Thermodynamics of Nucleotide Base Pairing

Thermodynamics plays a major role in the structure and stability of nucleic acid molecules. The core mechanism of nucleic acid thermodynamics centers on the hydrogen-bonding capabilities of the nucleotides. The stability of these interactions not only influences the formation and stability of duplex nucleic acids but also impacts the structure and catalytic characteristics of single-stranded nucleic acids through intramolecular base pairing. In addition to these physiological functions, the phenomenon of complementary base pairing profoundly impacts clinical diagnostic assay development. Prior to the advent of clinical molecular diagnostic testing, many diagnostic tests required obtaining an antibody to identify or detect a target protein. The procedures for generating and validating diagnostic antibodies required extensive time and expense. The application of techniques utilizing the capability of two molecules to form a base pair as the basis for detection and characterization of target nucleic acids has greatly facilitated diagnostic test development. The formation of hydrogen bonding between two pieces of nucleic acid is called hybridization, or annealing, and the disruption of the hydrogen bonds holding two nucleic acid molecules together is called denaturation, or melting. The fact that molecular diagnostic tests use hybridization techniques based on A :T and G: C base pairing underscores the necessity for understanding the thermodynamics of hydrogen base pairing of nucleic acids.

Short pieces of DNA or RNA called probes, or primers, that contain a specific sequence complementary to a disease-related region of DNA or RNA from a clinical specimen are frequently used in the molecular pathology laboratory. To achieve hybridization of a DNA or RNA probe to genomic DNA for a diagnostic test, the two genomic DNA strands must be separated, or denatured, prior to probe hybridization. Increasing the temperature of a DNA molecule is one mechanism for disrupting the hydrogen bonds between the DNA base pairs and denaturing double-stranded DNA into single-stranded form. The temperature at which 50% of the double-stranded DNA molecules separate into single-stranded form constitutes the melting temperature (Tm). The shorter the two complementary DNA molecules are, the easier it is to calculate the Tm. This primarily results from the decreased likelihood of nonspecific intramolecular annealing or base pairing compared to inter- and intramolecular base pairing. The simplest and least accurate formula for determining the Tm for short double-stranded DNA multiplies the sum of the G: C base pairs by 4 and multiplies the sum of the A: T base pairs by 2 and then adds these numbers together.

Although this is the least accurate method for calculation of the Tm of a double-stranded DNA molecule, it mathematically illustrates that G: C bonds are roughly twice the strength of A:T bonds. This formula works fairly well for short DNA molecules (i.e., <18bp); however, as the length of the DNA molecule increases to 100bp,the nearest neighbor Tm calculation for DNA and RNA is more accurate.6,7

Table 1-3. Melting-Temperature Calculations for Short Oligomers

where

AH = enthalpy of the nucleic acid fragment AS = entropy of the nucleic acid fragment R = 1.987 calK-1 mol-1 Ct = total strand concentration

For longer sequences (>100bp), the most accurate formula for calculation of Tm is as follows:8

Tm = 81.5 + 16.6 log [NA] + 0.41 [%G = %C} - 0.65 (% formamide) - 675/length - % mismatch

Table 1-3 demonstrates the effect of increasing the relative amounts of G:C base pairs on the Tm using these formulas.

Intramolecular base pairing also generates complex three-dimensional forms within single-stranded nucleic

Table 1-3. Melting-Temperature Calculations for Short Oligomers

Total

Number of

Number of

A:T +

Length

G:C

A:T

T *

%G:Cf

G:C*

30

30

0

106.2

100.0

100.0

30

25

5

101.2

93.2

100.0

30

20

10

89.5

79.5

90.0

30

10

20

83.4

72.7

80.0

30

0

30

71.6

59.0

60.0

20

20

0

90.4

88.8

80.0

20

10

10

72.7

65.1

60.0

20

0

20

55.9

47.8

40.0

* Nearest neighbor calculation of Tm.6 tTm method for sequences over 100 bases.8

* Nearest neighbor calculation of Tm.6 tTm method for sequences over 100 bases.8

acid molecules. As a result, the single-stranded nature of eukaryotic RNA molecules affords great structural diversity via intramolecular base pairing. These conformations strain the linear RNA molecule and produce chemically reactive RNA forms. Catalytic RNA molecules play pivotal roles in cellular functions and in gene-targeting therapies.

Intra- and intermolecular base pairing can negatively affect hybridizations. Dimers, bulge loops, and hairpin loops exemplify some of these interactions. Hairpins inhibit plasmid replication and attenuate bacterial gene expression.2 These detrimental effects may also include initiation of spurious nonspecific polymerization, steric hindrance of hybridization of short stretches of nucleic acids (i.e., 10 to 30 base pieces of single-stranded nucleic acids, known as oligomers or primers), and depletion of probes or primers away from the specific target by either primer dimerization or other mechanisms. These interactions can result in poor sensitivity or poor specificity for diagnostic molecular tests.

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