Enzymatic Synthesis of Redox‐Labeled RNA and Dual‐Potential Detection at DNA‐Modified Electrodes

Abstract

Ferrocene and anthraquinone were used to label RNA (red, dark blue), which could then be detected at an electrode surface bearing complementary DNA oligonucleotide probes (light blue). This approach is an electrochemical analog of popular fluorescence methods for gene expression analysis.

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates

“…Most of the previous studies were performed only with 5-modified UTPs [25][26][27][28][29][30][31][32][33][34][35][36][37] and with just a few examples of 5-modified CTPs. [38][39][40][41] Our current work for the first time compares the same substituents directly linked (without any flexible View Article Online tether) to different NTPs. This not only confirms the previous findings [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] that the pyrimidine NTPs bearing even bulkier groups at position 5 are good substrates for T7 RNA polymerase, but also shows that the corresponding 7-substituted 7-deazapurine NTPs (with smaller or mid-sized groups up to benzofuryl) can be reasonably efficiently incorporated into RNA.…”

“…1, Table S1 in the ESI †). The transcription has been performed in analogy to literature procedures [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]54 using [α-32 P]-GTP or [α-32 P]-ATP and the RNA products were analyzed by PAGE and quantified using QuantityOne software (Fig. 2, means from 2-3 independent experiments).…”

“…Only a few scattered examples have been published on the incorporation of modified cytidines. [38][39][40][41] Modified adenine NTPs were never studied systematically and just a few examples of successful incorporation of 7-aryl-42 and 7-ethynyl-7-deazaadenine 43 or -8-aza-7-deazaadenine 44 nucleotides were reported. In addition, fluorescent analogues of NTPs (including GTP) 45 and even formylpyrrole [46][47][48] …”

Direct Modification of mRNA by Ferrocenyl Carbodiimide and Its Application to Electrochemical Detection of mRNA

“…4 Recently, the usefulness of electrochemical gene expression analysis has been demonstrated. 5,6 For example, King and coworkers demonstrated the model experiment of electrochemical gene expression analysis using cRNAs labeled with ferrocene and anthraquinone, which are constructed by indirect methods (mRNA → cDNA → cRNA). 6 However, no one succeeded in the direct electroactive labeling of mRNA.…”

“…5,6 For example, King and coworkers demonstrated the model experiment of electrochemical gene expression analysis using cRNAs labeled with ferrocene and anthraquinone, which are constructed by indirect methods (mRNA → cDNA → cRNA). 6 However, no one succeeded in the direct electroactive labeling of mRNA. Using our reagents, 1 and its derivative, electrochemical gene expression analysis should be achieved by the use of mRNAs differently labeled with reagents having two different redox potentials.…”