Synthesis and incorporation of a furan-modified adenosine building block for DNA interstrand crosslinking

Abstract

2 0 -O-(3-(Furan-2-yl)propyl)adenosine was synthesized and evaluated for interstrand crosslink (ICL) formation in DNA duplexes. In situ oxidation of the furan moiety with NIS showed rapid crosslink formation to dA and dC, while dT and dG were inactive.Oligonucleotides that form interstrand crosslinks (ICLs) have found widespread applications in chemical biology research areas. Therefore, chemical and enzymatic methods have been developed to incorporate crosslinks into helical regions of DNA and RNA. 1 Examples include the synthesis of a duplex incorporating a preformed crosslinked dinucleotide 2,3 or postsynthetic modification of duplexes by bifunctional crosslinking reagents. 4,5 However, in some cases, the site-specific introduction of a crosslink is problematic due to formation of a mixture of monoadducts, intrastrand and interstrand crosslinks. Introduction of reactive moieties at a specific position within the duplex can circumvent the selectivity issue as is shown for photocrosslinking with 4-thiouridine, 6 5-bromouridine, 7 5-methyleneaminouridine 8 or 8-azidoadenosine. 9 Furthermore, a thio-modified oligonucleotide (ON) can form disulfide bonds post-synthetically. 10,11 However such ICLs are highly dependent on the proper positioning of thiol groups and, more importantly, require modification of both strands. More recent studies on site-specific ICL formation involve modified phenylselenyl derivatives of thymidine or 5-methyl-2 0 -deoxycytidine, 12 1,4-dioxobutane abasic lesion, 13 alkyl-connected 2-amino-6-vinylpurine 14 and 4-amino-6-oxo-2-vinylpyrimidine. 15 We have earlier developed a complementary methodology incorporating furan-modified 2 0 -amido-uridine and an acyclic building block to give a site-specific ICL upon oxidation with Nbromosuccinimide (NBS). 16,17 First crosslinking results obtained for the furan-modified 2 0 -amido-uridine with complementary adenine (A) were further extended into a more detailed selectivity study against all canonicals using a more synthetically accessible acyclic furanmodified building block. In the latter case, strong selectivity for crosslinking to its opposite canonical A or cytidine (C) was observed, without formation of crosslinks to neighboring or distant bases. 17 In this context, furan-modification on other canonicals than uridine and the potential impact on the crosslinking process have not been evaluated yet. From this point of view, we became interested in evaluating the scope of furan-modified purines for ICL. We here wish to report on the synthesis and incorporation of 2 0 -O-(3-(furan-2-yl)propyl)adenosine as a building block for crosslinking of DNA duplexes.From a synthetic point of view, 2 0 -modification was an obvious choice as nucleobase modification will in most cases be unfavourable for hybridization. Taking into account that 2,2 0 -anhydrointermediates are only feasible for pyrimidines, a 2 0 -amido modification of adenosine is difficult to achieve. Therefore, we decided to take advantage of our previously developed 2 0 -O-alkylation strategy ...

Pyrrolidinyl Peptide Nucleic Acid Probes Capable of Crosslinking with DNA: Effects of Terminal and Internal Modifications on Crosslink Efficiency

“…On the other hand, the internally modified acpcPNA could only form the ICL when an unpaired nucleobase was available to react with the activated furan moiety, as in the case of the DNA target carrying a C‐insertion close to the furan modification site, despite the low duplex stability. The results are in good agreement with other studies on furan‐modified DNA probes, whereby the crosslinking efficiency was low when the target nucleobase was involved in base pairing [19,20] . However, the effect is more exaggerated in the case of PNA‐DNA duplexes since acpcPNA binds to DNA much more strongly than normal DNA.…”

“…According to a previous study by Madder, [20] cytosine was found to be the most susceptible nucleobase for the ICL by furan‐modified DNA. To investigate the effect of switching of the probe from DNA to acpcPNA, we performed a preliminary crosslinking experiment between the terminally furan‐modified PNA(T) and its complementary DNA (DNA−com).…”

“…[19] The crosslinking reaction occurred specifically with the A and C nucleobases of the DNA targets in a 13-57 % yield. [20] Similar crosslinking reactions were also applicable to RNA targets, and a specific crosslinking product was observed with a complementary C base in a 42 % yield. [21] Furthermore, crosslinkable ODN probes were applied to target double-stranded DNA by triplex formation.…”

“…In 2012, Op de Beeck and Madder studied a light‐induced ICL of DNA with furan‐modified oligodeoxynucleotides (ODNs by using methylene blue as a photosensitizer) [19] . The crosslinking reaction occurred specifically with the A and C nucleobases of the DNA targets in a 13–57 % yield [20] . Similar crosslinking reactions were also applicable to RNA targets, and a specific crosslinking product was observed with a complementary C base in a 42 % yield [21] .…”

DNA Interstrand Cross‐Link Formation Using Furan as a Masked Reactive Aldehyde

“…In view of the general stability of the furan moiety before oxidation, handling during synthesis and binding events is straightforward and no special protection strategies or precautions need to be considered. This has led to the synthesis and evaluation of several alternative furan‐modified ODNs, allowing the fine‐tuning of yield and selectivity (Halila et al, ; Stevens and Madder, ; Jawalekar et al, ; Op de Beeck and Madder, , ; Stevens et al, ).…”