8.1 Nucleotides and Nucleic Acids: the Basics

The characteristic bases and pentoses

A nucleotide is a base plus a pentose plus one or more phosphates, while base and sugar alone give a nucleoside
Phosphorylating a nucleoside gives a nucleotide, so ATP with three phosphates is still a nucleotide
Purines are adenine and guanine with fused two-ring structures
Pyrimidines are cytosine, thymine and uracil with a single six-membered ring
DNA and RNA share A, G and C, with T standard in DNA and U in RNA, though modified bases prevent absolute generalisation
The RNA sugar is ribose with a 2-prime OH, and the DNA sugar is 2-prime deoxyribose with H at that position
Sugar carbons are numbered 1-prime to 5-prime, with 1-prime for base attachment, 3-prime OH for chain extension and 5-prime for phosphate linkage
The 2-prime OH aids folding but can act as a nucleophile on the adjacent phosphate, which makes RNA alkali-labile
Adenine with ribose gives adenosine, adding one phosphate at 5-prime gives AMP, and the DNA notation is dAMP
Purine N9 and pyrimidine N1 form the beta-N-glycosidic bond with sugar C1-prime

A phosphodiester bond is one phosphate esterified to oxygen atoms from the hydroxyls of two different sugars
Most links join the 3-prime position of one residue to the 5-prime of the next, giving a 3-prime to 5-prime phosphodiester bond
The sugar-phosphate backbone is hydrophilic and carries negative charge near neutral pH
Sequences are written 5-prime to 3-prime, with a 5-prime phosphate or 5-prime OH at one end and usually a 3-prime OH at the other
The claim that every 5-prime end must carry a phosphate is false
A linear strand of n residues has n minus 1 phosphodiester bonds, so ten residues give nine
A 10 bp linear duplex has 2(10 minus 1) = 18 internal links
Cellular synthesis uses dNTPs or NTPs, and the 3-prime OH of the growing chain attacks the alpha phosphate to form the new bond and release pyrophosphate
The reaction needs enzyme and metal ions, so the idea that two free nucleotides simply lose one water is inaccurate
Internal phosphate linkages carry roughly a single negative charge, making long nucleic acids polyanions
Charge depends on pH, terminal phosphorylation and chemical modification
In alkali the RNA 2-prime OH can attack the adjacent phosphate to give cleavage and a 2-prime,3-prime cyclic phosphate, and though DNA cannot follow this path easily it is not absolutely stable under all conditions

Purines and pyrimidines have nearly planar rings with conjugated electron systems
Stacking of adjacent bases combines dispersion forces, the hydrophobic effect and electrostatic interactions
Stability depends on sequence, neighbouring pairs, ions and water rather than hydrogen bond count alone
Canonical pairs have two hydrogen bonds for A-T and three for G-C, with A-U in RNA
One purine with one pyrimidine keeps the duplex width relatively constant
Bond count alone cannot fix the melting temperature of a whole DNA molecule
The strands are antiparallel, so 5-prime ATGCTAGC 3-prime pairs with 3-prime TACGATCG 5-prime, written 5-prime GCTAGCAT 3-prime
In a duplex the counts of A and T are equal, as are those of G and C
Higher GC content tends to raise stability, though base order and solution conditions matter too
Single-stranded RNA can still form stem-loops and hairpins through complementary segments
Absorbance at 260 nm is used for quantitation, and loss of stacking on denaturation gives a hyperchromic effect
High absorbance does not guarantee a pure sample

A 25-residue linear strand has 24 internal links, and a 25 bp linear duplex has 48
Terminal phosphorylation and the number of internal links are separate questions
The complement of 5-prime AGTCCA 3-prime is 3-prime TCAGGT 5-prime, written 5-prime TGGACT 3-prime
A simple complement must be distinguished from a reverse complement
The canonical hydrogen bond total is 3 per GC pair plus 2 per AT pair, so 1,000 bp at 40 percent GC gives 400 x 3 plus 600 x 2, that is 2,400
That number is not a thermodynamic measure of whole-molecule stability
With epsilon 1.50e4 L per mol per cm, path length 1.00 cm and A 0.300, the concentration is 2.00e-5 M, that is 20.0 micromolar
The coefficient is a practice assumption, not a universal conversion for real samples

Identify molecules from the base, pentose and phosphate count on cards
Check directionality and bond count geometrically with a linear duplex model
Estimate concentration from a given molar extinction coefficient using A = epsilon l c
Argue how structural differences between DNA and RNA lead to different chemical behaviour

References

**표 8.1-1**Purine and pyrimidine bases
Class	Base	Standard base in DNA	Standard base in RNA
Purine	Adenine and guanine	A, G	A, G
Pyrimidine	Cytosine	C	C
Pyrimidine	Thymine	T	Replaced in principle by U
Pyrimidine	Uracil	Replaced in principle by T	U

**표 8.1-2**Nucleoside and nucleotide names by base
Base	Ribonucleoside	Typical 5-prime monophosphate	DNA notation
Adenine	Adenosine	AMP	dAMP
Guanine	Guanosine	GMP	dGMP
Cytosine	Cytidine	CMP	dCMP
Uracil	Uridine	UMP	none
Thymine	Ribothymidine, rare	none	dTMP

**표 8.1-3**Key relations for nucleic acid calculations
Item	Relation	Condition to check
Links in one linear strand	n minus 1	Check terminal modification and circularity separately
Links in a linear duplex	2(n minus 1) for n bp	n residues per strand with no nicks
GC content	GC bp divided by total bp times 100	Applies to a fully complementary duplex
Model absorbance	A = epsilon l c	State epsilon, path length and wavelength