Nucleic acids. DNA. RNA

This topic draws on two lectures. From the DNA lecture: how the function and the structure of DNA were discovered, the structure of nucleotides and polynucleotide chains, complementarity, denaturation, the three forces that stabilise the double helix, base stacking and the PAH world hypothesis, intercalation, the three helix types, supercoiling, and the sense and antisense strands. From the RNA lecture: the secondary and tertiary structures of RNA, the regions of messenger RNA, the organisation of ribosomal RNA, transfer RNA, examples of ribozymes, of a large non-coding RNA, of small nuclear and small cytoplasmic RNAs, and how gene silencing occurs through RNA.

Nucleic acids are macromolecules responsible for the storage, transmission and retrieval of genetic information.

There are two types:

• Deoxyribonucleic acid (DNA) - encodes genetic information;
• Ribonucleic acid (RNA) - decodes genetic information.

DNA was discovered in 1869 by Friedrich Miescher, then a 25-year-old newly graduated doctor. It was named nuclein, and a little later nucleic acid, because it was found in the nucleus.

By the late nineteenth century the role of the nucleus in heredity had become known. The nuclear localisation of nucleic acid made Walter Flemming and others suggest a role in heredity. Most scientists, including Miescher, dismissed these ideas. Proteins were already known as the key compounds of life, and everyone expected the substance of heredity to be a protein too.

Since 1924 the Feulgen reaction, which stains DNA pink, has been used for the specific cytochemical detection of DNA. It was found in all nuclei and all chromosomes, with the quantity varying twofold in somatic cells and reduced in gametes. This was strong indirect evidence for the importance of DNA - the amount behaved exactly as a hereditary substance should.

Bacterial transformation was discovered in 1928 by Frederick Griffith.

He infected mice with two strains of pneumococci (Streptococcus pneumoniae):

• one strain was easily destroyed by the immune system and therefore non-virulent;
• the other was virulent, because a capsule over the cell wall protected it from host defence.

Griffith found that living non-virulent and killed virulent bacteria, though harmless when given separately, were lethal if injected together. From the dead mice, live virulent bacteria were isolated.

He concluded that some component of the dead bacteria had "transformed" the living ones, making them able to make a capsule and hence virulent.

In 1944, Oswald Avery, Colin MacLeod and Maclyn McCarty set out to find which substance was responsible for bacterial transformation.

They selectively removed or purified individual components of virulent pneumococci and checked each resulting extract for transforming activity.

Of all the cell components, DNA alone proved able to convert non-virulent bacteria into virulent ones with capsules.

The role of DNA was confirmed by Alfred Hershey and Martha Chase in 1952.

They cultured two groups of bacteriophages in the presence of ³⁵S and ³²P, labelling proteins and DNA respectively. The labelled phages were then added to bacteria.

After stirring and centrifugation, the pellet of bacterial cells was radioactive only in the labelled phosphorus group. That meant the protein part of the phages had not entered the host cells at all.

After culture, the phage progeny was obtained, and it too was radioactive only in the labelled phosphorus group.

In 1950, Erwin Chargaff found that in the DNA of any cell or organism, the amount of adenine equals that of thymine, and the amount of cytosine equals that of guanine: %A = %T, %G = %C. These are Chargaff's rules.

Meanwhile James Watson and Francis Crick were trying to reveal the structure of DNA. They had good ideas but needed X-ray crystallographic data.

Rosalind Franklin (1920-1958) was their ideal complement: she was excellent at crystallography, though less so at theory. However, Watson and Crick were unable to team with a woman.

In early 1953, Franklin's colleague Maurice Wilkins took a photograph from her desk without her knowledge and showed it to Watson and Crick. Together with Chargaff's rules, it allowed them to create the molecular model of DNA.

Watson, Crick and Wilkins received the Nobel Prize in 1962. Franklin had died in 1958.

Nucleic acids are polymers of nucleotides.

Each nucleotide consists of:

• a nitrogenous base;
• a pentose;
• at least one phosphate group.

The bases are related to the parent compounds pyrimidine and purine.

Note that the carbon atoms of the pentose are numbered from 1' to 5' - the primes exist to keep them distinct from the numbering of the base.

• DNA has A, T, C, G and deoxyribose.
• RNA has A, U, C, G and ribose.

