When you think about the biological macromolecules, you generally have a solid idea as to what each one generally does—which molecules have catalytic, binding, energy storage, structural or information storage roles.

But what happens when you throw decidedly non–natural molecules into the biochemical mix?

Let's find out.

The paper for this week is Nguyen-Hackley DH, Ramm E, Taylor CM, Joung JK, Dervan PB and Pabo CO (2004) Allosteric inhibition of zinc–finger binding in the major groove of DNA by minor–groove binding ligands Biochemistry 43 3880–90.

As a general introduction, the authors are working with a zinc–finger protein known as Zif268 (the PDB accession code for a structure of this protein is 1AAY), a member of the Cys2His2 class of zinc finger proteins.

Having written zinc–finger multiple times already, I would guess that you are most curious about that, right?

Alright, there is the zinc. Yes, these proteins bind to a zinc cation (two Cys and two His residues) to stabilize the structure. They are, in isolation, rather small proteins. More often an isolated zinc–finger is a domain (section) from a larger protein. A larger protein which binds to DNA.

Oh, the finger part. Well, the first zinc–finger was identified in TFIIIA from the African clawed frog and was suggested to have a long, thin structure. Kind of like ... you guessed it, a finger. The actual structure looks a bit more like your index finger if you are making the letter x in American Sign Languauge. So it is still aptly named.

The other two molecules in these studies are DNA (that's the theme) and a small organic hairpin polyamide. These molecules are polymers, similar to proteins (amide bonds join the monomers) but not made of amino acids, which also bind to DNA (DNA is often called the noble ligand as so many things bind to it well).

The catch is that zinc–fingers bind in the major groove of DNA (typical of DNA binding proteins) while the polyamides bind in the minor groove (atypical DNA binding).

As to that major/minor groove thing. DNA has two grooves that spiral around the DNA continuously on the long axis. The major groove is the larger one which provides the greatest access to the nucleotide sequence. The minor groove is the smaller one presenting mostly sugars and phosphates. When you see a small section of DNA (like Figure 1 in the paper), if you're looking at the major groove, the minor groove is in the back of the molecule. If you're looking at the minor groove, the major groove is in the back. Or a search on the topic might provide an image which makes it abundantly clear.

Words. Picture. One thousand.

Again your pre–writing assignment is to, in no more than 250 words, summarize this work. Think about the fundamental question at play, the system used to address that question, the results and what those results mean with regard to fundamental question.

The target for DNA isolation is the strawberry fruit.

"Why the strawberry fruit?"

Glad you asked. The short answer is that the strawberry fruit is octoploid.

"What does that mean?"

Again, glad you asked. Ploidy is the biological term for the number of complete copies of genetic information in a cell. For a mammalian vertebrate (you're one) a typical set of genetic information is described as diploid. Two sets of genetic information, one from dad, one from mom. And yes, you guessed it, germ cells are haploid (one copy) such that the joining of two gametes makes a new diploid organism.

While the situation for human somatic cells is pretty stable at diploid (there are some genetic disorders like Kleinfelter or Down syndrome in which there is an extra copy of one chromosome, humans have twenty–three, but you don't generally find higher than diploid numbers in humans), polyploidy (more than two copies) is common in plants. For us, the cultivated strawberry fruit is octoploid, eight copies of each of the seven chromosomes. And, if you'd like to purify a large amount of something, you either need to start with a large amount of tissue, or go for tissue that has a large amount of what you're after. Thus the strawberry fruit is an ideal starting point for DNA purification.

1. Tare a weigh boat and add strawberry pieces until you have 2 to 3 grams of strawberry fruit, pieces are just fine and it doesn't have to be all from the same berry.
2. Add the strawberry to a semi-micro blender along with 10 mL of lysis buffer.
3. Blend the strawberry and lysis buffer for 60 seconds.
4. Allow the pulverized strawberry to rest in lysis buffer for 2 minutes.
5. Vacuum filter the homogenate over Whatman #1 filter paper
6. Discard the filter paper and fibrous strawberry remnants.
7. Add 30 mL of ice cold ethanol to a 50 mL conical tube.
8. Add the filtrate from the strawberry homogenate to the conical tube of cold ethanol and allow it to sit for five minutes. During this time the DNA will condense (precipitate) and rise to the surface.
9. Transfer the DNA mass (catching it with a spatula works well) to a second 50 mL conical tube and wash with 30 mL of cold ethanol. The purpose of this rinse is to remove red pigment from the fruit. Allow the DNA to sit in this ethanol for five minutes.
10. Remove the DNA to a 15 mL conical tube containing 10 mL cold ethanol.

1. The physical bases for isolating DNA was changing the solvent from water to ethanol (CH3CH2OH), which precipitates the DNA. Considering the structure of DNA shown below, why might you expect it to be less soluble in a less polar solvent? (Remember the mantra of high school chemistry—Like dissolves like.)
   
2. The above question has some subtelties as the monomer unit of DNA (nucleotide monophosphate, lower left above) has both polar and nonpolar portions. Rank the three portions (nitrogenous base, phosphate and sugar) from most polar to least polar. In the structure of double strand DNA, where do you find the more polar parts (inside or outside), where do you find the less polar parts?
3. While DNA is soluble in aqueous solution, double strand DNA has issues in distilled water. Why?

Provide answers to the questions above.