Linearization and Labeling of Single-Stranded DNA for Optical Sequence Analysis

• Published in: The journal of physical chemistry letters 2019-02, Vol.10 (3), p.316-321
• Main Authors: Basak, Rajib, Liu, Fan, Qureshi, Sarfraz, Gupta, Neelima, Zhang, Ce, de Vries, Renko, van Kan, Jeroen A, Dheen, S. Thameem, van der Maarel, Johan R. C
• Format: Article
• Language: English
• Subjects: Benzoxazoles - chemistry; DNA - chemistry; DNA - metabolism; DNA Probes - metabolism; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; Microscopy, Atomic Force; Nucleic Acid Denaturation; Physical Chemistry and Soft Matter; Quinolinium Compounds - chemistry; Sequence Analysis, DNA; VLAG
• ISSN: 1948-7185; 1948-7185
• DOI: 10.1021/acs.jpclett.8b03465

Genetic profiling would benefit from linearization of ssDNA through the exposure of the unpaired bases to gene-targeting probes. This is compromised by ssDNA’s high flexibility and tendency to form self-annealed structures. Here, we demonstrate that self-annealing can be avoided through controlled coating with a cationic–neutral diblock polypeptide copolymer. Coating does not preclude site-specific binding of fluorescence labeled oligonucleotides. Bottlebrush-coated ssDNA can be linearized by confinement inside a nanochannel or molecular combing. A stretch of 0.32 nm per nucleotide is achieved inside a channel with a cross-section of 100 nm and a 2-fold excess of polypeptide with respect to DNA charge. With combing, the complexes are stretched to a similar extent. Atomic force microscopy of dried complexes on silica revealed that the contour and persistence lengths are close to those of dsDNA in the B-form. Labeling is based on hybridization and not limited by restriction enzymes. Enzyme-free labeling offers new opportunities for the detection of specific sequences.