Manipulation of nucleic-acids is based on their physical & chemical properties in turn reflected in their biological function; intrinsically DNA is a very stable molecule; it has been recovered from frozen mammoths in high-enough quality to be cloned; this stability is provided by the strong repetitive phosphate-sugar backbone in each strand. Controlled degradation of DNA requires enzymes like nucleases that break the covalent bonds in the structure; others attack the linkages between the base-sugar residues, some of which are non-specific & lead to generalized destruction of the molecule. The discovery of restriction endonucleases that incise DNA-strands at precise positions opened up the possibility of recombinant DNA technology (genetic-engineering’) together with DNA-ligases capable of joining 2 double-stranded (ds) DNA molecules.
RNA which contains the ribose sugar instead of deoxyribose, is much less stable than DNA partially due to its greater susceptibility to nuclease attack, but is also more readily degraded by chemicals esp by alkaline conditions.
Since the 2DNA strands in a double helix are complementary, there’s no information in the second strand that cannot be deduced from the first one; owing to the base-pairing arrangement, the strands can be separated intact in vivo & in vitro under moderate conditions that disrupt hydrogen (H) bonds between the bases, referred to as denaturation & unlike that of many proteins it’s reversible; due to the complementarity of the base-pairs (bps), both strands will readily associate & renature. Since DNA is effortlessly denatured by heating, the denaturation process is frequently denoted as melting even when it’s accomplished by other means, & occurs over a short temperature range, the midpoint of which is defined as the melting temp(Tm) that’s influenced by base-composition; guanine-cytosine (GC) bps have 3H-bonds, so melt less easily than adenine-thymine (AT) bps that have 2H-bonds, hence the Tm of a DNA fragment can be estimated if the sequence is known.
Along with H-bonds, the duplex DNA structure is maintained by hydrophobic-interactions amongst the bases; their hydrophobicity implies that a single-stranded structure, in which the bases are exposed to the aqueous environment, is unstable thus pairing enables them to be removed from contact with surrounding water. In contrast to H-bonding, these interactions are relatively nonspecific; accordingly, nucleic-acid strands will tend to combine even in the absence of specific base-pairing although this reinforces the association.
Single-stranded (Ss) forms of DNA also exist as in some viruses; these tend to fold up on themselves to form localized double-stranded regions, including stem-loop structures & hairpins, with the effect of isolating the bases from the enclosing water. At room- temperature in the absence of denaturing agents, an Ss nucleic-acid normally consists of a complex set of such secondary structure elements confined to a small area that’s esp evident with transfer & ribosomal RNA; this can also occur to a limited extent with dsDNA where short sequences tend to loop out of the regular double-helix & have a role in regulating gene-expression + initiating DNA replication since they make it accessible for enzymes to unwind the molecule & separate the strands.
The strong negative charge on phosphate-groups in the nucleic-acid backbone works in the opposite direction to H-bonds & hydrophobic interactions, causing electrostatic-repulsion that tends to repel the 2strands; in salt presence, this effect is counteracted by the formation of a cloud of counterions encircling the molecule, neutralizing the negative charge, however if the salt concentration is reduced, any weak contact between the strands will be disrupted by the repulsion & so low salt conditions can be used to increase the specificity of hybridization.
Chemically, RNA is very similar to DNA; the fundamental difference is the presence of ribose instead of deoxyribose lacking the hydroxyl group on C2, & has a powerful effect on some properties of the molecule esp on its stability, hence RNA is readily destroyed by exposure to high pH in which DNA is stable. Although the strands will separate but will remain intact + capable of renaturation when the pH is lowered again; an another dissimilarity between RNA & DNA is that the former contains uracil in place of thymine.
Most of the RNA consists of a single polynucleotide strand, though folding of Ss nucleic-acids does occur; nonetheless this distinction amongst RNA & DNA is not an inherent property of the molecules themselves but is a reflection of their natural roles in the cell, & the mode of production. In all cellular organisms (i.e. excluding viruses) DNA is the inherited material responsible for the cell’s genetic-composition, & the replication process that has evolved is based on a double-stranded molecule; the function of RNA does not require a second complementary strand, & indeed its presence would preclude its role in protein synthesis. Conversely some viruses have ds-RNA as their genetic material, & some replicate via Ss-DNA.
DNA can be denatured + renatured, deformed + reformed & still retain unaltered function which is a crucial feature as this large structure will need to be packaged in order to fit within the cell it controls. The stretched-out DNA of a human chromosome would be several centimeters long, thus cells are dependent on DNA-packaging into modified configurations for their very existence. In its relaxed state, ds-DNA usually exists as a right-handed double-helix with one complete turn per 10bps & is known as the B-form; hydrophobic-interactions between consecutive bases on the strand contribute to thus winding of the helix, as they’re brought closer together enabling a more effective exclusion of water from contact with the hydrophobic bases.
Other forms of duplex DNA exist especially the A-form that’s also right-handed but more compact with 11bps per turn, & the Z-form, a left-handed helix with a more irregular appearance (zigzag structure hence its designation). Certain sections of DNA-sequence can initiate a localized switch between the B & Z form; however, natural DNA resembles most closely to B-form for the majority of its length.
There are furthermore higher orders of conformation; the double-helix is in turn coiled on itself; the effect is known as supercoiling. DNA in vivo is constrained, the ends are not free to rotate as in circular structures like bacterial plasmids. Enzymes called topoisomerases (including DNA gyrase) break the strands, effectively rotate the ends & then reseal them; this alters the degree of helix-winding thus affecting DNA supercoiling.
If one of the strands of a naturally-supercoiled plasmid is broken at any point, DNA is then free to rotate & can relax into the typical B-form of the helix, resulting in an open circular arrangement; the plasmid will also be in a relaxed form following insertion of a foreign DNA fragment, or other manipulations. Although each DNA incision is resealed, supercoiling of the molecule will not occur until it’s reinserted into a bacterial cell, hence some of the properties of manipulated plasmid, like transforming ability & its mobility on an agarose gel, are not the same as those of the native one isolated from the cell.
