1 RNA structure basics (lecture 1)
1.1 Building block of RNA
RNA vs DNA
- RNA has Uracil as nucleotide, DNA has Thymine
- DNA lacks the 2’ hydroxyl group (ribose vs deoxyribose)
- RNA is single stranded
- RNA has non-standard nt
- RNA is unstable under alkaline conditions
- In RNA the A-form helix is most abundant
Building blocks of RNA: bases
4 bases found in RNA structure: adenine, guanine (purines) & uracil, cytosine (pyrimidines)
Building blocks of RNA: non-standard nucleotides
RNA has non-standard nucleotides such as tRNA, rRNA or residues such as inosine (structure similar
to adenosine but not identical), pseudo-uridine (structural the same to uridine, but differs in linkage;
found in t-RNA and r-RNA; linked through nitrogen instead of carbon), t-dihydrouridine.
These differences aren’t restricted to the bases, it can also be the 2’ hydroxyl that for e.g. can
be methylated.
1.2 Primary structure of RNA
RNA is synthesized from 5’ to 3’ using DNA as a template (in most cases) the nucleotides are then
added to each other using an RNA polymerase.
The single RNA strand tends to assume a right-handed helical conformation dominated by base-
stacking interactions (pi-stacking) Right handed vs left handed helix structure trick; go along the
helix with your thumb and the direction your thumb goes is the orientation. Or you can pretend it is a
staircase and see where you need to hold the ‘leuning’.
Hydrolysis of RNA
RNases are always around, so if you study RNA you have to prevent the hydrolysis by RNase. RNases
are everywhere, even on your hands.
- RNA is unstable under alkaline conditions (base-catalyzed hydrolysis of RNA)
- Hydrolysis is also catalyzed by enzymes (ribonuclease; RNase)
- RNase enzymes are abundant around us:
o S-RNase in plants prevents inbreeding
o RNase P is a ribozyme (enzyme made of RNA) that processes tRNA precursors
o Dicer is an enzyme that cleaves dsRNA into oligonucleotides
Protection from viral genomes
RNA interference
o RNA exosome is a ubiquitois complex of 3’-5’ exoribonucleases
RNA is unstable under alkaline conditions (DNA isn’t), this due to that bases can easily deprotonate
the hydrogen from the hydroxyl group on the 2’-carbon atom. This deprotonation causes the oxygen
to become negatively charged leading to a nucleophilic attack on the adjacent phosphate atom
1
,leading to the cleavage of the phosphopentose backbone of RNA. The resultant 2',3'-cyclic phosphate
is further hydrolyzed to 2' or to 3' phosphate leaving RNA fragments or free ribonucleotides with 5’-
OH and 3’-phosphates, depending on the level of degradation. Conversely the lack of a 2’-OH in DNA
prevents cleavage of the phosphate backbone making DNA relatively stable at high pH.
1.3 Secondary and tertiary structure of RNA
Secondary structures of RNA
- Hairpin: nr of nt in the loop are not more than 5-6
- Stem-loop: a bigger hairpin; two-stem, three-stem, four-stem (junctions)
- Bulge: single nucleotide vs. multiple nucleotide bulge
- Internal loops (a bulge where there are in both strands unpaired residues)
o Asymmetric: unequal number of non-base paired residues in both strands
o vs. symmetric: equal number of ‘’.
Base pairing leads to double-stranded helices; in RNA the A-form helix is most abundant:
- A-form helix is wider in diameter.
- Angle of the base pair with the axis is smaller within the B-form
Major groove vs. minor groove:
- Accessibility bases is better in minor groove.
Secondary structure of mRNA’s: more RNA secondary structure in untranslated regions (UTR) of
mRNAs. interaction with regulator protein are related to the fact that these are found here more.
Phylogenetic conservation of base-pairing:
To what extent are molecules conserved during evolution? There are multiple factors that determine
conservation. Secondary structure determines sequence or structural conservation bc base pairs
have to be formed. The RNA secondary structure present in RNase P RNA is very similar in different
organisms; some residues are fully conserved, some in a less percentage. by looking at sequence
conservation in many species can also support information or the presence of secondary structure
elements in a particular RNA molecule.
Unusual base pairing:
In RNA sometimes there can also be unusual base pairs (non-watsoncrick). The hydrogen bond
doesn’t match so it not adequate. But the forming of the hydrogen bond can happen.
