Cardset

1. The ability to carry information

2. The ability toaccurately replicate itself

Genotype and phenotype

• Bind small metabolites

• Act as ribozymes

• RNA splicing reactions

• Ribosome function

• Spliceosome function

• Telomerase function

• DNA regulation

Lack of enzymes (Chicken and Egg)

1. RNA forms ribose

2. RNA learns to copy itself

3. RNA synthesises proteins that act as catalysts

4. RNA and proteins replicate and synthesise proteins more efficiently and make ds RNA, later DNA

5. DNA takes over

Primordial world: RNA -> RNA

Classical picture: DNA -> RNA -> Protein

Modern picture (in eukaryotes): DNA -> RNA -> Protein with ncRNA as transcriptional regulator between DNA and RNA

No, not without any doubts

However, it is very likely

DNA is genotype and primary structure

RNA is both

Protein is phenotype and secondary/tertiary structure

Purines:

• Adenine + Guanine

• Two circuits

Pyrimidines:

• Cytosine + Thymine/Uracil

• One circuit

DNA: C2-endo (S-shaped)

RNA: C3-endo (N-shaped)

only Z-DNA is syn, all relevant ones are anti

B form:

• more elongated

• wide major groove

A form:

• shorter

• deep, narrow major groove

A- -T or C- - -G

via H bonds

• all combinations of A-G

• all combinations of C-U

• random coil (polar/ vdW)

• base stacking (delocalised pi-electron)

• base pairing (still have stacking interactions, further stabilisation)

• A-form helix (shorter, thicker than B-form)

• sugar in C3-endo

• right-handed, anti-parallel

a: hairpin loop

b: internal loop

c: bulge

d: junction

e: stem

f: free end

g: joint

molecular signature

• interactions between helices

• interactions between helices and unpaired elements

• interactions between unpaired sequences

• coaxial stacking of helices

• RNA cleaves target, usually another stem, from the ribozyme

• possible because stacking -> more stability

bakbones contact between two parallel helices

Three nucleotides bind in a three-way situation

• Metal ions stabilize the structure

• Allows for tight packing

loop-loop interaction between two stem loops

stabilized by h-bonds and base stacking

one RNA sequence forms a “knot”

stabilized by h-bonds and base stacking

4 x 4 Gs, often at the end of telomers

4 stacked tetraeders with a metal ion in the middle

protein recognition motif

very stable

Secondary: clover leaf with 4 stems, 3 hairpin loops

Tertiary: L-shape with coaxial stacking

stabilize the lowest free energy structure

• First, it gets faster, then slower again and last one is slowest

• due to free ends folding fast and stacking slower

describes mfe needed to go into better structure, “cloud” form

RNA intrinsic interactions:

• polar, vdW interactions

• h-bonds, delocalized pi-electrons

Interactions between RNA and environment:

• water

• cations

• interactions with proteins, DNA, other RNA

slowest step in folding

1. conformational search

2. forming metal ion sites

3. kinetic trap

1. hairpin ribozyme

2. tetrahymena ribozyme

3. ribosome

• experimentally (eg. Probing, Cross-linking, NMR, Probing with antisense ODN)

• computationally (secondary, tertiary structures)

• expose RNA to chemical in modification step

• then cleavage

1. Folding is determined by thermodynamics alone

2. Sequence dependence of thermodynamics was completely understood

   Goal: structure with min. mfe

   Solution: dynamic programming

1. RNA will basepair into the most negative free energy state

2. Various interactions are additive

   Flaws: not always true, condition dependent!

Tools

• mfold

• RNAfold

Displays

• Structure drawings

• Dot-bracket schemes

• alternative folds

• fold probability

Yes

• considers representative of Boltzmann ensemble of structures for given sequence

• more meaningful than mfe

much more difficult, needs 2d structure information

…explored with increasing success using experimental and computational methods

1. naturally occurring vs artificial

2. Complementary vs non-complementary

3. RNA-only based functions vs functions in concert with proteins

4. Catalytic vs non-catalytic

5. Protein-coding vs non-coding

• only few RNA functions are exclusively depending on complementary

• most important ones: antisense RNAs

sense + antisense hybridise and form a duplex

no proteins needed but sometimes can facilitate such effect

G2, where the sequence-unspecific precomplex turns into a sequence-specific complex

1. Specificity for human genome already given

2. Thermodynamically stable

3. Most secondary and tertiary structures can be realised with rather short sequences

4. More agile

1. Control of plasmid replication

2. Control of bacteriophage development

3. Regulation of transpositions

4. Inhibition of bacterial gene expression

Extremely fast binding

No, as they depend on sophisticated ss and ts

• either antisense from same gene locus

• or distinct gene locus

• A to I RNA editing

• Duplex destabilization

• Changes in coding sequences

• Nucleus

• Cytoplasm

• kissing complex formation

• prokaryotes and eukaryotes

• Via formation of dsRNA blocking translation

• triggers various downstream effects

fast-hybridising asRNAs

• small ribozymes of microbial origin

• can cleave and ligate nucleic acids in cis or trans

1. hairpin ribozyme

2. hammerhead ribozyme

3. HDV ribozyme

4. varkud satellite

5. GlmS ribozyme

• cis: target gets cleaved at the end of sequence

• trans: target gets cleaved in the middle of sequence

• single trans cleaving ribozyme can gradually bind and cleave multiple targets, exhibiting catalytic turnover

