DNA and RNA Structure — ASCP MB — Technologist in Molecular Biology

Overview

Every molecular test in the laboratory — extraction, amplification, electrophoresis, sequencing — acts on the physical and chemical properties of two molecules: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Before any technique makes sense, you need to know what these molecules are made of and why they behave the way they do. This lesson builds that foundation from the single repeating unit up to the full strand, and connects each structural fact to a behavior you will rely on at the bench.

DNA and RNA are nucleic acids: long, unbranched polymers built from repeating subunits called nucleotides. The order of those subunits encodes genetic information ¹.

The nucleotide

A nucleotide has three parts: a five-carbon (pentose) sugar, one or more phosphate groups, and a nitrogen-containing base ². A unit with the base and sugar but no phosphate is a nucleoside; add at least one phosphate and it becomes a nucleotide.

        Base
         |
   Phosphate -- Sugar          (sugar + base        = nucleoside)
                               (sugar + base + Pi   = nucleotide)

The pentose sugar: ribose vs. deoxyribose

The sugar is what gives each nucleic acid its name. The five carbons of the sugar are numbered 1’ through 5’ (read “one prime” through “five prime”); the prime marks distinguish them from the atoms of the base, which use unprimed numbers.

In RNA the sugar is ribose, which carries a hydroxyl (-OH) group on the 2’ carbon.
In DNA the sugar is 2’-deoxyribose: the 2’ position has only a hydrogen (-H) instead of -OH. The prefix deoxy- means exactly this missing oxygen ².

That single difference at the 2’ carbon — one oxygen atom — is responsible for much of how the two molecules differ in stability, which is discussed below.

        2'-deoxyribose (DNA)            ribose (RNA)
             base                          base
              |                             |
        5'   1'                       5'   1'
   HO-CH2-O                      HO-CH2-O
         \  /                          \  /
        4'    \                       4'    \
          \    3'   2'                  \    3'   2'
           ---C----C---                  ---C----C---
              |    |                        |    |
             OH    H  <- 2'-H              OH    OH <- 2'-OH

The phosphate

Phosphate groups attach to the 5’ carbon of the sugar. A free nucleotide may carry one, two, or three phosphates (mono-, di-, or triphosphate). The triphosphate forms — for example dATP, dGTP, dCTP, dTTP for DNA — are the energy-rich precursors that polymerases use to build new strands; cleaving the high-energy phosphate bonds drives synthesis forward ¹. The phosphate group also carries a negative charge at the pH of the cell and of laboratory buffers, a property with direct consequences for the lab (see Chemical properties relevant to the lab).

The five bases

The bases fall into two chemical families:

Purines — a fused double-ring structure. The two purines are adenine (A) and guanine (G).
Pyrimidines — a single ring. The pyrimidines are cytosine (C), thymine (T), and uracil (U) ².

DNA and RNA share three bases — A, G, and C. The fourth base is where they differ: DNA uses thymine (T), whereas RNA uses uracil (U) in its place. Thymine is simply uracil with an extra methyl group, so the two are closely related chemically ¹.

   Purines (double ring)        Pyrimidines (single ring)
   ---------------------        -------------------------
   A  adenine    (DNA, RNA)     C  cytosine   (DNA, RNA)
   G  guanine    (DNA, RNA)     T  thymine    (DNA only)
                                U  uracil     (RNA only)

The phosphodiester backbone

Nucleotides are joined into a strand by phosphodiester bonds. Each bond links the 5’-phosphate of one nucleotide to the 3’-hydroxyl of the next. The result is a repeating sugar-phosphate-sugar-phosphate backbone, with the bases projecting off to the side ².

Because the linkage always runs from a 3’ carbon to a 5’ phosphate, the strand has a chemical direction, or polarity. One end terminates in a free 5’-phosphate (the 5’ end) and the other in a free 3’-hydroxyl (the 3’ end). By universal convention, sequences are written and read 5’ to 3’ (5’→3’). This directionality is not a bookkeeping detail: DNA and RNA polymerases add new nucleotides only to the 3’ end, so synthesis always proceeds 5’→3’ ¹.

   5' end
    |
    P
    |
   [sugar]---Base
    |
    P            phosphodiester bond joins
    |            3'-OH of one sugar to
   [sugar]---Base   5'-phosphate of the next
    |
    P
    |
   [sugar]---Base
    |
   3' end (free 3'-OH)

Antiparallel strands

Double-stranded DNA consists of two strands wound around each other. The strands run in opposite directions: where one strand runs 5’→3’, its partner runs 3’→5’. They are said to be antiparallel ¹. This arrangement is required for the bases on opposite strands to pair correctly, a topic taken up in the next lesson on base pairing and the double helix. RNA, by contrast, is usually single-stranded, though it commonly folds back on itself to form local double-stranded regions.

   5'--A--T--G--C--3'
       |  |  |  |        (the two strands are antiparallel)
   3'--T--A--C--G--5'

Chemical properties relevant to the lab

Three properties follow directly from the structure above and underlie everyday laboratory practice.

The backbone is negatively charged. Every phosphate in the backbone carries a net negative charge at physiological and buffer pH. A nucleic acid is therefore a polyanion — uniformly negative along its length, roughly in proportion to its size. This is why, in gel electrophoresis, nucleic acids of any sequence migrate toward the positive electrode (the anode), and why separation depends mainly on size rather than charge ³.

RNA is less stable than DNA because of the 2’-OH. The 2’-hydroxyl that ribose carries and deoxyribose lacks makes RNA chemically reactive: under alkaline (basic) conditions the 2’-OH can attack the neighboring phosphodiester bond and cleave the backbone. DNA, lacking that 2’-OH, is resistant to this alkaline hydrolysis. This alkaline lability is one reason RNA is more fragile and must be handled with greater care — and it is also exploited deliberately, for example to remove RNA from a DNA preparation ³.

Nucleic acids absorb ultraviolet light at 260 nm. The ring structures of the bases absorb UV light, with a peak near a wavelength of 260 nm. This makes it possible to detect and quantify nucleic acid spectrophotometrically by measuring absorbance at 260 nm (A260). Proteins, by comparison, absorb most strongly near 280 nm, and the ratio of the two absorbances (A260/A280) is used to judge the purity of a preparation ³. The techniques that use these measurements are developed later in the program.

DNA vs. RNA at a glance

The differences covered above can be summarized compactly:

   Property        DNA                     RNA
   --------        ---                     ---
   Sugar           2'-deoxyribose          ribose (has 2'-OH)
   Bases           A, G, C, T              A, G, C, U
   Strandedness    typically double        typically single
   Stability       stable                  less stable (alkaline-labile)
   Main role       long-term info storage  expression / regulation

Both molecules share the same essential plan — a sugar-phosphate backbone with bases attached, read 5’→3’ — and differ in a small number of well-defined ways: the sugar’s 2’ position, thymine versus uracil, and (usually) one strand versus two. Keeping these distinctions clear is the foundation for everything that follows, beginning with how the bases pair to form the double helix.