Procedure by in situ activation of the microRNA. This is specifically achieved by taking advantage of the fact that miRNAs, unlike other RNAs, are 5′-phosphorylated. The reaction is template-directed (and thus sequence specific) and can be performed together with enzymatic 3′-extension/labeling, either in solution or on a support. The short DNA labeling strand may feature one of a variety of different labels, such as a biotin group or a fluorophore.
pROcEDuRE First, a microarray is spotted with capture sequences for the miRNAs of interest. These capture sequences have attached to their 3′ terminus, a short sequence that is complementary to a labeling oligo. The labeling oligo is short only 8 nucleotides in length and contains a 5′ label such as biotin or a fluorescent dye, and on the 3′ terminus, a 2′,3′-dideoxy-3’amino dT (3′-Amino-dT) nucleoside. When the miRNAs and labeling oligo are hybridized onto the capture sequence, the 5′-phosphate of the miRNA is brought into close proximity to the 3′-amino of the labeling oligo, as shown on the left side of Figure 3. By activating the phosphate in situ with 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC), the 3′-amino group of the labeling oligo reacts to form a stable phosphoramidate linkage, chemically ligating the miRNA and the labeling oligo. Because the Tm of the ligated labeling oligo is significantly higher than the non-ligated labeling oligo, the unreacted labeling oligo can easily be washed away, affording a very low background signal, as seen in the pseudocolor image of the microarray in Figure 3. This procedure makes microarrays an attractive tool for the detection of these medically relevant species.209783-80-2 medchemexpress Figure 3 details the result of a microRNA detection assay performed with a biotinylated labeling strand on a microarray.60857-08-1 Formula The microRNA let7d was labeled by chemical ligation with subsequent staining of the biotins via Cy5-bearing streptavidin. The fluorescence scan shows little background and exquisite3 discrimination against other RNAs, including closely related microRNAs.PMID:20301623 For the differentiation between microRNAs that only differ in a base at the 5′-terminus, false positive signals obtained for enzymatic labeling could be avoided with chemical 5′-labeling. Chemical ligation at the 5′ terminus of miRNA has also been successfully combined with enzymatic 3′-labeling using an on-chip protocol, with reactions performed directly on microarrays. It was found that the dual labeling with two different dyes greatly improves selectivity when detecting closely sequence-related microRNAs and can be used to massively reduce background via subtraction of unmatched signals.
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3′-Amino-dT CPG 4, (1) in Figure 4, may also be used to prepare aminoterminal oligonucleotides for other applications, such as chemical primer extension.5 OlIGO SyNThESIS AND DEpROTEcTION To obtain an oligo modified with a 3′-amino-dT residue, the Richert group developed the 3′-amino-dT CPG, (1) in Figure 4.4 After standard synthesis and labeling of the 5′ terminus with a fluorophore or biotin, the 3′-amino-dT labeled oligo is isolated on deprotection with ammonium hydroxide. Following standard purification protocols using Glen-Pak cartridges or RP HPLC, the labeling strand can be used without special steps or precautions. Notes: 1. This support is not compatible with modifiers requiring mild or UltraMild deprotection and it is not compatible with UltraFast deprotection with AMA. ORDERING INFORMATION
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