Novel Near-Infrared Cyanine Dyes for DNA Sequencing

High-performance fluorescent dyes, serving as essential components for DNA base labeling, are recognized as critical elements for enhancing the accuracy and efficiency of the sequencing processes. However, current sequencing dyes are constrained by limitations, including insufficient fluorescence brightness and compromised photostability, which adversely affect the sequencing accuracy and efficiency. Herein, a series of novel near-infrared cyanine dyes with N-position and 3-position substitutions of the indole ring of the Cy5 scaffold and corresponding reversible terminators are designed and tested by a systematic evaluation of their spectral properties and DNA sequencing capabilities. The novel dyes exhibited enhanced quantum yields of fluorescence (as high as 0.29 vs 0.23 for the control) while maintaining comparable maximum excitation/emission wavelengths relative to commercial Cy5 counterparts. DNA sequencing validation of the developed terminators demonstrated an excellent performance. Surprisingly, trimethylamino alkyl-substituted variants (dGTP-L-WHD Cy5-N1 and dGTP-L-WHD Cy5-N2) demonstrate significantly enhanced sequencing signal intensities compared to controls, whereas sulfonic alkyl groups (traditionally used) were found to exert inhibitory effects. These systematic investigations establish a structure-performance framework that provides theoretical guidance for the rational design of advanced fluorescent labeling materials and innovative DNA sequencing methodologies.