Base pairs, which form between specific nucleobases, are the building blocks of the DNA double
helix and contribute to the folded structure of both DNA and RNA. Dictated by specific
hydrogen bonding patterns, Watson-Crick base pairs allow the DNA helix to maintain a regular
helical structure that is subtly dependent on its nucleotide sequence. The complementary
nature of this based-paired structure provides a backup copy of all genetic information encoded
within double-stranded DNA. The regular structure and data redundancy provided by the DNA double
helix make DNA well suited to the storage of genetic information, while base-pairing
between DNA and incoming nucleotides provides the mechanism through which DNA polymerase
replicates DNA, and RNA polymerase transcribes DNA into RNA. Many DNA-binding proteins can
recognize specific base pairing patterns that identify particular regulatory regions of
genes. Intramolecular base pairs can occur within
single-stranded nucleic acids. This is particularly important in RNA molecules, where Watson-Crick
base pairs permit the formation of short double-stranded helices, and a wide variety of non-Watson-Crick
interactions allow RNAs to fold into a vast range of specific three-dimensional structures.
In addition, base-pairing between transfer RNA and messenger RNA forms the basis for
the molecular recognition events that result in the nucleotide sequence of mRNA becoming
translated into the amino acid sequence of proteins.
The size of an individual gene or an organism's entire genome is often measured in base pairs
because DNA is usually double-stranded. Hence, the number of total base pairs is equal to
the number of nucleotides in one of the strands. The haploid human genome is estimated to be
about 3.2 billion base pairs long and to contain 20,000–25,000 distinct protein-coding genes.
A kilobase is a unit of measurement in molecular biology equal to 1000 base pairs of DNA or
RNA.
Hydrogen bonding and stability
Hydrogen bonding is the chemical interaction that underlies the base-pairing rules described
above. Appropriate geometrical correspondence of hydrogen bond donors and acceptors allows
only the "right" pairs to form stably. DNA with high GC-content is more stable than DNA
with low GC-content, but, contrary to popular belief, the hydrogen bonds do not stabilize
the DNA significantly, and stabilization is mainly due to stacking interactions.
The larger nucleobases, adenine and guanine, are members of a class of double-ringed chemical
structures called purines; the smaller nucleobases, cytosine and thymine, are members of a class
of single-ringed chemical structures called pyrimidines. Purines are complementary only
with pyrimidines: pyrimidine-pyrimidine pairings are energetically unfavorable because the
molecules are too far apart for hydrogen bonding to be established; purine-purine pairings
are energetically unfavorable because the molecules are too close, leading to overlap
repulsion. Purine-pyrimidine base pairing of AT or GC results in proper duplex structure.
The only other purine-pyrimidine pairings would be AC and GT and UA; these pairings
are mismatches because the pattern of hydrogen donors and acceptors do not correspond. The
GU pairing, with two hydrogen bonds, does occur fairly often in RNA.
Paired DNA and RNA molecules are comparatively stable at room temperature but the two nucleotide
strands will separate above a melting point that is determined by the length of the molecules,
the extent of mispairing, and the GC content. Higher GC content results in higher melting
temperatures; it is, therefore, unsurprising that the genomes of extremophile organisms
such as Thermus thermophilus are particularly GC-rich. On the converse, regions of a genome
that need to separate frequently — for example, the promoter regions for often-transcribed
genes — are comparatively GC-poor. GC content and melting temperature must also be taken
into account when designing primers for PCR reactions.
Examples The following DNA sequences illustrate pair
double-stranded patterns. By convention, the top strand is written from the 5' end to the
3' end; thus, the bottom strand is written 3' to 5'.
A base-paired DNA sequence: ATCGATTGAGCTCTAGCG
TAGCTAACTCGAGATCGC
The corresponding RNA sequence, in which uracil is substituted for thymine where uracil takes
its place in the RNA strand: AUCGAUUGAGCUCUAGCG
UAGCUAACUCGAGAUCGC
Base analogs and intercalators
Chemical analogs of nucleotides can take the place of proper nucleotides and establish
non-canonical base-pairing, leading to errors in DNA replication and DNA transcription.
This is due to their isosteric chemistry. One common mutagenic base analog is 5-bromouracil,
which resembles thymine but can base-pair to guanine in its enol form.
Other chemicals, known as DNA intercalators, fit into the gap between adjacent bases on
a single strand and induce frameshift mutations by "masquerading" as a base, causing the DNA
replication machinery to skip or insert additional nucleotides at the intercalated site. Most
intercalators are large polyaromatic compounds and are known or suspected carcinogens. Examples
include ethidium bromide and acridine. Unnatural base pair
An unnatural base pair is a designed subunit of DNA which is created in a laboratory and
does not occur in nature. DNA sequences have been described which use newly created nucleobases
to form a third base pair, in addition to the two base pairs found in nature, A-T and
G-C. A few research groups have been searching for a third base pair for DNA, including teams
led by Steven A. Benner, Philippe Marliere, Floyd Romesberg and Ichiro Hirao. Some new
base pairs have been reported. In 1989 Steven Benner, then at the Swiss Federal
Institute of Technology in Zurich, and his team led with modified forms of cytosine and
guanine into DNA molecules in vitro. The nucleotides, which encoded RNA and proteins, were successfully
replicated in vitro. Since then, Benner's team has been trying to engineer cells that
can make foreign bases from scratch, obviating the need for a feedstock.
