Interaction of fluorescently labeled pyrrole-imidazole polyamide probes with fixed and living murine and human cells

Abstract

Pericentromeric heterochromatin plays important roles in controlling gene expression and cellular differentiation. Fluorescent pyrrole-imidazole polyamides targeting murine pericentromeric DNA (major satellites) can be used for the visualization of pericentromeric heterochromatin foci in live mouse cells. New derivatives targeting human repeated DNA sequences (α-satellites) were synthesized and their interaction with target DNA was characterized. The possibility to use major satellite and α -satellite binding polyamides as tools for staining pericentromeric heterochromatin was further investigated in fixed and living mouse and human cells. The staining that was previously observed using the mouse model was further characterized and optimized, but remained limited regarding the fluorophores that can be used. The promising results regarding the staining in the mouse model could not be extended to the human model. Experiments performed in human cells showed chromosomal DNA staining without selectivity. Factors limiting the use of fluorescent polyamides, in particular probe aggregation in the cytoplasm, were investigated. Results are discussed with regards to structure and affinity of probes, density of target sites and chromatin accessibility in both models.

Compound Shape Effects in Minor Groove Binding Affinity and Specificity for Mixed Sequence DNA

“…4–6 Although several groups have worked with these synthetic polyamides, solution difficulties, aggregation, and poor cell uptake have limited their applications. 7–9…”

Small Sequence‐Sensitive Compounds for Specific Recognition of the G⋅C Base Pair in DNA Minor Groove

“…The extra nitrogen in N ‐methylimidazole relative to pyrrole should allow that group to H bond with the G‐NH 2 that protrudes into the minor groove. Although several groups have worked with synthetic polyamides, solution difficulties have limited their applications …”

“…In making these small molecules we used readily available starting materials, avoided using palladium‐catalyzed expensive organometallic reactions and the final products were obtained after few synthetic steps. There is also a general improvement in cell uptake with smaller compounds . A problem with DB2429, however, is that its ratio of binding to a target single G⋅C bp sequence over an equivalent pure AT sequence is only about a factor of 10.…”

“…Although severalg roups have workedw ith synthetic polyamides,s olution difficultiesh ave limited their applications. [16][17][18] Our goal wast op reparee ntirely new types of compounds without the amide group that could bind to mixed A·T and G·C bps containing sequences but would also have varied chemicala nd structural properties that would allow them to specifically bind to target sequences and effectively targeta varietyo fd ifferent cell types. The initial research in the area has produced quite different compounds that are undergoing additional development to increase affinity and selectivity for the target sequencet ype with as ingle G·C flanked by A·T bps.…”

“…There is also ag eneral improvement in cell uptake with smaller compounds. [30,[16][17][18] A problemw ithD B2429, however,i st hat its ratio of binding to a target single G·C bp sequence over an equivalent pure AT sequencei so nly about af actor of 10. Ac oncept for improving this ratio is that by designing DB2429 analogsw ith av ariety of substituents, local variations in DNA microstructure could be recognized betterw ith higherbinding selectivity.…”