Reversibly locked thionucleobase pairs in DNA to study base flipping enzymes

Abstract

SummaryCovalently interstrand cross-linked DNA is an interesting tool to study DNA binding proteins that locally open up the DNA duplex by flipping single bases out of the DNA helix or melting whole stretches of base pairs to perform their function. The ideal DNA cross-link to study protein–DNA interactions should be specific and easy to synthesize, be stable during protein binding experiments, have a short covalent linker to avoid steric hindrance of protein binding, and should be available as a mimic for both A/T and G/C base pairs to cover all possible binding specificities. Several covalent interstrand cross-links have been described in the literature, but most of them fall short of at least one of the above criteria. We developed an efficient method to site-specifically and reversibly cross-link thionucleoside base pairs in synthetic duplex oligodeoxynucleotides by bisalkylation with 1,2-diiodoethane resulting in an ethylene-bridged base pair. Both linked A/T and G/C base pair analogs can conveniently be prepared which allows studying any base pair-opening enzyme regardless of its sequence specificity. The cross-link is stable in the absence of reducing agents but the linker can be quickly and tracelessly removed by the addition of thiol reagents like dithiothreitol. This property makes the cross-linking reaction fully reversible and allows for a switching of the linked base pair from locked to unlocked during biochemical experiments. Using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as example, we demonstrate that the presented cross-linked DNA with an ethylene-linked A/T base pair analog at the target position is a useful tool to determine the base-flipping equilibrium constant of a base-flipping enzyme which lies mostly on the extrahelical side for M.TaqI.

Alkyne–Alkyne Photo-cross-linking on the Flipping-out Field

“…This cross-linking can fix the flipped out structure. Thus, this product might be a unique tool for the study of the enzymes which induce the base flipping . Furthermore, the cross-linking reactions and cross-linked products have been utilized for various applications such as a terminus-free siRNA, DNA-encoded dynamic library, anti-miRNA, and DNA origami. − Generally, the photo-cross-linking is performed using alkenes, − psoralen, and thionucleotides , for the [2 + 2] cycloaddition reaction, and photocaged or photoactivated compounds. − Our alkyne–alkyne photo-cross-linking would be a new candidate to form cross-linked DNAs.…”

Metal–Organic Framework MIL-101(Cr) as an Efficient Heterogeneous Catalyst for Clean Synthesis of Benzoazoles

“…15 Compounds 8f – h contain an adenine moiety, which it connected to spacer using the sulfur group in 6-position of the adenine ring. 16 In fact, 6-thio adenine (9 H -purine-6-thiol) was used as the starting material for the synthesis of these compounds according to the reaction pathway designed in this study. When 6-thio adenine was reacted with bromo-substituted aldehyde 6 (Scheme 3), it underwent reaction by SH group and connected to the aldehyde backbone via the C–S bond.…”

“…These compounds containing a benzoazole ring and also a theophylline heterocycle, both of which are important in medicinal applications . Compounds 8f – h contain an adenine moiety, which it connected to spacer using the sulfur group in 6-position of the adenine ring . In fact, 6-thio adenine (9 H -purine-6-thiol) was used as the starting material for the synthesis of these compounds according to the reaction pathway designed in this study.…”

Close encounters for the first time: Helicase interactions with DNA damage

“…The ICL substrate may be particularly useful for study of helicases such as UvrD which unwind one base pair at a time [78]. Technical developments have been made in approaches to make chemically defined DNA cross-links [79]. Up to this point, such ICL DNA substrates have not been used to study DNA helicase unwinding mechanisms, but it seems likely this experimental strategy is on the horizon with the rapid development of single-molecule studies.…”