In 2002, Ichiro Hirao’s group in Japan developed an unnatural base pair between 2-amino-8-(2-thienyl)purine
and pyridine-2-one that functions in transcription and translation, for the site-specific incorporation
of non-standard amino acids into proteins. In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine
and pyrrole-2-carbaldehyde as a third base pair for replication and transcription. Afterward,
Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole was discovered as a high fidelity pair in
PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by in
vitro selection and demonstrated the genetic alphabet expansion significantly augment DNA
aptamer affinities to target proteins. In 2012, a group of American scientists led
by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego,
California, published that his team designed an unnatural base pair. The two new artificial
nucleotides or Unnatural Base Pair were named d5SICS and dNaM. More technically, these artificial
nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form
a complex or base pair in DNA. His team designed a variety of in vitro or "test tube" templates
containing the unnatural base pair and they confirmed that it was efficiently replicated
with high fidelity in virtually all sequence contexts using the modern standard in vitro
techniques, namely PCR amplification of DNA and PCR-based applications. Their results
show that for PCR and PCR-based applications, the d5SICS–dNaM unnatural base pair is functionally
equivalent to a natural base pair, and when combined with the other two natural base pairs
used by all organisms, A–T and G–C, they provide a fully functional and expanded six-letter
"genetic alphabet". In 2014 the same team from the Scripps Research
Institute reported that they synthesized a stretch of circular DNA known as a plasmid
containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's
laboratory had designed, and inserted it into cells of the common bacterium E. coli that
successfully replicated the unnatural base pairs through multiple generations. The transfection
did not hamper the growth of the E. coli cells, and showed no sign of losing its unnatural
base pairs to its natural DNA repair mechanisms. This is the first known example of a living
organism passing along an expanded genetic code to subsequent generations. Romesberg
said he and his colleagues created 300 variants to refine the design of nucleotides that would
be stable enough and would be replicated as easily as the natural ones when the cells
divide. This was in part achieved by the addition of a supportive algal gene that expresses
a nucleotide triphosphate transporter which efficiently imports the triphosphates of both
d5SICSTP and dNaMTP into E. coli bacteria. Then, the natural bacterial replication pathways
use them to accurately replicate a plasmid containing d5SICS–dNaM. Other researchers
were surprised that the bacteria replicated these human-made DNA subunits.
The successful incorporation of a third base pair is a significant breakthrough toward
the goal of greatly expanding the number of amino acids which can be encoded by DNA, from
the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential
for living organisms to produce novel proteins. The artificial strings of DNA do not encode
for anything yet, but scientists speculate they could be designed to manufacture new
proteins which could have industrial or pharmaceutical uses. Experts said the synthetic DNA incorporating
the unnatural base pair raises the possibility of life forms based on a different DNA code.
Length measurements The following abbreviations are commonly used
to describe the length of a D/RNA molecule: bp = base pair(s)— one bp corresponds to
approximately 3.4 Å of length along the strand, or to roughly 618 or 643 daltons for DNA and
RNA respectively. kb = kilo base pairs = 1,000 bp
Mb = mega base pairs = 1,000,000 bp Gb = giga base pairs = 1,000,000,000 bp.
For case of single-stranded DNA/RNA units of nucleotides are used, abbreviated nt, as
they are not paired. For distinction between units of computer storage and bases kbp, Mbp,
Gbp, etc. may be used for base pairs. The centimorgan is also often used to imply
distance along a chromosome, but the number of base pairs it corresponds to varies widely.
In the Human genome, the centimorgan is about 1 million base pairs.
See also List of Y-DNA single-nucleotide polymorphisms
References
Further reading Watson JD, Baker TA, Bell SP, Gann A, Levine
M, Losick R. Molecular Biology of the Gene. Pearson Benjamin Cummings: CSHL Press.
Astrid Sigel, Helmut Sigel, Roland K. O. Sigel, ed.. Interplay between Metal Ions and Nucleic
Acids. Metal Ions in Life Sciences 10. Springer. doi:10.1007/978-94-007-2172-2. ISBN 978-9-4007-2171-5.
Clever, Guido H.; Shionoya, Mitsuhiko. "Chapter 10. Alternative DNA
Base-Pairing through Metal Coordination". Interplay between Metal Ions and Nucleic Acids.
pp. 269–294. doi:10.1007/978-94-007-2172-2_10. Megger, Dominik A.; Megger, Nicole; Mueller,
Jens. "Chapter 11. Metal-Mediated Base Pairs in Nucleic Acids with Purine and Pyrimidine-Derived
Neucleosides". Interplay between Metal Ions and Nucleic Acids. pp. 295–317. doi:10.1007/978-94-007-2172-2_11.
External links DAN—webserver version of the EMBOSS tool
for calculating melting temperatures