TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

Abstract

Fluorophore-linked, sequence-specific DNA binding reagents can visualize sequence information on a large DNA molecule. In this paper, we synthesized newly designed TAMRA-linked polypyrrole to visualize adenine and thymine base pairs. A fluorescent image of the stained DNA molecule generates an intensity profile based on A/T frequency, revealing a characteristic sequence composition pattern. Computer-aided comparison of this intensity pattern with the genome sequence allowed us to determine the DNA sequence on a visualized DNA molecule from possible intensity profile pattern candidates for a given genome. Moreover, TAMRA-polypyrrole offers robust advantages for single DNA molecule detection: no fluorophore-mediated photocleavage and no structural deformation, since it exhibits a sequence-specific pattern alone without the use of intercalating dyes such as YOYO-1. Accordingly, we were able to identify genomic DNA fragments from Escherichia coli cells by aligning them to the genomic A/T frequency map based on TAMRA-polypyrrole-generated intensity profiles. Furthermore, we showed band and interband patterns of polytene chromosomal DNA stained with TAMRA-polypyrrole because it prefers to bind AT base pairs.

Nanochannel-Confined TAMRA-Polypyrrole Stained DNA Stretching by Varying the Ionic Strength from Micromolar to Millimolar Concentrations

“…Figure 1 compares surface-tethered DNA molecules dependent on the staining reagents. First of all, TAMRA-polypyrrole stained DNA ( 2 in Figure 1) stretched up to 14.03 ± 0.54 µm ( X / L = 86% ± 3%) at 100 µL/min within 100 µm high flow cell [29]. Previously, we calculated the force of 0.79 pN applied to the λ DNA in the same flow cell (100 µL/min) [27].…”

“…For this experiment, we used T4 DNA instead of λ DNA, because high A/T content in T4 DNA provided more precise visualization of entire molecules. TAMRA-polypyrrole generally stains AT-rich regions specifically via hydrogen bonding on minor groove [29]. Therefore, λ DNA is not an appropriate model molecule because the first half of the λ DNA has CG-rich regions, showing dim DNA backbone (see Figure 2B) [29], which is practically unfavorable in a nanochannel.…”

“…TAMRA-polypyrrole generally stains AT-rich regions specifically via hydrogen bonding on minor groove [29]. Therefore, λ DNA is not an appropriate model molecule because the first half of the λ DNA has CG-rich regions, showing dim DNA backbone (see Figure 2B) [29], which is practically unfavorable in a nanochannel. However, DNA stretch is independent on the length and species.…”

“…Recently, we have developed several novel DNA staining reagents, such as fluorescent protein-DNA binding peptides (FP-DBPs) [21,27,28] and TAMRA-polypyrrole [29]. They consist of a fluorophore and a DNA binding moiety, which makes the fluorophore well separated from the DNA backbone.…”

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer

“…Large DNA molecules have been a versatile platform to investigate a variety of genomic, epigenetic, biochemical, and biophysical studies 1 . For genome analysis, Optical Mapping and nanocoding have been representative single-molecule analytical systems to obtain DNA physical maps 2–6 . They have been applied for many genome projects to aid genome assembly with sequencing 7,8 , as well as to reveal large scale structural variations of the genome 9,10 .…”