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

The answer might be the interaction between the biotin and DNA is very unstable due to which the melting temperature is unchanged. The Kd at 25oC is 0.5uM, so at 72oC biotin is easily displaced off the DNA sequence due it which there is no change in the Tm before and after binding of biotin.

Related Questions

Navigate

• Browse (All)
• Browse A
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.