Modifying the N-terminus of polyamides: PyImPyIm has improved sequence specificity over f-ImPyIm.
Seven N-terminus modified derivatives of a previously published minor-groove binding polyamide (f-ImPyIm, 1) were synthesized and the biochemical and biophysical chemistry evaluated. These compounds were synthesized with the aim of attaining a higher level of sequence selectivity over f-ImPyIm (1), a previously published strong minor-groove binder. Two compounds possessing a furan or a benzofuran moiety at the N-terminus showed a footprint of 0.5microM at the cognate ACGCGT site (determined by DNase I footprinting); however, the specificity of these compounds was not improved. In contrast, PyImPyIm (4) produced a footprint of 0.5microM but showed a superior specificity using the same technique. When evaluated by thermal melting experiments and circular dichroism using ACGCGT and the non-cognate AAATTT sequence, all compounds were shown to bind in the minor-groove of DNA and stabilize the cognate sequence much better than the non-cognate (except for the non-amido-compound that did not bind either sequence, as expected). PyImPyIm (4) was interesting as the DeltaT(m) for this compound was only 4 degrees C but the footprint was very selective. No binding was observed for this compound with a third DNA (non-cognate, ACCGGT). ITC studies on compound 4 showed exothermic binding with ACGCGT and no heat change was observed for titrating the compound to the other two DNA sequences. The heat capacity (DeltaC(p)) of the PIPI/ACGCGT complex calculated from the hydrophobic interactions and SASA calculations was comparable to the experimental value obtained from ITC (-146calmol(-1)K(-1)). SPR results provided confirmation of the sequence specificity of PyImPyIm (4), with a K(eq) value determined to be 7.1x10(6) M(-1) for the cognate sequence and no observable binding to AAATTT and ACCGGT. Molecular dynamic simulations affirmed that PyImPyIm (4) binds as a dimer in an overlapped conformation, and it fits snugly in the minor-groove of the ACGCGT oligonucleotide. PyImPyIm (4) is an especially interesting molecule, because although the binding affinity is slightly reduced, the specificity with respect to f-ImPyIm (1) is significantly improved.
Brown
Toni
T
Department of Chemistry, Furman University, Greenville, SC 29613, USA.
Mackay
Hilary
H
Turlington
Mark
M
Sutterfield
Arden
A
Smith
Traci
T
Sielaff
Alan
A
Westrate
Laura
L
Bruce
Chrystal
C
Kluza
Jerome
J
O'Hare
Caroline
C
Nguyen
Binh
B
Wilson
W David
WD
Hartley
John A
JA
Lee
Moses
M
D001483:Base Sequence; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000737:chemistry; D005663:Furans / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D009757:Nylons / Q000138:chemical synthesis / Q000737:chemistry*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D020349:Surface Plasmon Resonance; D013997:Time Factors
D005663:Furans; D007093:Imidazoles; D009757:Nylons; D003850:Deoxyribonuclease I