posted on 2022-12-30, 19:38authored bySally
B. Morton, L. David Finger, Roxanne van der Sluijs, William D. Mulcrone, Michael Hodskinson, Christopher L. Millington, Christina Vanhinsbergh, Ketan J. Patel, Mark J. Dickman, Puck Knipscheer, Jane A. Grasby, David M. Williams
DNA interstrand cross-links (ICLs) prevent DNA replication
and
transcription and can lead to potentially lethal events, such as cancer
or bone marrow failure. ICLs are typically repaired by proteins within
the Fanconi Anemia (FA) pathway, although the details of the pathway
are not fully established. Methods to generate DNA containing ICLs
are key to furthering the understanding of DNA cross-link repair.
A major route to ICL formation in vivo involves reaction
of DNA with acetaldehyde, derived from ethanol metabolism. This reaction
forms a three-carbon bridged ICL involving the amino groups of adjacent
guanines in opposite strands of a duplex resulting in amino and imino
functionalities. A stable reduced form of the ICL has applications
in understanding the recognition and repair of these types of adducts.
Previous routes to creating DNA duplexes containing these adducts
have involved lengthy post-DNA synthesis chemistry followed by reduction
of the imine. Here, an efficient and high-yielding approach to the
reduced ICL using a novel N2-((R)-4-trifluoroacetamidobutan-2-yl)-2′-deoxyguanosine
phosphoramidite is described. Following standard automated DNA synthesis
and deprotection, the ICL is formed overnight in over 90% yield upon
incubation at room temperature with a complementary oligodeoxyribonucleotide
containing 2-fluoro-2′-deoxyinosine. The cross-linked duplex
displayed a melting transition 25 °C higher than control sequences.
Importantly, we show using the Xenopus egg extract system that an
ICL synthesized by this method is repaired by the FA pathway. The
simplicity and efficiency of this methodology for preparing reduced
acetaldehyde ICLs will facilitate access to these DNA architectures
for future studies on cross-link repair.