Nucleic Acid Structure

What defines the molecular structures of DNA and RNA?

• Nucleotides are the building blocks of DNA and RNA. Nucleotides consist of a nitrogenous base, a ribose or deoxyribose sugar, and one or more phosphoryl groups.

• DNA is formed from nucleotides containing the bases adenine, thymine, guanine, and cytosine. RNA is formed from nucleotides containing the bases adenine, uracil, guanine, and cytosine. RNA contains ribose sugars, whereas DNA contains 2′-deoxyribose sugars.

• The DNA double helix is formed through base pairing of nucleotide bases. Adenine bases hydrogen bond with thymine bases. Guanine bases hydrogen bond with cytosine bases. The two strands of DNA are oriented in an antiparallel fashion. Base pairing provides the specificity required for genetic information transfer in DNA replication and RNA synthesis.

• Stacking of adjacent base pairs in a helix stabilizes the helix through van der Waals interactions and the hydrophobic effect. Base stacking is the primary source of stabilization for the DNA double helix.

• DNA can adopt three double-helical conformations: A-DNA, B-DNA, and Z-DNA. B-DNA is the primary conformation under most physiologic conditions.

• Denaturation and renaturation refer to the process of separating and reannealing, respectively, two strands of double-helical DNA.

• The melting temperature (Tm) of DNA is influenced by its base composition, the ionic strength of the solution, and the length of the DNA molecule.

• The absorbance of nucleotides at 260 nm can be used to monitor the transition between the single-stranded and double-stranded states.

• The coiling of a coiled (helical) DNA structure results in a supercoil. Supercoils can be described by their linking number (Lk), which is the sum of twist (Tw) and writhe (Wr) values.

• The Lk is defined as the number of times a strand of DNA winds in the right-handed direction around the helix axis when the axis lies in a two-dimensional imaginary plane. The Tw value is the number of times one DNA strand crosses over the other in the DNA double helix in the imaginary plane. The Wr value is the number of times the double-stranded DNA crosses over itself, which can be the result of a supercoil.

• DNA supercoiling is induced by DNA replication and RNA synthesis; it is relieved by topoisomerases. Type I topoisomerases only break one strand of DNA at a time. Type II topoisomerases cleave both strands of DNA.

• The 2′-hydroxyl group on RNA can lead to an autocleavage reaction. This property makes RNA unsuitable as a storage form for genetic material. DNA, which lacks the 2′-hydroxyl group, is a chemically more stable molecule.

• Catalytic RNA molecules are called ribozymes; RNaseP is an example of a ribozyme.

• Common secondary structures in RNA include duplexes, bulges, loops, and hairpins.

• RNA molecules, excluding mRNA, often contain modified nucleotides that aid in adoption of the correct three-dimensional structure or perform other functional roles.

• Two DNA quadruplex structures have been characterized in vitro and in vivo: the G-quadruplex structure consists of four guanine bases connected by hydrogen bonds, and the intercalated-motif (I-motif) structure consists of hydrogen bonds between hemiprotonated cytosine residues.

• DNA and RNA binding proteins can bind in either a sequence-independent or sequence-dependent manner. Proteins largely contact the DNA or RNA backbone through sequence-independent interactions. In the case of sequence-dependent interactions, specific contacts are made between the protein and the DNA or RNA nucleotide bases.

How are genomes organized and how can genomics be used as a forensic tool?

• Condensation of genomic DNA differs in prokaryotes and eukaryotes but is necessary in both to compact the DNA within a cell.

• Histones facilitate the condensation of eukaryotic DNA. Nucleosomes are DNA–histone complexes, which can be further condensed to form chromatin.

• Chromatin can be found as either euchromatin (less condensed) or heterochromatin (more condensed). The switch between these states is influenced by covalent modifications of the histones or DNA.

• DNA in chromosomes is condensed more than 10,000-fold compared with the extended double-helical structure. A mitotic chromosome consists of a central region of heterochromatin called the centromere.

• The ends of chromosomes contain telomeres, which are repetitive DNA sequences. Telomeres function to maintain the length of chromosomes after replication.

• Genes contain regulatory elements and transcribed regions that encode an RNA transcript.

• Prokaryotic genes may have a single promoter but multiple coding regions, which often encode gene products that have related functions.

• Each coding sequence in eukaryotes is under the control of a separate promoter. Regulatory sequences in eukaryotes are generally more extensive than those in prokaryotes.

• Eukaryotic genes often contain both noncoding regions (introns and gene regulatory sequences) and coding regions (exons). RNA processing of eukaryotic transcripts refers to maturation of the primary RNA transcript to generate mRNA that has been modified by 5′ capping, 3′ polyadenylation, and splicing.

• Bioinformatic tools can be used to compare the genomes of different organisms and predict the structure of a gene or the function of a protein.

• Single nucleotide polymorphisms (SNPs) in DNA can be used to diagnose disease and to explore genetic diversity.

What are some of the most common methods in nucleic acid biochemistry?

• Plasmids are circular DNA molecules that are self-replicating in bacteria and normally encode antibiotic-resistance genes as a self-defense mechanism. Engineered plasmids are used for gene cloning methods.

• Bacteria contain enzymes that protect against bacteriophage infection: DNA sequence–specific DNA methylases and restriction endonucleases. DNA methylation blocks restriction endonuclease cleavage at recognition sites; thus, bacteriophage DNA is cleaved, but the bacterial DNA is protected.

• Restriction endonucleases can leave three different classes of DNA termini after cleavage: 5′ overhangs, 3′ overhangs, or blunt ends.

• Genes can be inserted into plasmids by cleaving the DNA with restriction endonucleases and resealing with DNA ligase. Bacterial colonies containing the recombinant plasmid are identified by genetic screening.

• The Sanger DNA sequencing method is based on chain termination through the incorporation of dideoxynucleotides, which lack a hydroxyl group at the deoxyribose 3′-position and block phosphodiester bond formation.

• PCR is an automated method of amplifying DNA sequences. Temperature cycling is used to denature and reanneal the DNA, whereas thermostable DNA polymerases extend the DNA primers.

• The CRISPR-Cas9 system is an RNA-guided DNA targeting tool that was developed using components of a bacterial adaptive immune response. The bacterial CRISPR-Cas9 system consists of an RNA–protein complex that recognizes target DNA by use of a complementary nucleotide RNA sequence of at least 20 nucleotides.

• Components of the CRISPR-Cas9 system required for sequence-specific genome editing in a variety of organisms are (1) single-guide RNA (sgRNA), (2) Cas9 endonuclease, and (3) a PAM sequence consisting of three to five nucleotides in the region of the target DNA.