1. A nitrogenous base binds to the 1' atom of the pentose, forming a nucleoside. For example, adenine plus ribose form adenosine.
2. A phosphate reacts with the 5' hydroxyl group of the pentose, forming a nucleotide. Such nucleotides are called nucleoside monophosphates; the example is adenosine monophosphate (AMP).
3. One or two more phosphates may join the first, forming a nucleoside diphosphate and a nucleoside triphosphate. These are nucleotides as well.

Nucleic acids are composed of polynucleotide chains, as proteins are composed of polypeptide chains.

To form a polynucleotide, the phosphate at the 5' atom of one pentose reacts with the 3' hydroxyl group of another pentose, creating a covalent phosphodiester bond between them.

• If the chain has two free ends, they are designated 5' and 3'. DNA chains are often circular.
• Because of the phosphates, DNA and RNA are acids. The bases are too weak to make a difference.
• The alternating pentoses and phosphates form a uniform backbone, from which the nitrogenous bases protrude. Their unique arrangement determines the sequence, that is the primary structure, of the nucleic acid.

Nitrogenous bases bind specifically by hydrogen bonds, according to the complementarity rule:

• A with T or U - 2 bonds;
• C with G - 3 bonds.

To interact in this way, the nucleotides must be in an antiparallel position, that is, their 5' ends must point in opposite directions.

Complementary binding defines the secondary structure of nucleic acids.

DNA is double-stranded, composed of two complementary antiparallel chains called strands, bound by numerous hydrogen bonds. The two backbones are at the edges and the bases are in the middle.

A consequence worth holding on to: DNA regions that must open easily - where replication or transcription begins - are AT-rich, because an A-T pair costs only two hydrogen bonds to break.

Heating above 70 °C causes full separation of the two strands. This is called denaturation, and unlike protein denaturation it is reversible.

As the temperature increases, regions of local unwinding occur, preferentially in regions enriched in A/T base pairs connected by only 2 hydrogen bonds. In G/C-rich regions the strands are held together more strongly, so they do not unwind until higher temperatures.

Incidentally, even at normal cell temperatures the DNA "breathes", and local regions become temporarily unwound. As you would expect, A/T-rich regions are more likely to open than G/C-rich ones. This is one of the reasons why transcription initiation bubbles and DNA replication origins are often A/T rich.

1. Base pairing by hydrogen bonds. Note that this connection does not force the bases to stand perpendicular to the backbone of the chain.
2. Base stacking. This connection does force the bases to stand nearly perpendicular to the backbone.
3. Ionic interactions.

Base stacking:

• is based on ionic and other weak interactions between the bases, and also between them and the surrounding molecules;
• depends on the base couples involved;
• because it pushes water molecules out of the space between the hydrophobic bases, it makes the greatest contribution to stabilising DNA structure - greater than the hydrogen bonding that gets all the attention;
• contributes to DNA-protein interactions.

Nitrogenous bases are fairly hydrophobic and minimise their contact with water.

As a result, when two complementary strands bind, their bases stack together like cards to exclude water molecules. This forces the backbones to bend spirally, forming a double-stranded helix.

In other words the helix is not an aesthetic fact about DNA; it is the geometric consequence of hydrophobic bases packing to avoid water. The stacking itself results from hydrophobic, Van der Waals and other interactions between bases.

Because base stacking is sequence dependent, the shape of the double helix varies along the DNA.

• Between GC-GC couples the strength of stacking is highest.
• Between AT-AT couples it is lowest.
• In other combinations it is intermediate.

Some features of stacking also make the spiral uneven, producing a major and a minor groove. The reason is a small difference between the distance separating adjacent bases measured at the centre of the helix and the distance measured at its periphery.

Polycyclic aromatic hydrocarbons (PAHs) are a class of chemicals occurring naturally in coal, crude oil and petrol.

According to astrophysics, the infrared spectra of many galactic and extragalactic objects are due to PAHs. In other words, PAHs are abundant in the universe: more than 20% of the carbon in the universe may be associated with them. The green light of the Cat's Paw Nebula, in Scorpio, is due to a high concentration of PAHs.

The hypothesis: PAHs were abundant in the primordial soup of the early Earth and played a major role in the origin of life, by mediating the synthesis of RNA molecules and so leading into the RNA world.

