- A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR-Cas9
-
Several genome engineering applications of CRISPR-Cas9, an RNA-guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small-molecule systems lack wide dynamic range, have background activity in the absence of the small-molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high-intensity light. We previously reported a small-molecule-controlled Cas9 system with some dosage and temporal control. By photocaging this Cas9 activator to render it biologically inert and photoactivatable, and employing next-generation protein engineering approaches, we have built a system with a wide dynamic range, low background, and fast photoactivation using a low-intensity light while rendering the small-molecule activator biologically inert. We anticipate these precision controls will propel the development of practical applications of Cas9.
- Manna, Debasish,Maji, Basudeb,Gangopadhyay, Soumyashree A.,Cox, Kurt J.,Zhou, Qingxuan,Law, Benjamin K.,Mazitschek, Ralph,Choudhary, Amit
-
-
Read Online
- Synthesis of photolabile 2-(2-nitrophenyl)propyloxycarbonyl protected amino acids
-
The 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) group has been introduced as a photolabile amino protecting group for amino acids to be used as building blocks in photolithographic solid-phase peptide synthesis. NPPOC-protected amino acids were found to be cleaved in the presence of UV light about twice as fast as the corresponding o-nitroveratryloxycarbonyl (NVOC)-protected amino acids.
- Bhushan, Kumar R.,DeLisi, Charles,Laursen, Richard A.
-
-
Read Online
- METHODS OF SYNTHESIZING A POLYNUCLEOTIDE ARRAY USING PHOTOACTIVATED AGENTS
-
Described herein are methods for the synthesis of DNA polynucleotides and polynucleotides, as well as methods for their deprotection and methods for the use of said compounds and compositions comprising said compounds. In particular, such compounds and compositions comprising them are used in methods for light-directed synthesis of DNA microarrays.
- -
-
-
- Caged glutathione - Triggering protein interaction by light
-
Light, GSH, action! Glutathione (GSH) fulfills a universal role as redox factor, scavenger of reactive oxygen species, and as an essential substrate in the conjugation, detoxification, and reduction reactions catalyzed by glutathione S-transferase (GST). A photoactivatable glutathione allows the GSH-GST network to be triggered by light. GST fusion proteins can be assembled in situ at variable density and structures by laser-scanning activation. Copyright
- Gatterdam, Volker,Stoess, Tatjana,Menge, Clara,Heckel, Alexander,Tampe, Robert
-
p. 3960 - 3963
(2012/06/15)
-
- Photolabile peptide nucleic acid monomers: Synthesis and photodeprotection
-
The photolabile 2-(2-nitrophenyl)propyloxy carbonyl (NPPOC) group has been introduced as an amino protecting group for peptide nucleic acids (PNA) to be used as building blocks in photolithographic solid-phase PNA synthesis. NPPOC-protected PNA derivative
- Bhushan, Kumar R.
-
p. 2130 - 2132
(2008/02/05)
-
- Photogenerated reagents
-
This invention describes reagent precursors and methods for chemical and biochemical reactions. These reagent precursors that can be activated in solution upon irradiation to generate reagents required for the subsequent chemical reactions. Specifically, photogenerated reagents (PGR) are useful for controlling parallel combinatorial synthesis and various chemical and biochemical reactions.
- -
-
Page/Page column 17
(2008/06/13)
-
- New Types of Very Efficient Photolabile Protecting Groups Based upon the [2-(2-Nitrophenyl)propoxy]carbonyl (NPPOC) Moiety
-
Based upon the photolabile [2-(2-nitrophenyl)propoxy]carbonyl group (NPPOC), a large number of modified 2-(2-nitrophenyl)propanol derivatives substituted at the phenyl ring (see 23-34 and 57-76) as well as at the side-chain (see 85-92 and 95-98) were synthesized to improve the photoreactivity of this new type of photolabile entity. The phenyl moiety was also exchanged by the naphthalenyl group (see 102, 103, 105, 108, 110, 113, and 114), the thienyl substituent (see 115, 117, 118, and 120), and the benzothienyl substituent (see 121). The 2-(2-nitroaryl- and heteroaryl) propanols were converted with diphosgene into the corresponding carbonochloridates, which reacted subsequently with thymidine to the thymidine 5′-(protected carbonates) 123-178 as the main reaction products. In several cases, the corresponding 3′-carbonates and 3′,5′ -dicarbonates 179-212 were also isolated and characterized. Photolysis studies under standardized conditions (see Table) indicated that the rate of photocleavage varies in a broad range depending on the substituents. So far, the thymidine 5′-[2-(5-halo-2-nitrophenyl)propyl carbonates] 127-129, 5′-[2-(nitro[1,1′-biphenyl]3-yl)propyl carbonates] 136-139, 5′-{2-[2-nitro-5-(thianthren-1-yl)phenyl]propyl carbonate} (140), 5′-[2-(5-naphthalenyl-2-nitrophenyl)propyl carbonates] 141 and 142, and 5′-[2-(2-nitro-5-thienylphenyl)propyl carbonates] 143 and 144 showed the best properties regarding fast and uniform deprotection. Since the nucleobases of 213-215 do not influence the photocleavage features, in general, the new type of photolabile building blocks allows in form of their 3′ -phosphoramidites the photolithographic formation of high-quality biochips.
