Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA

TY - JOUR

T1 - Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA

AU - Demidov, V V

AU - Frank-Kamenetskii, M D

AU - Nielsen, Peter E.

PY - 1998/1/15

Y1 - 1998/1/15

N2 - Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially. Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude. Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300. Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.

AB - Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially. Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude. Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300. Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.

KW - Animals

KW - Base Sequence

KW - Cations

KW - Cytosine/analogs & derivatives

KW - DNA/chemistry

KW - DNA, Complementary/chemistry

KW - Electrochemistry

KW - Kinetics

KW - Male

KW - Nucleic Acids/chemistry

KW - Osmolar Concentration

KW - Peptides/chemistry

KW - Salmon

KW - Sodium/pharmacology

KW - Structure-Activity Relationship

M3 - Journal article

C2 - 9421519

SN - 0305-1048

VL - 26

SP - 582

EP - 587

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 2

ER -