• Data from the Hubble Space Telescope show that in interstellar environments, PAHs are transformed by hydrogenation, oxygenation and hydroxylation into more complex molecules. This can be a step on the path to amino acids and nucleotides.
• In general PAHs are not very soluble in water, but when exposed to radiation such as UV light they become much more soluble, because their outer hydrogen atoms are replaced by hydroxyl groups.
• These modified PAHs are amphiphilic, having both hydrophilic and hydrophobic parts. When in solution they form stacks, to keep their hydrophobic portions out of contact with water - exactly the behaviour of the bases above.
• In a self-assembling stack, the distance between adjacent rings is the same as that between adjacent RNA and DNA nucleotides: 0.34 nm.
• In stacks, the PAH rings tend to rotate on top of each other, forming double helices. This makes the attachment of flat molecules such as pyrimidines and purines to the stack very likely - and those bases are also amphiphilic, so they too tend to incorporate into twisted stacks.

The explanation for the origin of RNA in the primary soup is based on chemical experiments, and runs in three steps.

How the backbone was created. After the nitrogen bases were attached to the stacks of PAHs by hydrogen bonds, linker molecules, also taken from the primary soup, stood between the bases. These molecules linked the bases through covalent bonds, creating a flexible backbone. The linkers were initially not pentoses, as in DNA and RNA, but derivatives of formaldehyde; ribose and phosphate were added later.

How single-stranded RNA detached from the scaffold. A temporary change in the pH of the environment, for example from volcanic ejection of acid gases, allowed the bases to break away from their PAH scaffold, forming RNA-like molecules.

How ribozymes were created. Once long RNA-like single strands had detached, they became able to fold back on themselves in search of a thermodynamically stable shape. Stability is provided by the pairing of complementary sequences through hydrogen bonds, so partially double-stranded RNA-like structures were created.

Some compounds with flat ring molecules can intercalate, that is insert themselves, between the stacked bases.

Such compounds have two faces:

• they are good cytochemical stains for nucleic acids, usually fluorochromes;
• they are also mutagens and carcinogens.

Examples:

• Propidium iodide, used to stain DNA red, as in dividing plant cells with tubulin labelled green.
• Ethacridine lactate (Rivanol), whose disinfectant properties are based on its intercalation into bacterial DNA.
• Actinomycin D, an antibiotic with antibacterial and antitumour activity, which is an intercalator.

There are three types of nucleic acid double helix: A, B and Z.

B DNA is the main helix type of DNA. It is right-handed, with a diameter of 2 nm and about 10 nucleotides per turn. Most of the DNA, most of the time, is in the B form, and it is thermodynamically the most stable. The helix forms two grooves, major and minor, and proteins use them to bind DNA - a transcription-regulating protein typically binds in the major groove.

A DNA forms when DNA is dehydrated. It is similar to B DNA but with a larger diameter. Under natural conditions the A helix is characteristic not of DNA but of the double-stranded parts of RNA and of the hybrid DNA-RNA intermediate formed during transcription. The reason is chemical: the 2' OH group of ribose does not allow the formation of a B helix.

Z DNA is named for the zig-zag its backbone forms. It can appear in small parts of the DNA molecule with a special primary structure, namely a CGCGCG... sequence. This sequence is found associated with some genes, located close to the start point of transcription, and it is thought that some transcription regulator proteins recognise and bind Z DNA helices.

The helices described above were under no torsion stress. Such DNA is called relaxed.

Torsion force can be applied in the same direction as the helix itself (+) or in the opposite direction (-).

If the DNA is circular, or if the ends of a linear DNA are fixed, torsion stress makes the axis of the DNA helix bend spirally. This is DNA supercoiling.

• Positive supercoiling - the strand is overwound compared with the relaxed state.
• Negative supercoiling - the strand is underwound compared with the relaxed state.

The functional consequence is the point: negative supercoiling facilitates separation of the two strands, while positive supercoiling makes separation more difficult.

Supercoiling occurs when the right-handed double helix is twisted tighter, overwound in a right-handed mode, until the helical structure becomes distorted and develops into a level of "knot". Supercoiling is relaxed by the topoisomerase enzymes.

To identify DNA strands, they are described as sense and antisense.

• Sense strand = positive = coding strand. Its nucleotide sequence corresponds directly to the sequence of the RNA transcript that is translated, provided the Ts in the DNA are replaced by Us in the RNA.
• Antisense strand = negative = template strand. It is complementary both to the positive-sense strand and to the RNA transcript.

Two refinements matter:

• The terms are relative only to a particular RNA transcript, not to the DNA strand as a whole. Either strand can be the sense or the antisense strand. Most organisms with sufficiently large genomes use both strands as template for different RNA transcripts, in different places along the same DNA molecule.
• Ambisense - single-stranded genomes can use the same strand as both sense and antisense. Some viruses have ambisense genomes.

