targeting probes and the corresponding duplexeswith cDNA (only one probe-target duplex is shown). Pseudocomplementarybase-pairs are shown in red. Droplets denote intercalators. Note,the large difference in energy between pc-Invader:cDNA duplexes andpc-Invader probe duplexes. (c) Illustration of Invader-mediated recognitionof dsDNA target regions.
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fig1: (a) Structures of monomers used herein. (b) Representationof energylevels of different dsDNA-targeting probes and the corresponding duplexeswith cDNA (only one probe-target duplex is shown). Pseudocomplementarybase-pairs are shown in red. Droplets denote intercalators. Note,the large difference in energy between pc-Invader:cDNA duplexes andpc-Invader probe duplexes. (c) Illustration of Invader-mediated recognitionof dsDNA target regions.

Mentions: Probes capable of recognizingspecific mixed-sequence double-strandedDNA (dsDNA) regions have been long-sought-after as they can be developedinto tools that enable modulation of gene expression at the transcriptionallevel, gene editing, and detection of specific genetic signatures.Early examples of dsDNA-targeting probes include triplex-forming oligonucleotides1 (TFOs) and peptide nucleic acids2,3 (PNAs), as well as minor groove binding polyamides.4,5 Unfortunately, only a subset of the possible target regions is availablefor recognition by these probes due to requirements for exclusivepurine content (TFOs/PNAs) or short target regions (polyamides). Consequentially,significant efforts have been devoted to develop alternative approaches,which has resulted in TFOs and PNAs with reduced target site limitations.6−11 More recently, engineered proteins12,13 such as zincfinger nucleases, transcription activator-like effector nucleases(TALENs) and—in particular—CRISPR/Cas9 systems,14 have gained a tremendous amount of attention,despite mounting concerns regarding recognition specificity and cellulardelivery.15 Another class of compoundsthat has emerged from these efforts are the so-called pseudocomplementaryDNA and PNA (pcDNA/pcPNA),16−18 in which a short DNA or PNA duplexis modified to contain pseudocomplementary base pairs between 2-thiothymineand 2,6-diaminopurine (Figure 1a). The steric clash between the 2-thio and the 2-amino groupperturbs and weakens the hydrogen bonding between these moieties,resulting in a destabilized probe duplex. In contrast, 2-thiothymineand 2,6-diaminopurine form stable base pairs with canonical adenineand thymine, respectively. The difference in thermodynamic stabilitybetween probe duplexes and duplexes between individual probe strandsand complementary DNA (cDNA) allows for double-duplex invasion ofdsDNA target regions under certain conditions (Figure 1b). Thus, pcDNA can recognize terminal targetregions, while pcPNA also recognize internal target regions of DNAduplexes, albeit at low ionic strengths. However, a recent study hassuggested that recognition of mixed-sequence dsDNA regions by pcPNAmay be possible under the highly viscous conditions found in the nucleus.19


Merging Two Strategiesfor Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes
(a) Structures of monomers used herein. (b) Representationof energylevels of different dsDNA-targeting probes and the corresponding duplexeswith cDNA (only one probe-target duplex is shown). Pseudocomplementarybase-pairs are shown in red. Droplets denote intercalators. Note,the large difference in energy between pc-Invader:cDNA duplexes andpc-Invader probe duplexes. (c) Illustration of Invader-mediated recognitionof dsDNA target regions.
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Related In: Results  -  Collection

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fig1: (a) Structures of monomers used herein. (b) Representationof energylevels of different dsDNA-targeting probes and the corresponding duplexeswith cDNA (only one probe-target duplex is shown). Pseudocomplementarybase-pairs are shown in red. Droplets denote intercalators. Note,the large difference in energy between pc-Invader:cDNA duplexes andpc-Invader probe duplexes. (c) Illustration of Invader-mediated recognitionof dsDNA target regions.
Mentions: Probes capable of recognizingspecific mixed-sequence double-strandedDNA (dsDNA) regions have been long-sought-after as they can be developedinto tools that enable modulation of gene expression at the transcriptionallevel, gene editing, and detection of specific genetic signatures.Early examples of dsDNA-targeting probes include triplex-forming oligonucleotides1 (TFOs) and peptide nucleic acids2,3 (PNAs), as well as minor groove binding polyamides.4,5 Unfortunately, only a subset of the possible target regions is availablefor recognition by these probes due to requirements for exclusivepurine content (TFOs/PNAs) or short target regions (polyamides). Consequentially,significant efforts have been devoted to develop alternative approaches,which has resulted in TFOs and PNAs with reduced target site limitations.6−11 More recently, engineered proteins12,13 such as zincfinger nucleases, transcription activator-like effector nucleases(TALENs) and—in particular—CRISPR/Cas9 systems,14 have gained a tremendous amount of attention,despite mounting concerns regarding recognition specificity and cellulardelivery.15 Another class of compoundsthat has emerged from these efforts are the so-called pseudocomplementaryDNA and PNA (pcDNA/pcPNA),16−18 in which a short DNA or PNA duplexis modified to contain pseudocomplementary base pairs between 2-thiothymineand 2,6-diaminopurine (Figure 1a). The steric clash between the 2-thio and the 2-amino groupperturbs and weakens the hydrogen bonding between these moieties,resulting in a destabilized probe duplex. In contrast, 2-thiothymineand 2,6-diaminopurine form stable base pairs with canonical adenineand thymine, respectively. The difference in thermodynamic stabilitybetween probe duplexes and duplexes between individual probe strandsand complementary DNA (cDNA) allows for double-duplex invasion ofdsDNA target regions under certain conditions (Figure 1b). Thus, pcDNA can recognize terminal targetregions, while pcPNA also recognize internal target regions of DNAduplexes, albeit at low ionic strengths. However, a recent study hassuggested that recognition of mixed-sequence dsDNA regions by pcPNAmay be possible under the highly viscous conditions found in the nucleus.19

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.