Inducible alkylation of DNA by a quinone methide-peptide nucleic acid conjugate

Abstract

The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate, but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alternatively, alkylation of noncomplementary sequences is only possible when a template strand is present to colocalize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self-adducts formed between the PNA and its attached QM remained active and reversible over more than 8 days in aqueous solution prior to reaction with a chosen target added subsequently.

Figure 1

DNA alkylation by the PNA conjugate pPNA-QMP. (A) Alternate [³²P]-labeling of the target OD1 and pPNA-QMP generates the same product with an intermediate electrophoretic mobility after initiating reaction with fluoride and incubating the mixture for the indicated time under ambient conditions. (B) Alkylation of [³²P]-OD1 with pPNA-QMP was quantified and fit to a first order process. The error represents the range of two or more independent determinations (see Figure S1).

Figure 2

Competition for alkylation by pPNA-QMP between targets containing no mismatches (OD1) and a single mismatch (OD2). Selectivity for [³²P]-OD2 was measured in the presence of 0 – 10 equivalents of OD1 and 1.1 equivalents of pPNA-QMP. Conversely, the selectivity for [³²P]-OD1 was measured in the presence of 0 – 10 equivalents of OD2 and 1.1 equivalents of pPNA-QMP. Reaction mixtures were incubated for 192 h in the presence of MES (130 mM, pH 7.0), NaCl (130 mM) and KF (130 mM) under ambient conditions.

Figure 3

Template-dependent alkylation of target DNA. pPNA-QMP (3.3 μM) and the template OD3 (3.0 μM) were incubated for 192 h under ambient conditions in MES (130 mM pH 7) and NaCl (130 mM) after reaction was initiated by addition of KF (130 mM). OD6 (lane 1) and OD7 (lane 3) were then added to this mixture and incubated for another 192 h prior to electrophoretic analysis. Alternatively, pPNA-QMP, OD3 and either OD6 (lane 2) or OD7 (lane 5) were mixed together under equivalent conditions prior to addition of KF and then incubated for 192 h. pPNA-QMP and either OD6 (lane 3) or OD7 (lane 6) were also mixed together in the absence of the template OD3 under equivalent conditions prior to addition of KF and then incubated for 192 h.

Figure 4

DNA alkylation by the pPNA-QM self adduct. The self-adduct was prepared in situ by addition of KF (130 mM) to a solution pPNA-QMP (3.3 μM) in MES (130 mM pH 7) and NaCl (130 mM) and incubated under ambient conditions for 24 h. The target [³²P]-OD1 (3.0 uM) was then added and incubation was continued for the indicated time before quantifying the alkylated product (see Figure S5). The average of two independent determinations was plotted and fit to a first order process. The error represents the range of the observed yields.

Figure 5

Persistence of the pPNA-QM self adduct. The self adduct was formed as described in Figure 4 and then incubated under ambient conditions for an additional 0 – 168 h prior to addition of [³²P]-OD1. Incubation was then continued for 168 h before quantifying the alkylated product (see Figure S6). The average of two independent determinations was plotted and fit to a first order process. The error represents the range of the observed yields.

Scheme 1

Generation of a quinone methide conjugate, its subsequent reversible formation of a self adduct through intrastrand reaction and ultimate interstrand transfer of the quinone methide for target alkylation.

Scheme 2

Synthesis of pPNA-QMP.

Scheme 3

Template directed alkylation of a non-complementary target.

Scheme 4

Irreversible trapping of a self adduct.