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Enhancing the cellular uptake of Py-Im polyamides through next-generation aryl turns.

Meier JL, Montgomery DC, Dervan PB - Nucleic Acids Res. (2011)

Bottom Line: Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude.Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation.Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

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Schematic diagram of eight-ring Py–Im polyamides targeting the sequence 5′-WGWWCW-3′ (W = A/T). Dashed lines indicate hydrogen bonds between the polyamide and DNA base pairs. The γ-aminobutyric acid turn unit enforces an antiparallel hairpin configuration, and codes for W (16). Substitution of the chiral turn functionality can have substantial effects on DNA-binding and biological activity.
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gkr970-F1: Schematic diagram of eight-ring Py–Im polyamides targeting the sequence 5′-WGWWCW-3′ (W = A/T). Dashed lines indicate hydrogen bonds between the polyamide and DNA base pairs. The γ-aminobutyric acid turn unit enforces an antiparallel hairpin configuration, and codes for W (16). Substitution of the chiral turn functionality can have substantial effects on DNA-binding and biological activity.

Mentions: Hairpin pyrrole–imidazole (Py–Im) polyamides are a class of heterocyclic amino acid oligomers that can be programmed to bind a wide repertoire of DNA sequences with high affinity and specificity (1,2). Sequence-selective recognition of the minor groove of DNA is achieved through side-by-side stacked ring pairings: Im/Py distinguishes G·C from C·G, while Py/Py is degenerate for T·A and A·T. In recent years, our group has focused on the biological evaluation of eight-ring polyamides arranged in a hairpin configuration through a γ-aminobutyric acid linker (Figure 1) (3,4). These compounds are of modest (~1300 Da) molecular weight and recognize 6 bp of DNA, similar to the size of many eukaryotic transcription factor binding motifs (5). When applied to living cells, hairpin polyamides can disrupt protein–DNA interactions and modulate the expression of genes induced by many transcription factors, including the ligand-activated nuclear receptors glucocorticoid receptor (GR) and androgen receptor (AR) (6–10). However, one persistent challenge encountered when applying Py–Im polyamides to new biological systems is cellular uptake. Previous studies have shown that the nuclear localization of fluorescently labeled polyamides can be influenced by several variables including molecular weight, modifications to the C-terminal moiety, and composition of Py/Im content (11–13). In particular, Py–Im polyamides incorporating multiple (>2–3) N-methylimidazole subunits show reduced nuclear localization, limiting the ability to target GC rich sequences in vivo (14). Therefore, new solutions for enhanced uptake are important for advancing Py–Im polyamides as probes of transcription factor binding and, potentially, as therapeutic agents.Figure 1.


Enhancing the cellular uptake of Py-Im polyamides through next-generation aryl turns.

Meier JL, Montgomery DC, Dervan PB - Nucleic Acids Res. (2011)

Schematic diagram of eight-ring Py–Im polyamides targeting the sequence 5′-WGWWCW-3′ (W = A/T). Dashed lines indicate hydrogen bonds between the polyamide and DNA base pairs. The γ-aminobutyric acid turn unit enforces an antiparallel hairpin configuration, and codes for W (16). Substitution of the chiral turn functionality can have substantial effects on DNA-binding and biological activity.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300022&req=5

gkr970-F1: Schematic diagram of eight-ring Py–Im polyamides targeting the sequence 5′-WGWWCW-3′ (W = A/T). Dashed lines indicate hydrogen bonds between the polyamide and DNA base pairs. The γ-aminobutyric acid turn unit enforces an antiparallel hairpin configuration, and codes for W (16). Substitution of the chiral turn functionality can have substantial effects on DNA-binding and biological activity.
Mentions: Hairpin pyrrole–imidazole (Py–Im) polyamides are a class of heterocyclic amino acid oligomers that can be programmed to bind a wide repertoire of DNA sequences with high affinity and specificity (1,2). Sequence-selective recognition of the minor groove of DNA is achieved through side-by-side stacked ring pairings: Im/Py distinguishes G·C from C·G, while Py/Py is degenerate for T·A and A·T. In recent years, our group has focused on the biological evaluation of eight-ring polyamides arranged in a hairpin configuration through a γ-aminobutyric acid linker (Figure 1) (3,4). These compounds are of modest (~1300 Da) molecular weight and recognize 6 bp of DNA, similar to the size of many eukaryotic transcription factor binding motifs (5). When applied to living cells, hairpin polyamides can disrupt protein–DNA interactions and modulate the expression of genes induced by many transcription factors, including the ligand-activated nuclear receptors glucocorticoid receptor (GR) and androgen receptor (AR) (6–10). However, one persistent challenge encountered when applying Py–Im polyamides to new biological systems is cellular uptake. Previous studies have shown that the nuclear localization of fluorescently labeled polyamides can be influenced by several variables including molecular weight, modifications to the C-terminal moiety, and composition of Py/Im content (11–13). In particular, Py–Im polyamides incorporating multiple (>2–3) N-methylimidazole subunits show reduced nuclear localization, limiting the ability to target GC rich sequences in vivo (14). Therefore, new solutions for enhanced uptake are important for advancing Py–Im polyamides as probes of transcription factor binding and, potentially, as therapeutic agents.Figure 1.

Bottom Line: Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude.Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation.Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

Show MeSH