Watson-Crick = usual Wobble:
- G-C - G-U
- A-U
Can also occur in RNA molecules but unusual: A-A, G-G, A-G, C-C, C-U, U-U
2
,5 most common noncanonical base-pars in RNA:
- scheared GA (bp on the 6-ring and 5-ring)
- GA imino (bp on the 6-ring)
- AU reverse Hoogsteen (not Watson-crick bp)
- GU wobble
- AC wobble (only when adenine is protonated at N1)
Unusual are generally less stable and mostly happen at secondary structure borders. = tertiary
structures
Tertiary structure
From secondary to tertiary structure in RNA
Formation of double strands will be accompanied by electrostatic repulsion due to the negative
charges in the strands between the phosphates. There will also be positive contribution due to the
base paring interaction. You can compensate the electrostatic repulsion with the presence of cations,
this will stabilize the strand.
Tertiary structures:
Strategies used for assembling RNA tertiary structures
- Long-range base-pairing
- Coaxial-stacking
o Short helices are often stacked on top of each other
Kissing-loop & pseudoknot also do this
- Hydrogen bonding
o Base triples (hoogsteen base-pairing = AU reversed can also interact with an extra A)
o Ribose zippers (involvement of ribose 2’ -OH groups in hydrogen bonding)
- Metal ions
o Positively charged metal ions help negatively charged RNA strands to associate into
helices and also to bind to specific sites within RNA tertiary structures to hold RNA
helices together. Potassium is the most present monovalent ion in RNA because
potassium concentrations are higher inside cell vs. higher concentrations of sodium
outside cells
- RNA G quadruplex structures, very specific structures in RNA
o repeated stretches of guanine-rich sequences can form quadruple base pairs that
stack on each other to form quadruplexes stabilized by potassium ions forming
the structures (purple)
- Ribose zippers
o 2’-oh groups can act as a H-bond donor and as acceptor, it is a stabilizing component
in many tertiary interactions, since it can make two hydrogen bonds.
Surface of an RNA molecule is much more complex than that of a DNA molecule, this explains why
the functions of RNAs can be quite different ex. Enzymes, etc.
3
, 2 RNA methodologies (lecture 2)
2.1 Why measure RNA?
Research: Clinic:
- As a proxy of protein - Therapeutics with RNA as a marker
- Fundamental science: understand - Biomarker
gene regulation - Virus detection
- To determine cell identity (scRNA-Seq)
2.2 RNA analysis
RNA isolation and quality check Other analysis of individual genes
RNA isolation - Sequence analysis (cDNA) of individual
genes
- Total RNA
- Localization of individual RNAs in the
- RNA subclass
cell
- RNA tagging
RNA structure determination
Agarose/polyacrylamide gel electrophoresis
- Enzymatic and chemical structure
- Ethidium bromide; SYBR green
probing
Gene quantification / 5’ end determination - FRET (Froster/fluorescence resonance
individual genes energy transfer)
- X-ray crystallography and NMR
- RT-PCR
- Northern blot hybridization Large scale determination and quantification
- Nuclease protection analysis of RNA molecules
- Primer extension
- RNA-seq
- RNA-seq for alternative usage
- scRNA-seq
Ethidium bromide & SYBR green are nonspecific fluorescent dyes used for detection of dsRNA. They
bind to dsRNA because they contain a lot of aromatic rings, they are very flat , so these molecules
can easily intercalate between the ds bases. The intensity of the fluorescens indicates the proxy for
the visualisation of ds regions. (Intercalation is the reversible inclusion or insertion of a molecule (or
ion) into materials with layered structures.)
2.3 RNA isolation and quality check
1. Difficulty in RNA isolation: most ribonucleases are very stable and there are active enzymes
that require no cofactors to function first step isolation: lysis of the cell in chemical
environment which results in denaturation of ribonuclease.
2. RNA is fractionated from the other cellular macromolecules under conditions that limit or
eliminate any residual RNase activity.
The cell type from which RNA is to be isolated and the eventual use determines which procedure
is appropriate. ! always watch out for contamination (containing RNase)
The eventual use of the RNA determines whether total RNA is suitable or a subclass of RNAs or a
specific RNA is required. Total RNA can be fractionated based upon subclass-specific features (e.g.
poly(A) tail for mRNAs or a specific molecular feature introduced by RNA tagging).