RNA molecules are much smaller than DNA, and they are linear.

RNA is generally single-stranded but often has double-stranded parts called stem-loops or hairpin loops. These are formed by complementary binding within the same chain, after a 180° turn.

Besides the secondary structure determined by the loops, RNA undergoes additional three-dimensional folding, its tertiary structure. As in proteins, secondary and tertiary structure are based on primary structure.

Some RNAs have catalytic activity and are called ribozymes, a name implying both their similarity to and their difference from enzymes. They are not to be confused with ribosomes.

The complementarity rule allows RNA molecules to replicate like DNA. It is thought that billions of years ago life existed as self-replicating ribozymes, the RNA world theory. Today, replicating RNAs are the genomes of RNA viruses.

RNAs involved in transcription or translation:

• mRNA, messenger RNA - encodes the amino acid sequence of a polypeptide.
• tRNA, transfer RNA - brings amino acids to the ribosomes during translation.
• rRNA, ribosomal RNA - with the ribosomal proteins, makes up the ribosomes.
• Signal recognition particle RNA - allows the ribosome to recognise membranes, and so allows the protein to cross them.

RNAs involved in post-transcriptional modification or DNA replication:

• Small nuclear RNAs (snRNA), average length about 150 nucleotides, taking part in mRNA processing. They associate with specific proteins into complexes called small nuclear ribonucleoproteins (snRNP), often pronounced "snurp".
• Small nucleolar RNAs (snoRNA), taking part in rRNA processing.
• Ribonuclease P, a ribozyme taking part in tRNA processing, cleaving an extra sequence off pre-tRNA molecules.
• Telomerase RNA component.

Regulatory RNAs: antisense RNA, micro RNA, small interfering RNA, CRISPR.

Parasitic RNAs: viral genomic RNA, single-stranded but sometimes double-stranded; viroid RNA; retrotransposons.

Unlike the other RNAs, messenger RNA has no remarkable spatial structure. It is its primary structure that matters - a linear script.

Each mRNA is a copy of a gene encoding a protein, and it brings the message for the protein sequence to the ribosomes where it will be synthesised.

• The coding region consists of nucleotide triplets called codons, recognised by the tRNA anticodons. The first and last codons are special and are called the start and stop codons.
• The coding region is flanked by the 5' and 3' untranslated regions.
• The 5' untranslated region contains a ribosome recognition site binding to the small subunit.
• The 3' untranslated region is important for the life span of the mRNA.

Most rRNAs are large. Much of the rRNA molecule is engaged in hairpin loops, and it has a structural role in the formation of the ribosomal subunits - the human small subunit rRNA, 18S, is the standard illustration of that folding.

The largest rRNA is a ribozyme. The largest rRNA, embedded in the large ribosomal subunit, is not only a structural scaffold but also a catalyst. It catalyses the formation of peptide bonds and is called peptidyl transferase. Two amino acids brought by tRNA converge in the ribosome's active centre, and the RNA joins them.

Transfer RNAs are small, for macromolecules. They have 4 loops: 3 large and 1 small. The secondary structure resembles a clover leaf, and the tertiary structure is L-shaped.

The function of tRNA is to match amino acids to the programme provided by mRNA during protein synthesis.

• Each tRNA is specific for a particular amino acid, which binds covalently to the 3' end.
• Binding of tRNA to mRNA is complementary and based on 3 nucleotides. This triplet is the anticodon, and it is located in one of the large loops.

Non-coding RNAs are divided into two groups by length:

• small non-coding, fewer than 200 nucleotides;
• long non-coding, more than 200 nucleotides.

The small non-coding RNAs are ribosomal RNA (rRNA), small nuclear RNA (snRNA), transfer RNA (tRNA), micro RNAs, small interfering RNAs, and telomeric RNAs.

The long non-coding RNAs include long intergenic RNA (lincRNA), intronic RNA, enhancer RNA (eRNA), natural antisense transcripts (NAT), and circular RNA (circRNA).

Xist RNA is responsible for the inactivation of one of the X chromosomes in female mammals.

The gene for this RNA is located on the X chromosome. Xist is produced by one X chromosome, remains attached to it, and inactivates it. That chromosome then moves to the periphery of the nucleus.

The more Xist accumulates on the X chromosome, the more compacted it becomes.

In the interphase nucleus of a female cell, Xist RNA can be visualised accumulated around the inactivated X (Xi) but not around the active one (Xa), even though the Xist gene locus is present on both.

