**4. Peptide nucleic acids (PNA) as sequence targeted radiosensitizers**

In searching for new RT approaches to inflict heavy DNA damage (specific, repair-refractive and lethal) we developed the concept of cell radiosensitization by non-covalently bound DNA radiosensitizers. Our original idea was to use *semi*-complementary BrdU-substituted oligonucleotide vectors which would hybridize to specific genomic sequences and create a mismatch at the site of the bromouracil. In theory, the sequences of the BUdR-loaded oligonucleotide vectors could be designed to efficiently form crosslinks with the target DNA upon radiation, since, as discussed above, the crosslinking efficiency is dependent on the target sequence. However, the use of oligonucleotides as vectors to bring BrUdR close to cellular DNA has many pitfalls (similarly to the antisense RNA applications) and instead we focused on peptide nucleic acids (PNA) as vectors, because PNAs are resistant to degradation and are able to invade DNA duplexes under physiological conditions. To our surprise, PNA were found to efficiently form crosslinks with DNA under ionizing radiation even without bearing halogenated bases.

> R1-ATG-CCG-ATC-GTA-R2 3'-TAC-GGC-TAG-CAT-5' PNA: DNA:

R1 = Acetyl (Ac) N-methylmorpholinium (MeMor) L-Lysine (K)

Fig. 3. The 12-mer PNA-DNA heteroduplex sequence (top) used in -radiation experiments to induce ICL and the variable N- and C-capping groups (R1 and R2) on PNA.

PNAs are nucleic acid analogues with an uncharged peptide-like backbone (Nielsen, 1995; Porcheddu & Giacomelli, 2005; Pellestor *et al*., 2008). PNAs bind strongly to complimentary DNA and RNA sequences. Originally designed as ligands for the recognition of double stranded DNA (Egholm *et al.*, 1993; Demidov *et al.*, 1995) their unique physicochemical properties allow them to recognize and invade complimentary sequences in specific genes and to interfere with the transcription of that particular gene (antigene strategy) (Nielsen *et al.*, 1994; Ray & Norden, 2000; Pooga *et al.*, 2001; Cutrona, *et al.*, 2000; Doyle *et al.*, 2001; Romanelli *et al.*, 2001; Kaihatsu *et al.*, 2004). The introduction of a bulky charged amino acid (*e.g.* lysine, Fig. 3) improves binding specificity, solubility and cell uptake (Menchise *et al.*, 2003). PNAs have several advantages over oligo-deoxyribonucleotides including: greater chemical and biochemical stability (PNAs are not substrates for proteases, peptidases and nucleases), greater affinity towards targets (lack of electrostatic repulsion between hybridizing strands, Fig. 4), and more sequence-specific binding.

In searching for new RT approaches to inflict heavy DNA damage (specific, repair-refractive and lethal) we developed the concept of cell radiosensitization by non-covalently bound DNA radiosensitizers. Our original idea was to use *semi*-complementary BrdU-substituted oligonucleotide vectors which would hybridize to specific genomic sequences and create a mismatch at the site of the bromouracil. In theory, the sequences of the BUdR-loaded oligonucleotide vectors could be designed to efficiently form crosslinks with the target DNA upon radiation, since, as discussed above, the crosslinking efficiency is dependent on the target sequence. However, the use of oligonucleotides as vectors to bring BrUdR close to cellular DNA has many pitfalls (similarly to the antisense RNA applications) and instead we focused on peptide nucleic acids (PNA) as vectors, because PNAs are resistant to degradation and are able to invade DNA duplexes under physiological conditions. To our surprise, PNA were found to efficiently form crosslinks with DNA under ionizing radiation

O <sup>O</sup>

R1 = Acetyl (Ac) N-methylmorpholinium (MeMor) L-Lysine (K)

<sup>N</sup> <sup>O</sup>

H2N

to induce ICL and the variable N- and C-capping groups (R1 and R2) on PNA.

hybridizing strands, Fig. 4), and more sequence-specific binding.

Fig. 3. The 12-mer PNA-DNA heteroduplex sequence (top) used in -radiation experiments

PNAs are nucleic acid analogues with an uncharged peptide-like backbone (Nielsen, 1995; Porcheddu & Giacomelli, 2005; Pellestor *et al*., 2008). PNAs bind strongly to complimentary DNA and RNA sequences. Originally designed as ligands for the recognition of double stranded DNA (Egholm *et al.*, 1993; Demidov *et al.*, 1995) their unique physicochemical properties allow them to recognize and invade complimentary sequences in specific genes and to interfere with the transcription of that particular gene (antigene strategy) (Nielsen *et al.*, 1994; Ray & Norden, 2000; Pooga *et al.*, 2001; Cutrona, *et al.*, 2000; Doyle *et al.*, 2001; Romanelli *et al.*, 2001; Kaihatsu *et al.*, 2004). The introduction of a bulky charged amino acid (*e.g.* lysine, Fig. 3) improves binding specificity, solubility and cell uptake (Menchise *et al.*, 2003). PNAs have several advantages over oligo-deoxyribonucleotides including: greater chemical and biochemical stability (PNAs are not substrates for proteases, peptidases and nucleases), greater affinity towards targets (lack of electrostatic repulsion between

