Three‐Dimensional Control of DNA Hybridization by Orthogonal Two‐Color Two‐Photon Uncaging

Abstract

We successfully introduced two‐photon‐sensitive photolabile groups ([7‐(diethylamino)coumarin‐4‐yl]methyl and p‐dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three‐dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double‐strand displacement in a hydrogel and in neurons. Orthogonal two‐photon uncaging of the two cages is possible, thus enabling complex scenarios of three‐dimensional control of hybridization with light.

Chromatic Selectivity of Coumarin‐Caged Oligonucleotides

“…“Orthogonal uncaging” was achieved in combination with a second cage excited in a one‐photon process at 420 nm. Ellis‐Davies and our own group combined two two‐photon photocages, releasing neurotransmitters or oligonucleotides between 840 and 980 nm [16,17] . However, two‐photon excitation requires very expensive laser setups and is restricted to femtoliter volumes – which can be both an advantage or a disadvantage.…”

Wavelength‐Dependent Stiffening of Hydrogel Matrices via Redshifted [2+2] Photocycloadditions

“…While their study demonstrates the potential of photocycloadditions in engineering biomaterial properties for in vitro cell study, certain cell types—such as hepatic stellate cells24—are not resistant toward the applied UV light. However, in the field of soft matter materials, the majority of studies on photocycloadditions are carried out on stilbene,25,26 coumarin,27–29 thymine,30,31 and cinnamic acid32 derivatives, which require activation by short wavelength UV light (note that we are referring exclusively to the bond forming photocycloaddition of coumarin, in the context of bond cleavage short wavelength blue light has been successfully used for coumarin‐based photocages) 33. To overcome limitations of applicability in biology‐related systems from UV light exposure,34 and broaden the synthetic toolbox of orthogonally addressable photoligation reactions,21,35,36 the development of redshifted photocycloadditions is key 37…”

Synthesis of Deoxypseudouridine 5′-Triphosphate Bearing the Photoremovable Protecting Group at the N1 Position Capable of Enzymatic Incorporation to DNA

“…Oligodeoxynucleotides (ODNs) incorporating the photocleavable protecting group (PPG), namely, photocaged ODN, have been widely studied to append photoswitchable properties to ODNs. There are many reports on photocaged DNA − and photoregulation of DNA functions, such as transcription, − DNA cleavage by restriction enzyme, , and recognition by DNA binding proteins such as MutS . In many photocaged DNAs, PPGs were introduced to the base moiety at the atoms involved in Watson–Crick base pairing, such as N 3 or O 4 of thymine, ,,− N 4 of cytosine, , N 6 of adenine, and N 1 or N 2 or O 6 of guanine. , However, such photocaged DNAs could only be chemically synthesized using the phosphoramidite method and not enzymatically because deoxynucleoside triphosphates (dNTPs) bearing the above-mentioned nucleobase cannot be substrates for DNA polymerase.…”

“…There are many reports on photocaged DNA − and photoregulation of DNA functions, such as transcription, − DNA cleavage by restriction enzyme, , and recognition by DNA binding proteins such as MutS . In many photocaged DNAs, PPGs were introduced to the base moiety at the atoms involved in Watson–Crick base pairing, such as N 3 or O 4 of thymine, ,,− N 4 of cytosine, , N 6 of adenine, and N 1 or N 2 or O 6 of guanine. , However, such photocaged DNAs could only be chemically synthesized using the phosphoramidite method and not enzymatically because deoxynucleoside triphosphates (dNTPs) bearing the above-mentioned nucleobase cannot be substrates for DNA polymerase. Considering the potential utility of enzymatic incorporation of modified dNTPs for the functionalization of ODNs, the development of photocaged dNTPs applicable to enzymatic DNA synthesis is necessary.…”