• The spliceosome includes several proteins and snRNA.
• The spliceosome members join the mRNA during its synthesis.
• The splice sites are recognised by snRNA - by base pairing, not by protein recognition.

Secreted and cell surface proteins are marked for export by a specific signal sequence at their N terminus. It is recognised by a ribonucleoprotein complex called the signal recognition particle (SRP).

SRP helps the ribosome dock onto the endoplasmic reticulum, and the nascent polypeptide is then pushed across the membrane.

The SRP is a complex of SRP-RNA and SRP-protein, and it changes conformation between its free state and its state bound to a signal peptide.

RNase P is a ribozyme. It cleaves RNA.

Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA molecules.

RNase P is one of only two known multiple-turnover ribozymes in nature - that is, one of two that act catalytically rather than being consumed.

These small RNAs are involved in the post-transcriptional regulation of gene expression. The selective blocking or destruction of mRNA transcripts from a particular gene is called post-transcriptional gene silencing.

Both miRNAs and siRNAs are 18 to 25 nucleotides long. The binding of the small RNA to the mRNA reduces the expression level of the protein, and how it does so depends on the fit:

• A perfect match between the small RNA and the mRNA results in cleavage of the mRNA by RISC.
• Incomplete complementarity leads to partial translational repression instead.

RISC, the RNA-induced silencing complex, is a multiprotein complex incorporating nucleases. The complementary connection between the small RNA and a short segment of the mRNA acts as the signal for destruction.

The major difference in their action:

• siRNA are highly specific, with only one mRNA target.
• miRNA have multiple targets and are not fully complementary to them.

The two pathways in detail.

siRNA: double-stranded RNA, either transcribed or artificially introduced, is processed by Dicer into siRNA, which is loaded into RISC. AGO2, a component of RISC, cleaves the passenger strand. The guide strand then guides the active RISC to the target mRNA, and the full complementary binding leads to cleavage of that mRNA.

miRNA: transcription of the miRNA gene is carried out by RNA polymerase II in the nucleus to give pri-miRNA, which is cleaved by Drosha to form pre-miRNA. The pre-miRNA is transported by Exportin 5 to the cytoplasm, where it is processed by Dicer into miRNA. The miRNA is loaded into RISC, the passenger strand is discarded, and the miRISC is guided by the remaining guide strand to the target mRNA through partially complementary binding. The target is then inhibited by translational repression, degradation or cleavage.

miRNAs are endogenous. They are associated with several physiological activities, such as tissue development, lipid metabolism, cell differentiation, apoptosis, and stem cell division.

In humans, approximately 70% of known miRNAs reside in non-protein-coding regions of the genome, while the remaining 30% are transcribed from intergenic regions or from the introns of genes.

miRNAs were initially identified for their central role in differentiation and development, and their functions are tissue-specific or tied to particular stages of development. In vertebrate embryos, individual microRNAs have a specific localisation and their activity is limited to the tissues and organs in which they are expressed:

• miR-206 is expressed in muscles;
• miR-126 in blood vessels and the heart;
• miR-200a in the lateral line and sensory organs;
• miR-30c in the kidney progenitor.

Abnormal expression of microRNAs contributes to the pathology of many diseases, including cancer.

Exogenous siRNA and miRNA can be used to block:

• Viral infection - by targeting viral RNAs, or by blocking the initial viral entry by targeting host cell genes.
• Cancer - by targeting cancer-related genes, that is, oncogenes.
• Neurological diseases - by targeting the genes responsible for products that disturb cell function, which can significantly reduce the amount of the molecule provoking the disease.

Translation can be blocked either before or after its start.

ASOs are short, synthetic, single-stranded oligonucleotides, designed to be a perfect complementary match to a specific target DNA or RNA sequence, such as an mRNA. Both RNA and DNA can be used to create synthetic ASOs.

Unlike siRNA and miRNA, ASOs do not require a RISC complex.

They can be used therapeutically by four mechanisms:

• Some ASOs attract the cellular enzyme RNase H, which then breaks down the targeted RNA, reducing the amount of protein produced.
• In some cases the process is mediated by siRNA, that is by RNA interference.
• ASOs can bind to pre-mRNA and alter how it is processed, causing an exon to be skipped or included, which changes the function of the protein.
• ASOs can bind to mature mRNA, physically blocking ribosomes from attaching to it.

