研究目的
To construct zipper-like chromophore-arrays in the major groove of duplex DNA using a post-synthetic strategy and characterize their properties.
研究成果
The post-synthetic CuAAC strategy successfully constructed chromophore-arrays in the major groove of DNA duplexes. TPE-arrays formed zipper-like structures with slight duplex destabilization and exhibited aggregation-induced emission, while Py-arrays did not form zipper-like structures, showed no stability loss, and had weak excimer emission due to free rotation. The formation of zipper-like arrays depends on chromophore size and structure.
研究不足
The yields of chromophore-labeled oligonucleotides were low (7-13%), which could limit scalability. The study focused on only two chromophores (TPE and Py), and the arrays were constructed in a specific DNA sequence context, potentially limiting generalizability. The MD simulations are computational models and may not fully capture all experimental nuances.
1：Experimental Design and Method Selection:
The study employed a post-synthetic labeling strategy using copper-catalyzed alkyne-azide cycloaddition (CuAAC) on solid-phase supports to incorporate chromophores into oligonucleotides. This method was chosen for its efficiency, regioselectivity, and mild conditions.
2：Sample Selection and Data Sources:
Oligonucleotides with self-complementary sequences (D1 and D2) containing 5-ethynyl uridine residues were synthesized on controlled-pore glass (CPG) supports using phosphoramidite chemistry. Chromophores (TPEN3 and PyN3) were prepared according to literature methods.
3：List of Experimental Equipment and Materials:
CPG supports, phosphoramidites, DMSO, CuSO4, L-ascorbic acid sodium salt, dichloromethane, acetonitrile, water, aqueous ammonia, acetic acid, HPLC system, MALDI-TOF/MS, spectrophotometer for absorption and fluorescence measurements, CD spectrometer.
4：Experimental Procedures and Operational Workflow:
Oligonucleotides were synthesized and dried. Labeling involved adding chromophore-azide in DMSO, CuSO4, and ascorbic acid to the CPG-bound oligonucleotide, stirring overnight, filtering, washing, cleaving with ammonia, purifying via HPLC, deprotecting DMT with acetic acid, and final purification. Characterization included melting temperature analysis, absorption and CD spectroscopy, fluorescence spectroscopy, and molecular dynamics simulations.
5：Data Analysis Methods:
Melting temperatures (Tm) were determined from thermal denaturation profiles. Absorption and CD spectra were analyzed for structural insights. Fluorescence quantum yields were calculated. MD simulations provided averaged structures.
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