- Buehler, Sigrid,Lagoja, Irene,Giegrich, Heiner,Stengele, Klaus-Peter,Pfleiderer, Wolfgang
-
p. 620 - 659
(2007/10/03)
-
- Nucleoside derivatives with photo-unstable protective groups
-
The present invention relates to nucleoside derivatives having photolabile protective groups of general formula (I) in which R1=H, NO2, CN, OCH3, halogen, an alkyl, alkoxy or alkoxyalkyl residue having 1 to 4 C atoms or an
- -
-
-
- 3'-nitrophenylpropyloxycarbonyl (NPPOC) protecting groups for high-fidelity automated 5' --> 3' photochemical DNA synthesis.
-
[structure: see text]. The most powerful DNA microarrays would be prepared by photolithography with free 3'-ends that could be processed enzymatically. A photoremovable group that could be removed in quantitative yield would ensure high purity of the synt
- Pirrung,Wang,Montague-Smith
-
p. 1105 - 1108
(2007/10/03)
-
- New photolabile protecting groups of the 2-(2- nitrophenyl)ethoxycarbonyl-and the 2-(2-nitrophenyl)ethylsulfonyl-type for the oligonucleotide synthesis
-
New photolabile blocking groups have been synthesized and introduced into the 5'-OH position of thymidine. The 5'-O-protected thymidines were irradiated at 365 nm under identical conditions and the half-lives and thymidine yields were determined to investigate the influence of different substituents in the two corresponding series.
- Buehler,Giegrich,Pfleiderer
-
p. 1281 - 1283
(2007/10/03)
-
- New photolabile protecting groups in nucleoside and nucleotide chemistry - Synthesis, cleavage mechanisms and applications
-
New photolabile protecting groups have been found in the 2-(2- nitrophenyl)ethoxycarbonyl and the 2-(2-nitrophenyl)ethylsulfonyl group, respectively. The influence of substituents at the phenyl ring as well as the side-chain has been investigated regarding the photolysis rates on irradiation at 365 mn. β-Branching in the side-chain leads to highly increased rates of photodeprotection. A new type of photocleavage mechanism consisting of a photoinduced β-elimination process is proposed.
- Giegrich,Eisele-Buehler,Hermann,Kvasyuk,Charubala,Pfleiderer
-
p. 1987 - 1996
(2007/10/03)
-
- Nucleoside derivatives with photolabile protective groups
-
The invention relates to nucleoside derivatives having photolabile protective groups of the general formula (I) STR1 in which R1 =H, NO2, CN, OCH3, halogen or alkyl or alkoxyalkyl having 1 to 4 C atoms R2 =H, OCH3 R3 =H, F, Cl, Br, NO2 R4 =H, halogen, OCH3, or an alkyl radical having 1 to 4 C atoms R5 =H or a usual functional group for preparing oligonucleotides R6 =H, OH, halogen or XR8, where X=O or S and R8 represents a protective group usual in nucleotide chemistry, B=adenine, cytosine, guanine, thymine, uracil, 2,6-diaminopurin-9-yl, hypoxanthin-9-yl, 5-methylcytosin-1-yl, 5-amino-4-imidazolcarboxamid-1-yl, or 5-amino-4-imidazolcarboxamid-3-yl, where in the case of B=adenine, cytosine or guanine, the primary amino function optionally exhibits a permanent protective group. These derivatives may be used for the light-controlled synthesis of oligonucleotides on a DNA chip.
- -
-
-
- Photolabile protecting groups for nucleosides: Synthesis and photodeprotection rates
-
o-Nitrobenzyloxycarbonyl and a number of related groups have been tested for the photolabile protection of nucleoside 5'-hydroxyls. The rates of photodeprotection were found to vary by approximately 17-fold in a series of 5'-O-protected thymidine derivati
- Hasan, Ahmad,Stengele, Klaus-Peter,Giegrich, Heiner,Cornwell, Paul,Isham, Kenneth R.,Sachleben, Richard A.,Pfleiderer, Wolfgang,Foote, Robert S.
-
p. 4247 - 4264
(2007/10/03)
-