Carbazole-Based Photoreactive Nucleotide for Sequence-Selective Crosslinking
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Solution Overview
Problem
Current photoreactive crosslinking agents, such as psoralen, are limited in their ability to target specific sequences and require short-wavelength light, which can cause DNA or cell damage during photoirradiation, and are unable to crosslink sequences that cannot be targeted by psoralen.
Innovation Solution
A novel photoreactive crosslinking agent with a carbazole structure and a vinyl group is developed, allowing for crosslinking of sequences that cannot be targeted by psoralen and using longer wavelengths, reducing the risk of DNA or cell damage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If psoralen is used as a photo-crosslinking agent, then crosslinking reaction can be carried out, but the target sequence is restricted to 5'-TA-3' sequences and short wavelength light (350 nm) must be used which causes DNA or cell damage
Solution Approach 1:
The patent changes the photophysical parameters of the crosslinking agent by using a different chromophore system (aminocoumarin or anthraquinone derivatives) that absorbs at longer wavelengths (400-500 nm) compared to psoralen (350 nm). This parameter change in absorption wavelength allows the agent to crosslink at sequences not targetable by psoralen while avoiding the harmful effects of short wavelength light on DNA and cells.
Solution Approach 2:
The patent employs small-molecule photoreactive agents with specific photophysical properties that can be easily incorporated into nucleic acids. These agents act as disposable crosslinking tools that are incorporated during synthesis and then activated by light, providing sequence-specific crosslinking without the need for complex enzymatic systems or prolonged exposure to harmful wavelengths.
2Productivity
If psoralen is used for photo-crosslinking, then crosslinking reaction occurs, but the reaction requires short wavelength light source which is not suitable for all sequence types
Solution Approach 1:
The patent modifies the photophysical parameters by selecting chromophores with different absorption spectra. The aminocoumarin and anthraquinone derivatives used in this patent absorb at longer wavelengths (400-500 nm) compared to psoralen, enabling crosslinking reactions at a broader range of sequences including those that psoralen cannot target, thus improving both productivity and adaptability.
Solution Approach 2:
The patent develops photoreactive agents that can universally crosslink at multiple sequence types through their extended wavelength absorption characteristics. The designed compounds with carbazole structures and vinyl groups can crosslink at various sequences (including 5'-GC-3', 5'-CG-3', and other combinations) that are not accessible to psoralen, providing multi-functional crosslinking capability.
3Object-affected harmful factors
If conventional photoreactive crosslinking agents are used, then crosslinking can be achieved, but they cannot crosslink sequences that are not targetable by psoralen
Solution Approach 1:
The patent changes the absorption wavelength parameter from 350 nm (psoralen) to 400-500 nm (aminocoumarin/anthraquinone derivatives), which minimizes DNA and cell damage while enabling crosslinking at a wider variety of sequences. This parameter shift allows the agent to target sequences that were previously inaccessible, achieving both reduced harm and increased versatility.
Solution Approach 2:
The patent employs composite molecular structures combining carbazole chromophores with vinyl groups and nucleic acid backbones. This composite design creates a photoreactive agent that integrates the photophysical properties of carbazole (absorption at 400-500 nm) with the nucleic acid framework, enabling crosslinking at diverse sequences while avoiding the harmful effects of shorter wavelength light.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The new agent enables crosslinking of specific sequences with high sequence-selectivity and can form crosslinks using longer wavelengths, minimizing damage to DNA or cells during photoirradiation, and allows for rapid reaction completion.
Implementation Method 1
a photoreactive crosslinking agent having a photoreactive nucleobase-like structure, which can be crosslinked with a nucleic acid compound
Implementation Method 2
the photocoupling wavelength for psoralen is 350 nm while its photocleavage wavelength is 250 nm
Data Source
AI summary
The present invention provides a photoreactive crosslinking agent that is capable of crosslinking a sequence which cannot be photo-crosslinked by psoralen, and is capable of photo-crosslinking using a light having a longer wavelength, as compared with psoralen. The present invention also provides a compound having a group represented by formula (I) coupled with a group represented by formula (II).