Most mRNA contain a poly(A) tail, while structural RNAs don’t enriched poly(A) section mRNA
4
1.1 Building block of RNA
RNA vs DNA
- RNA has Uracil as nucleotide, DNA has Thymine
- DNA lacks the 2’ hydroxyl group (ribose vs deoxyribose)
- RNA is single stranded
- RNA has non-standard nt
- RNA is unstable under alkaline conditions
- In RNA the A-form helix is most abundant
Building blocks of RNA: bases
4 bases found in RNA structure: adenine, guanine (purines) & uracil, cytosine (pyrimidines)
Building blocks of RNA: non-standard nucleotides
RNA has non-standard nucleotides such as tRNA, rRNA or residues such as inosine (structure similar
to adenosine but not identical), pseudo-uridine (structural the same to uridine, but differs in linkage;
found in t-RNA and r-RNA; linked through nitrogen instead of carbon), t-dihydrouridine.
These differences aren’t restricted to the bases, it can also be the 2’ hydroxyl that for e.g. can
be methylated.
1.2 Primary structure of RNA
RNA is synthesized from 5’ to 3’ using DNA as a template (in most cases) the nucleotides are then
added to each other using an RNA polymerase.
The single RNA strand tends to assume a right-handed helical conformation dominated by base-
stacking interactions (pi-stacking) Right handed vs left handed helix structure trick; go along the
helix with your thumb and the direction your thumb goes is the orientation. Or you can pretend it is a
staircase and see where you need to hold the ‘leuning’.
Hydrolysis of RNA
RNases are always around, so if you study RNA you have to prevent the hydrolysis by RNase. RNases
are everywhere, even on your hands.
- RNA is unstable under alkaline conditions (base-catalyzed hydrolysis of RNA)
- Hydrolysis is also catalyzed by enzymes (ribonuclease; RNase)
- RNase enzymes are abundant around us:
o S-RNase in plants prevents inbreeding
o RNase P is a ribozyme (enzyme made of RNA) that processes tRNA precursors
o Dicer is an enzyme that cleaves dsRNA into oligonucleotides
Protection from viral genomes
RNA interference
o RNA exosome is a ubiquitois complex of 3’-5’ exoribonucleases
RNA is unstable under alkaline conditions (DNA isn’t), this due to that bases can easily deprotonate
the hydrogen from the hydroxyl group on the 2’-carbon atom. This deprotonation causes the oxygen
to become negatively charged leading to a nucleophilic attack on the adjacent phosphate atom
1
,leading to the cleavage of the phosphopentose backbone of RNA. The resultant 2',3'-cyclic phosphate
is further hydrolyzed to 2' or to 3' phosphate leaving RNA fragments or free ribonucleotides with 5’-
OH and 3’-phosphates, depending on the level of degradation. Conversely the lack of a 2’-OH in DNA
prevents cleavage of the phosphate backbone making DNA relatively stable at high pH.
1.3 Secondary and tertiary structure of RNA
Secondary structures of RNA
- Hairpin: nr of nt in the loop are not more than 5-6
- Stem-loop: a bigger hairpin; two-stem, three-stem, four-stem (junctions)
- Bulge: single nucleotide vs. multiple nucleotide bulge
- Internal loops (a bulge where there are in both strands unpaired residues)
o Asymmetric: unequal number of non-base paired residues in both strands
o vs. symmetric: equal number of ‘’.
Base pairing leads to double-stranded helices; in RNA the A-form helix is most abundant:
- A-form helix is wider in diameter.
- Angle of the base pair with the axis is smaller within the B-form
Major groove vs. minor groove:
- Accessibility bases is better in minor groove.
Secondary structure of mRNA’s: more RNA secondary structure in untranslated regions (UTR) of
mRNAs. interaction with regulator protein are related to the fact that these are found here more.
Phylogenetic conservation of base-pairing:
To what extent are molecules conserved during evolution? There are multiple factors that determine
conservation. Secondary structure determines sequence or structural conservation bc base pairs
have to be formed. The RNA secondary structure present in RNase P RNA is very similar in different
organisms; some residues are fully conserved, some in a less percentage. by looking at sequence
conservation in many species can also support information or the presence of secondary structure
elements in a particular RNA molecule.
Unusual base pairing:
In RNA sometimes there can also be unusual base pairs (non-watsoncrick). The hydrogen bond
doesn’t match so it not adequate. But the forming of the hydrogen bond can happen.
