Bifunctional cross-linking approaches for mass spectrometry-based investigation of nucleic acids and protein-nucleic acid assemblies

Abstract

With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers.

Figure 1.

Denaturing PAGE analysis of cross-linking reaction mixtures. A) Reaction of 8-MOP with 17DNA(p:c) and 17RNA(p:c) (see Experimental for details). Irradiated DNA sample displayed a new band (xl-DS) with a relative mobility (Rf) of 0.69 versus the corresponding single-strands (SS) (lane 4), consistent with inter-strand conjugation. In contrast, no new bands were detected for the irradiated RNA sample, consistent with the lack of inter-strand conjugation. B) Reaction of NM with SL3, NC, and NC•SL3 complex. Crosslinked NC•SL3 complexes showed a new band (xl-NC•SL3) Rf = 0.34 versus SL3, which is consistent with the formation of a cross-linking product (lanes 6 and 9) (see Experimental). All gels were stained by using Stains-All, which can effectively stain both nucleic acid and protein moieties. It must be noted, however, that an isoelectric point of 9.9 units prevented free NC protein, which was neither bound nor conjugated to the cognate nucleic acid substrate, from migrating into the gel, thus eluding detection. No multimeric nucleic acids artifacts were ever detected under the denaturing conditions employed in these experiments.

Figure 2.

ESI-MS spectra obtained from reaction mixtures of A) 8-MOP and 17DNA(p:c); B) 8-MOP and 17RNA(p:c); and C) NM and NC•SL3 complex (see Experimental). The irradiated DNA sample displayed adducts with up to two 8-MOP equivalents, whereas its RNA counterpart did not react under identical conditions. The NM-treated sample displayed crosslinked products, together with type 0 adducts with a hydrolyzed 2-chloroethyl arm. The masses of the species displayed in this figure are provided in Table S1 of Supporting Information.

Figure 3.

ESI-MS analysis of 8-MOP adducts of 17DNA(p:c) obtained at A) 50V and b) 100V desolvation voltages to induce in-source dissociation (see Experimental). The formation of inter-strand crosslinks was revealed by the presence of intact type 2 products, which persisted at even higher desolvation voltages capable of inducing backbone fragmentation. The masses of the species displayed in this figure are provided in Table S1 (see Supporting Information).

Figure 4.

ESI-MS analysis of a digestion mixture of NC•SL3 complex treated with NM. The product mixture was digested with both RNase A and trypsin (see Experimental). The concerted digestion procedure produced both peptide (purple) and oligonucleotide (black) products, as well as their conjugates bridged by the NM cross-linker. The masses of the species displayed in this figure are provided in Table 2S of Supporting Information.

Figure 5.

MS/MS analysis of the precursor ion detected at m/z 914.23 in Figure 4. Panel A) was obtained in negative ion mode with 43 V activation voltage. Panel B) was obtained in positive ion mode with 51 V activation (see Experimental). Panel C) displays the fragmentation pattern produced by the putative structure assigned to this precursor ion.

Scheme 1.

Typical products generated by treating a putative duplex substrate with a bifunctional reagent.

Scheme 2.

Model substrates employed in the study. A) 17DNA(p:c) DNA duplex; B) 17RNA(p:c) RNA duplex; C) HIV-1 stemloop domain 3 (SL3 RNA); D) HIV-1 nucleocapsid protein (NC). Duplex samples were obtained by annealing corresponding complementary strands, whereas protein-nucleic acid complexes were obtained by simply mixing the desired components and monitoring the outcome by ESI-MS analysis under non-denaturing conditions (see Experimental).