A colleague has performed an in vitro selection experiment using a 40 nucleotide

Question

A colleague has performed an in vitro selection experiment using a 40 nucleotide random sequence DNA

to find a molecule that binds biotin with high affinity. They found a 30 nucleotide DNA that binds biotin with a K_d of 0.5 M at 25°C. They also measured the “melting temperature” (T_m) of the DNA by optical denaturation. They were initially surprised to find that the T_m was unchanged (~ 72°C) upon addition of biotin to this biotin-binding DNA. Normally small molecules that bind DNA raise the T_m of a DNA. Use the following data to show why biotin would not be expected to affect the T_m of this DNA. H°= + 20 kcal/mol for the reaction DNA•biotinDNA + biotin ... The measured T_m for 1M DNA at pH = 7 was 72°C and the T_m was the same when biotin was added at a 1:1 ratio of DNA:biotin. 1 cal = 4.184 J

Explanation / Answer

In case of Biotin binding of Oligonucleotide, There is incorporation of Biotin within DNA as dT-Biotin, due to which there is no significant changes ion the no. of hydrogen bonds, and hence there is no difference in the Tm.

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