Summarised, the main mechanisms of action of ASO and siRNA drugs are: RNA cleavage through the recruitment of endogenous enzymes; steric hindrance; splicing modulation; and activation of the RISC complex by double-stranded siRNA.

Several ASOs have been approved in the United States, the European Union and elsewhere, for Duchenne muscular dystrophy, familial hypercholesterolaemia and spinal muscular atrophy, among others.

CRISPR stands for clustered regularly interspaced short palindromic repeats, a family of DNA sequences within the genomes of prokaryotes.

These sequences are derived from DNA fragments of viruses that have previously infected the prokaryote, and they are used to detect and destroy DNA from similar viruses during subsequent infections. They therefore play a key role in the antiviral defence system of prokaryotes - a prokaryotic immune memory, written in DNA.

CRISPR sequences are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.

How the natural system works:

1. CRISPRs are composed of short DNA repeats and spacers.
2. When a previously unseen virus infects a bacterium, a new spacer derived from that virus is incorporated among the existing spacers.
3. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules.
4. The CRISPR RNA associates with and guides bacterial molecular machinery to a matching target sequence in the invading virus.
5. The machinery cuts up and destroys the invading viral genome, using the enzyme Cas9 to cut the DNA apart and disable the virus.

CRISPR-Cas9 was adapted to modify eukaryotic genomes.

Researchers create a small piece of RNA with a short guide sequence that binds to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA recognises the DNA sequence and the Cas9 enzyme most often cuts the DNA at the targeted location. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to replace an existing segment with a customised DNA sequence.

More precisely: a guide RNA hybridises to a 20-nucleotide DNA sequence immediately preceding the protospacer-associated motif (PAM), resulting in a double-strand break. The double-stranded breaks become substrates for the endogenous repair machinery, which catalyses non-homologous end joining (NHEJ) or homology-directed repair (HDR).

Uses. CRISPR technology can be used to inactivate genes in human cells, to change insect genomes so that they cannot transmit diseases, and to modify plant genomes.

Genome editing is of great interest in the prevention and treatment of human disease. Most current research is done to understand diseases using cells and animal models, and scientists are still working to determine whether the approach is safe and effective for use in people. It is being explored for single-gene disorders such as cystic fibrosis, haemophilia and sickle cell anaemia, and holds promise for more complex diseases such as cancer, heart disease and mental disorders.

In July 2019, doctors in Mississippi used CRISPR to treat a patient experimentally: a 34-year-old woman with sickle cell anaemia.

• Three experiments established that DNA is the hereditary material: Griffith (transformation exists), Avery, MacLeod and McCarty (the transforming substance is DNA), Hershey and Chase (only the phage DNA enters the cell).
• Chargaff's rules (%A = %T, %G = %C) plus Franklin's X-ray photograph gave Watson and Crick the model.
• A nucleotide is base + pentose + phosphate; the chain is joined 5' phosphate to 3' hydroxyl by a phosphodiester bond. The phosphates are why nucleic acids are acids.
• A-T is 2 hydrogen bonds, G-C is 3, and the strands are antiparallel. Origins of replication and transcription are AT-rich for that reason.
• Three stabilising forces: hydrogen bonding, base stacking (the largest contribution, because it excludes water), and ionic interactions. Stacking is what makes the molecule a helix.
• B DNA is the normal form (right-handed, 2 nm, ~10 bp per turn, major and minor grooves). A DNA appears when dehydrated and is the natural form of RNA duplexes and DNA-RNA hybrids, because ribose's 2' OH forbids B. Z DNA is a zig-zag over CGCGCG near transcription starts.
• Negative supercoiling helps the strands separate; positive supercoiling hinders it. Topoisomerases relax both.
• Sense and antisense are defined relative to one transcript, not to the whole strand.
• RNA is single-stranded with stem-loops; some RNAs are ribozymes - the peptidyl transferase of the large subunit and RNase P are the two to name.
• mRNA has a 5' UTR with the ribosome recognition site, a coding region between start and stop codons, and a 3' UTR that governs lifespan.
• tRNA: 4 loops, cloverleaf secondary, L-shaped tertiary, amino acid on the 3' end, anticodon in a large loop.
• Xist is the long non-coding RNA that coats and silences one X chromosome.
• siRNA is one target and cleaves; miRNA has many targets, binds partially, and usually represses. Both act through RISC.
• ASOs need no RISC. CRISPR is a bacterial immune memory repurposed as a genome editing tool, cutting at a site defined by a guide RNA and a PAM.