研究目的
To develop and characterize new DNA building blocks with tetrazole groups for fluorogenic photoclick labeling, enabling spatiotemporal control and reduced background fluorescence in molecular imaging applications.
研究成果
The new DNA building blocks enable efficient and fluorogenic photoclick labeling with high rate constants and up to 17-fold fluorescence enhancement, suitable for spatiotemporal control in molecular imaging, with applicability to various dyes and potential for use in living cells.
研究不足
The reaction yield is lower for internal DNA positions due to steric hindrance, and the fluorogenicity varies with the dye used, depending on spectral overlap. The bioorthogonality of the reaction may be borderline due to potential side reactions with nucleophiles.
1：Experimental Design and Method Selection:
The study designed two new 2'-deoxyuridine building blocks with tetrazole groups for DNA synthesis. The photoclick reaction was initiated by LED irradiation, and fluorogenicity was assessed through energy transfer.
2：Sample Selection and Data Sources:
DNA oligonucleotides were synthesized using solid-phase synthesis with modified building blocks. Samples included DNA1A, DNA1B, DNA2A, and DNA2B with tetrazole modifications.
3：List of Experimental Equipment and Materials:
LEDs (300 nm, 365 nm, 385 nm, 405 nm), HPLC for purification, MALDI-TOF mass spectrometry for identification, UV/Vis spectrophotometer for quantification, and various chemicals including maleimide-modified dyes (Cy3, Alexa Fluor 488, Sulfo-Cy5).
4：5). Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: DNA synthesis with extended coupling times, purification by HPLC, irradiation with LEDs in the presence of maleimide dyes, monitoring absorbance and fluorescence changes, and kinetic analysis via HPLC.
5：Data Analysis Methods:
Absorbance and fluorescence spectra were analyzed to track reaction progress, fluorogenicity was quantified as fluorescence enhancement (I/I0), and rate constants were determined using pseudo-first-order kinetics.
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