CH3

O

O

NH2

NH2

H2N

**4. Peptide nucleic acids (PNA) as sequence targeted radiosensitizers** 

even without bearing halogenated bases.

PNA:

DNA:

H3C

H2N

O

R1-ATG-CCG-ATC-GTA-R2

3'-TAC-GGC-TAG-CAT-5'

R2 = Amido (Amd) L-Lysine (K)

Fig. 4. Electrostatic potentials surrounding 10-mer 3D-models of (DNA)2 and DNA-PNA duplexes, calculated using Sybyl modeling interface and dielectric constant, D = 4. Colorcodes as indicated: electronegative potentials -3.5 eV, -1.7 eV (dark blue and yellow) and electropositive +0.2 eV (red). (A) Symmetric electronegative potential surfaces along the two strands of the DNA duplex expand over all backbone atoms (blue) and the two grooves (yellow). (B) Asymmetric isopotential surfaces around PNA-DNA duplex. The electropositive/neutral PNA backbone region (red) extends over the minor and major groove atoms (0 - 0.2 eV at distances ~ 0.1 Å), but the DNA backbone atoms remain enveloped by a negative surface (-3.5 eV). The resultant dipole momenta (green vectors) are 19.2 D and 60.4 D for the DNA-DNA and PNA-DNA duplexes, respectively. In the latter case it is oriented diagonally from PNA to DNA strand and can be a driving force for e¯aq interaction with accessible PNA backbone and groove atoms.

We have studied hybridization of DNA oligonucleotides with PNA, where PNA bear (or not) N- or C-terminal amino groups (-NH2, lysine, or methylmorpholinium) (Fig. 3, Gantchev *et al.*, 2009). After -irradiation (typically 750 Gy) of PNA-DNA heteroduplexes, those with PNA containing free amino group ends formed ICLs (Fig. 5). The multiple bands in each lane represent different crosslinked products and match the number of available amino groups in each heteroduplex. The ICL-formation efficiency is high, G = (5-8) x10-8 mol.J-1. This G-value even exceeds the ICL yields observed after irradiation BrdUsubstituted wobble DNA under identical conditions. Using selective scavengers it was shown that ICL formation in PNA-DNA heteroduplexes strongly depends on the availability of solvated electrons (e¯aq), but proceeds only with a concomitant presence of •OH radicals (Gantchev *et al.*, 2009). Thus, it appears that PNA-DNA ICLs arise in a concerted free radical mechanisms resembling those involved in DNA multiply damaged sites (MDS). By hybridizing 12-mer PNAs with shorter (11-mer), or longer (up to 16-mer) complementary oligo-deoxyribonucleotides thus creating unpaired (single-stranded) regions (deletions and overhangs) at one, or both duplex ends we compared sequence effects on the cross-linking reaction (*e.g.* dT vs. dA termini), the susceptibility of duplex ends to radiation damage, *etc*. The 3'- and 5'- DNA terminal dT nucleotides proved to be of most importance for the efficient ICL formation. Since hydrolysis of N-glycosidic bonds in -

DNA Radiosensitization: The Search for Repair Refractive

constituting a clustered type (MDS) lesion.

6B).

Lesions Including Double Strand Breaks and Interstrand Crosslinks 425

stand breaks and alkali-labile lesions were also induced at the halogenated site and at the flanking nucleotides. The conclusions from this study highlight the importance of the local DNA structure (wobble *vs.* normal duplex) in terms of Watson-Crick pairing restrictions that likely prevent the 5-uracil-yl σ-radical (in contrast to the aryl radical) interaction with the opposite strand bases to produce ICL. The chemical structures of several DNA interstrand crosslinks have been reported only recently. Several groups have focused on the identification of ICL structures synthesized in model systems using light- and/or oxidationsensitive precursors (Hong *et al.*, 2006; Weng *et al.*, 2007; Peng *et al.*, 2008; Kim & Hong, 2008; Op de Beeck & Madder, 2011). A few structures have been definitively identified in natural conditions and, to our knowledge, only one in cellular DNA (Regulus *et al.*, 2007) (Fig. 6C). However, the authors in Regulus *et al.*, (2007) failed to present evidence that this crosslink is exclusively an inter-strand crosslink in cells. Mechanistically, the sole ICL-structure that is exclusively generated with the participation of a primary radiation-induced 5-(2' deoxyuridinyl) methyl free radical, a product of •OH-induced hydrogen-atom abstraction from thymine, is the T[5m-6n]A crosslink (Ding & Greenberg, 2007; Ding *et al.*, 2008) (Fig.

