Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Two-step construction of base-modified nucleic acids for electrochemical DNA sensing
Electrochemical DNA sensing strategies, oriented towards development of novel biosensors and bioassays, use various label-free (based on intrinsic DNA electroactivity) as well as label-based detection principles (1). The latter appear better suited for sequence-specific DNA analysis when reliable recognition of a specific nucleotide sequence among others, or when a nucleotide at a specific position (as in single nucloeotide polymorphism typing), is desirable. Labeled nucleic acids have usually been prepared via multistep chemical synthesis of oligonucleotides (ON). We have introduced a novel two-step construction of modified ON, relying in direct aqueous cross-coupling of halogenated deoxynucleoside triphosphates (dNTP) with various functional groups and enzymatic incorporation of the nucleotide conjugates into ON by DNA polymerases (2-6) or by terminal nucleotidyl transferase (TdT) (7). Compared to the fully chemical ON synthesis, this approach is more versatile, allowing – once a set of modified dNTPs is available – an easy and instant construction of labeled ON bearing various tags (or their combinations) at specific positions in the nucloeotide sequence using standard molecular biological tools. Modified dNTPs, applicable as substrates for DNA polymerases or the TdT enzyme, were substituted with electrochemically active labels at 7-position of 7-deazapurines or 5-position of pyrimidines (8). The base-coupled tags include ferrocene (2), 3-nitro- or 3-aminophenyl groups (3) [M(bpy)3]2+ (M = Ru or Os) complexes (4), tetrathiafulvalene (5) as well as alkylsulfanylphenyl moieties possessing protected mercaptogroup (6). The correponding nucleotides were successfully incorporated into ONs, resuling in ON probes with distinct electrochemical properties owing to redox processes of the labels occuring at diverse potentials. Moreover, in some cases the electronic communication between the tags and nucleobases made their redox potentials responding to the conjugate nucleobase and to incorporation into DNA, thus offering another type of information to gain. We demonstrated applications of the modified dNTPs and ONs in sensing DNA hybridization, SNP typing and monitoring DNA-protein interactions. Further applications of the proposed strategy in nanotechnology, ON self-assembly, chemical biology and biosensing is anticipated.
This work was supported by the ASCR (Z4 055 0506, Z5 004 0507 and Z5 004 0702) GA ASCR (IAA400040901), GACR (203/09/0317, P301/11/2076) and MEYS CR (LC06035, LC512).
1 Institute of Biophysics, Academy of Sciences of the Czech Republic, CZ-61265 Brno, Czech Republic