Reaction biotin-BMCC

Figure 11.8 Biotin-BMCC provides sulfhydryl reactivity through its terminal maleimide group. The reaction creates a stable thioether linkage.

Biotin-BMCC is insoluble in water and must be dissolved in an organic solvent prior to addition to an aqueous reaction mixture. Preparing a concentrated stock solution in DMF or DMSO allows transfer of a small aliquot to a buffer reaction. The upper limit of biotin-BMCC solubility in DMSO is approximately 33 mM or 17 mg/ml. In DMF, it is only soluble to a level of about 7 mM (4 mg/ml). Upon addition of an organic solution of the reagent to an aqueous environment (do not exceed 10 percent organic solvent in the aqueous medium to prevent protein precipitation), biotin-BMCC may form a micro-emulsion. This is normal and during the course of the reaction, the remainder of the compound will be driven into solution as it couples or hydrolyzes. 

Reaction of Biotin-BMCC with Sulfhydryl-Modified DNA... 

Additional reaction components can be used that may enhance or be essential for catalytic activity in a particular system. These components may serve functions analogous to the cofactors used by protein catalysts (e. g., ATP, NADH, or metal ions). These components can be supplied free in solution or incorporated into the RNA library as previously described. As an example, Figure 8.5 oudines the RNA-catalyzed carbon-carbon bond-forming [4 + 2] cycloadclition reaction between a tethered diene substrate, (2E.4E)-hexa-2,4-clien-l-0-PEG (1), and 1-biotinamido-4-[A -(maleimidomethyl)cyclohexanecarboxamido] butane (BMCC-biotin, 2), a clienophile that is free in solution. The RNA catalyzes the formation of (3), which contains biotin for partitioning purposes. 

Fig. 8.5. Specific example of an RNA catalyzed reaction [4 + 2] Diels-Alder cycloaddition between an RNA-tethered diene and a biotin-linked maleimide dienophile (BMCC-biotin). Cycloaddition creates an affinity-tagged (biotin) RNA product that can be captured and purified.

Add BMCC-biotin to a final concentration of 100 pM (its addition accounts for the total amount of 2% DMSO in the reaction mix)... 

Dilute the reaction mixture by adding 4 volumes of H20. Remove unreacted BMCC-biotin through a 30kDaMWCO filter (Millipore) by centrifugation for 8 min at 11 000 x g. Repeat this step to ensure that all unreacted BMCC has been removed before proceeding to the partitioning step (Section 8.3.6). 

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