A common pathway for ICL formation in dsDNA is the condensation reaction between aldehydes (*e.g.* in abasic DNA sites) and exocyclic amines of opposite bases. Under radiation abasic (AP-apurinic/apyrimidinic) sites are generated either *via* direct H-atom abstraction by •OH radicals from 2'-deoxyribose or after oxidative base damage followed by N-glycosidic bond-cleavage. Oxidation of each of the five positions in 2'-deoxyribose in DNA is possible, but under -radiation the best known reactions involve H-atom abstraction at C1'-, C2'- and C4'-positions. The 4'-keto abasic site formed after C4'-oxidation (C4'-AP) is generally known as the "native" abasic site (Chen & Stubbe 2004). Subsequent interactions of sugar radicals with oxygen and/or elimination reactions give a variety of closedcycle/open-chain aldehydic products, accompanied (or not) by DNA strand-cleavage (Dedon, 2008). One of the first reported, and structurally identified DNA-ICL generated by -radiation and/or selective 4'-position 2'-deoxyribose oxidation by bleomycin in model systems and in cells (Fig. 6C; Regulus *et al.*, 2007; Cadet *et al.*, 2010) is produced *via* electrophilic interaction between 2'-deoxypentos-4-ulose abasic site (opened C4'-AP) and N4-dC. Formation of this ICL is accompanied by a DNA strand break. In a series of works, Greenberg and collaborators studied the ICL formation with participation of oxidized native C4'-AP (Sczepanski *et al.*, 2008, 2009a) and 5′-(2-phosphoryl-1,4-dioxobutane, DOB) (Guan & Greenberg, 2009) sites. DOB is produced concomitantly with a single-strand break byDNAdamaging agents capable of abstracting an H-atom from the C5′-position. When oxidized C4'-AP was opposed by dA, a single crosslink formation occurred exclusively with an adjacent dA on the 5′-side. The crosslink formation was attributed to condensation of C4'- AP with the N6-amino group of dA and less favorably with N4-amino group of dC, but not with dG or dT. Interestingly, C4'-AP produced ICLs in which both strands are either intact or ICLs, where the C4-AP containing strand was cleaved (3′ to the AP-site), while DOB-ICLs were always accompanied by an adjacent to the AP-site single-strand break, and thus

In contrast, following -radiation of BrdU-substituted wobble-DNA (scDNA) duplexes multiple crosslinked products were generated which impedes their chemical identification (Cecchini et al., 2005a; Dextraze et al., 2009). However, in DNA-PNA heteroduplexes, because the amino-groups attached to PNA are exogenous and could be omitted/varied,

damaged nucleotides and/or direct 2'-deoxiribose oxidation yields AP (apurinic/apyrimidinic) abasic sites as a common DNA lesion; we also assessed the role of AP-sites in the PNA-DNA ICL formation using synthetic AP-containing oligodeoxyribonucletides. We found that presence of AP-sites at different positions of the DNA strand (3'- or 5'-end, and/or penultimate to the ends) results in ICL formation without radiation, but instead required addition of a strong reductant, *e.g.* NaCNBH3. The electrophoretic gel-mobility of thus formed ICL bands resembled that of -radiation generated ones. Therefore, we concluded that AP-sites on the DNA strand are the likely partners of the free NH2, or α- and ε-amino groups of Lys at the PNA ends in the formation of DNA-PNA crosslinks *via* a Schiff-base reaction, followed by imine reduction.

Fig. 5. Electrophoretic migration of bands representing the covalent PNA-DNA dimers (ICL). Aqueous solutions of 32P-DNA hybridized with various PNA were -irradiated (750 Gy) under N2 – atmosphere and in the presence of 25 mM EDTA. Samples differ only by the type of capping groups on PNA ends (R1 and R2, Fig. 3). Lanes from left to right: (A) NH2-K-PNA; (B) NH2-PNA; (C) Ac-PNA; (D) MeMor-K-PNA; (E) NH2-PNA-K; (F) Ac-PNA-K; and (G) MeMor-PNA-K; (1- 4): different crosslinked products involving the different amino groups. No ICL are formed with Ac-PNA (C) and MeMor-K-PNA (D). Adapted from Gantchev *et al.*, (2009).