Watson-Crick = usual Wobble:
- G-C - G-U
- A-U
Can also occur in RNA molecules but unusual: A-A, G-G, A-G, C-C, C-U, U-U
2
,5 most common noncanonical base-pars in RNA:
- scheared GA (bp on the 6-ring and 5-ring)
- GA imino (bp on the 6-ring)
- AU reverse Hoogsteen (not Watson-crick bp)
- GU wobble
- AC wobble (only when adenine is protonated at N1)
Unusual are generally less stable and mostly happen at secondary structure borders. = tertiary
structures
Tertiary structure
From secondary to tertiary structure in RNA
Formation of double strands will be accompanied by electrostatic repulsion due to the negative
charges in the strands between the phosphates. There will also be positive contribution due to the
base paring interaction. You can compensate the electrostatic repulsion with the presence of cations,
this will stabilize the strand.
Tertiary structures:
Strategies used for assembling RNA tertiary structures
- Long-range base-pairing
- Coaxial-stacking
o Short helices are often stacked on top of each other
Kissing-loop & pseudoknot also do this
- Hydrogen bonding
o Base triples (hoogsteen base-pairing = AU reversed can also interact with an extra A)
o Ribose zippers (involvement of ribose 2’ -OH groups in hydrogen bonding)
- Metal ions
o Positively charged metal ions help negatively charged RNA strands to associate into
helices and also to bind to specific sites within RNA tertiary structures to hold RNA
helices together. Potassium is the most present monovalent ion in RNA because
potassium concentrations are higher inside cell vs. higher concentrations of sodium
outside cells
- RNA G quadruplex structures, very specific structures in RNA
o repeated stretches of guanine-rich sequences can form quadruple base pairs that
stack on each other to form quadruplexes stabilized by potassium ions forming
the structures (purple)
- Ribose zippers
o 2’-oh groups can act as a H-bond donor and as acceptor, it is a stabilizing component
in many tertiary interactions, since it can make two hydrogen bonds.
Surface of an RNA molecule is much more complex than that of a DNA molecule, this explains why
the functions of RNAs can be quite different ex. Enzymes, etc.
3
, 2 RNA methodologies (lecture 2)
2.1 Why measure RNA?
Research: Clinic:
- As a proxy of protein - Therapeutics with RNA as a marker
- Fundamental science: understand - Biomarker
gene regulation - Virus detection
- To determine cell identity (scRNA-Seq)
2.2 RNA analysis
RNA isolation and quality check Other analysis of individual genes
RNA isolation - Sequence analysis (cDNA) of individual
genes
- Total RNA
- Localization of individual RNAs in the
- RNA subclass
cell
- RNA tagging
RNA structure determination
Agarose/polyacrylamide gel electrophoresis
- Enzymatic and chemical structure
- Ethidium bromide; SYBR green
probing
Gene quantification / 5’ end determination - FRET (Froster/fluorescence resonance
individual genes energy transfer)
- X-ray crystallography and NMR
- RT-PCR
- Northern blot hybridization Large scale determination and quantification
- Nuclease protection analysis of RNA molecules
- Primer extension
- RNA-seq
- RNA-seq for alternative usage
- scRNA-seq
Ethidium bromide & SYBR green are nonspecific fluorescent dyes used for detection of dsRNA. They
bind to dsRNA because they contain a lot of aromatic rings, they are very flat , so these molecules
can easily intercalate between the ds bases. The intensity of the fluorescens indicates the proxy for
the visualisation of ds regions. (Intercalation is the reversible inclusion or insertion of a molecule (or
ion) into materials with layered structures.)
2.3 RNA isolation and quality check
1. Difficulty in RNA isolation: most ribonucleases are very stable and there are active enzymes
that require no cofactors to function first step isolation: lysis of the cell in chemical
environment which results in denaturation of ribonuclease.
2. RNA is fractionated from the other cellular macromolecules under conditions that limit or
eliminate any residual RNase activity.
The cell type from which RNA is to be isolated and the eventual use determines which procedure
is appropriate. ! always watch out for contamination (containing RNase)
The eventual use of the RNA determines whether total RNA is suitable or a subclass of RNAs or a
specific RNA is required. Total RNA can be fractionated based upon subclass-specific features (e.g.
poly(A) tail for mRNAs or a specific molecular feature introduced by RNA tagging).
Most mRNA contain a poly(A) tail, while structural RNAs don’t enriched poly(A) section mRNA
4
