DNA damage-site recognition by lysine conjugates

Abstract

Simple lysine conjugates are capable of selective DNA damage at sites approximating a variety of naturally occurring DNA-damage patterns. This process transforms single-strand DNA cleavage into double-strand cleavage with a potential impact on gene and cancer therapy or on the design of DNA constructs that require disassembly at a specific location. This study constitutes an example of DNA damage site recognition by molecules that are two orders of magnitude smaller than DNA-processing enzymes and presents a strategy for site-selective cleavage of single-strand nucleotides, which is based on their annealing with two shorter counterstrands designed to recreate the above duplex damage site.damage recognition ͉ double-stranded DNA cleavage ͉ phosphate recognition ͉ sequence-specific DNA modification A side from the common double helix, DNA forms a wide range of structural motifs, such as hairpin loops, triplex, tetraplex, and bulged structures, as well as nicks and gaps. The individual structural features of these motifs make them potential candidates for specific targeting (1-6). Among these structural elements, nicks and gaps are promising for the development of sequence-specific DNA cleavage, because they already feature a break in the phosphate backbone of DNA.Cleavage of the phosphate backbone of DNA can be caused by chemical reagents (7) such as radicals (8) and by radiation damage (9). To survive, cells develop enzymatic mechanisms for the repair that work efficiently on single-strand (ss) damage (10). Any further cleavage on the opposite strand at the damage site leads to ds cleavage, which is hard to repair (11). The ds cleavage requires either a bifunctional reagent (12-20) or detection and targeting of the damaged site. The only literature example of the latter is a complex natural antibiotic, bleomycin (21).In this paper, we show that simple lysine conjugates can identify ss damage sites with high selectivity and induce DNA cleavage at the strand opposite to the damage site (Scheme 1). Results and DiscussionWe used an internally 32 P-labeled ‡ 54-nt DNA (BW 54s, 5Ј-TAA TAC GAC TCA CTA TAG GCC CAG GGA AAA CTT GTA AAG GTC TAC CTA TCT *ATT; the 32 P label is indicated by an asterisk). This sequence incorporates several isolated guanine (G) nucleotides as well as GG diads, GGG triads, and an AT tract, the latter serving as a natural binding site for a family of lysine conjugates. We have shown that a combination of AT selectivity of binding and G selectivity of activation through photoinduced electron transfer (22-29) can be used for selective targeting of guanines flanking the AT tract (30).By annealing BW 54s with a variety of counterstrands, we built a family of constructs ( Fig. 1) inspired by a selection of sites that formed either in the process of chemical damage of DNA or, transiently, during enzymatic processing of DNA (31). Nicked DNA (constructs B and C) is involved in DNA topological transitions, DNA repair synthesis, and DNA replication of the lagging strand (32). Single-nucleotide gaps with 3Ј...

C-Lysine Conjugates: pH-Controlled Light-Activated Reagents for Efficient Double-Stranded DNA Cleavage with Implications for Cancer Therapy

“…This observation suggested that these photocleavers may damage DNA through base alkylation as well. Subsequent work confirmed that compound 4 in capable of localized DNA cleavage and the damage does not dissipate through hole hopping when the same single G site is chosen as a target using phosphate-lysine recognition 37. Furthermore, we also observed frank cleavage in 5’-(AATT) n GG(AT) m GG(AAATTT) l -3’ oligomers.…”

“…Several explanations are possible. For example, the remarkable ability of lysine to recognize a nick in DNA and facilitate a break of the complementary strand (the ss→ds cleavage conversion) discovered in our previous work37 may play some role in the high efficiency of the ds cleavage. However, the enhancement of this type of cleavage at the lower pH is an unprecedented phenomenon.…”

“…These reagents allowed for the first time to achieve a controlled photochemical transformation of the easily repairable ss DNA cleavage to the much more therapeutically important ds cleavage. 37…”

Opening Enediyne Scissors Wider: pH‐Dependent DNA Photocleavage by meta‐Diyne Lysine Conjugates

“…Not only do the positively charged amino acid residues allow to modulate binding with an anionic target such as DNA but they also allow to control photophysics of the enediyne chromophore via pH‐gated intramolecular photoinduced electron transfer (PET). These conjugates are able to selectively cleave at G‐sites flanking A‐T tracks , convert nicked (ss) DNA into linear (ds) DNA and induce intracellular DNA damage . Further modifications of the DNA binding and pH‐regulating amino acid part of the hybrid molecules lead to highly efficient ds DNA photocleavage .…”

Intracellular DNA Damage by Lysine‐Acetylene Conjugates

“…This dramatic increase of ds DNA cleavage at the lower pH renders these molecules more efficient ds DNA cleavers than calicheamicine under the conditions suitable for selective targeting of acidic cancer tissues ( Figure 2(a) ). We also found that the C-lysine conjugates bind selectively to nicks and gaps in a DNA duplex and, upon photochemical activation, transform the easily repairable ss-DNA damage into much more therapeutically important ds-DNA damage [ 20 ] ( Figure 2(b) ).…”