• targets cannot be translated anymore

because one ribozyme can cleave multiple mRNAs

nucleolytic ribozyme, of microbial origin

have to be active under sub-milimolar Mg2+ concentrattions

1. delivery into target cells in vivo

2. short half-life of RNA

3. careful design

• PCR

• sequencing

from the poly A RNA fraction

• one to multiple exons

• usually found in cytoplasm and extracellular vesicles

• poly A negative

• mainly exonic

• exonuclease-resistant

• varying sizes

• highly abundant

• evolutionary conserved

• found in extrcellular vesicles and cytoplasm

• probably generated via backsplicing

• found in extrcellular vesicles

• associated with human diseases

• explored as biomarker and drug

• most of our genome is transcribed

• most transcripts are non-coding

• evolution of RNA is ongoing

• asRNA: common in prokaryotes but meaning in eurkaryotes unclear

• ribozymes: much more common than initially thought, clease RNA substrates

• circRNA: highly abundant in cytoplasm, meaning and functions widely unknown, interesting as biomarkers

bacteria and archaea

• bacteria, archaea, eukaryotes

• common factor: RNA-based immune system

transcription forms hairpin

exact sequences split by different spacers

• bacteriophage binds to bacterial cell wall, injects his DNA

• expression of Cas proteins and CRISPR DNA

• crRNA/Cas complex binds the bacteriophage DNA adn destroys it

• infection is stopped before it starts

• (also works with no matching spacer, as a new one is inserted based on the invading DNA)

• -> CRISPR = adaptive immune system, unlike exo-siRNA RNAi

Just multiply, eg. 1/16 is equal to every 16 nucleotides

1. Cas9 screens for PAM presence

2. crRNA screens for target sequence match

point-mutations in CRISPR spacer sequences or PAM motifs

• a cis-regulatory segment of mRNA that binds small molecule resulting in a change in protein synthesis encoded by mRNA

• typically occur in 5’ untranslated region of bacterial mRNA, but also in plants and funghi

• allow them to react to environmental changes

• based on aptamer/expression function

• transcription control

• translation control

• splicing control

• no riboswitch as no aptamer

• temperative-sensitive ncRNA which regulates gene expression

• encodes heat shock proteins

• high surface-to-volume ratio in bacteria

• bacteria doesnt regulate their temperature and habve no other way to sense and react to temperature changes

• bacteria and archaea: CRISPR targeting DNA or RNA

• eukarya: RNAi targeting RNA

by complementary nucleid acids

Type 2

turn off (seldom turn on)

• cleave target RNA

• bind small molecules

Ribozyme

Formation of sequence-specific nucletion complex

• catalytic core

• target binding domains

increase it

• RNA-guided defence

• miRNA-guided post transcriptional regulation of gene expression

RNA-guided adaptive immune control of transposable elements in the germline

• short ncRNA

• length of 22nt

• posttranscriptional regulator

mature miRNA

• start at polymerase 2 promotors

• pri-miRNA, monocistronic, polycistronic

• includes 5-7-methyl-guanosine cap structure and 3-poly A tail

  
  

pri-miRNAs can harbor several mature miRNAs

• in nucleus: pri-miRNA with drosha, pasha combined to pre-miRNA

• in cytosol: dicer makes miRNA duplex from pre-miRNA, then miRNA

• miR very short, drosha recognizes structure, not sequence

• drosha as protein complex recognizes stem loops and cuts stem in distinct distance to the loop

• export mediated by exportin 5

• GTPase dependent

• TR: imperfect miRNA, one effector blocks 1 mRNA target

• TC: perfect miRNA, one effector cuts multiple mRNA targets

• only when mature miRNA is fully complimentary to target in Ago 2 RISC

• dominant translational block in animals

• target cleavage in plants

structure of effector duplex

• by the seed region, meaning positions 2-8 from 5’ end of mature miRNA

• problem: seeds are very short and match lots of times

• in human, guide RNA 5’ end

• in th. th. guide RNA 3’ end released differently

unilateral and double-negative feedback loop

• 3’ end modifications

• RBP facilitates miRISC binding

• strengething interactions between miRISC and downstream effectors

• counteracting miRISC function

• other proteins interfering with miRNAs

through:

• histone modification

• DNA methylation

• one mRNA can be regulated by several miRNAs

• one miRNA can regulate several mRNAs

  (n zu 1 und 1 zu n)

• within mRNA 3’ UTR

• allows precise and differentiated gene regulation

• forms RNA-protein complex with piwi protein

• found in nuclei and cytoplasm

• silences transposons and other stuff in germ line cells

• distinct biogenesis from miRNA and siRNA

• 5’ uridine and 3’ terminal 2’-O methylation

• transcription from piRNA clusters

• no dicer or drosha

• ping-pong cycle amplification

• secondary piRNAs can be reimported into nucleus

• small interfering RNA

• 15 to 25 nt RNA duplexes

• can be exo or endo, either cleavages dsRNA or is derived from transposable elements

• can also be artificial

• treats hATTR by knocking down responsible gene

• intravenous

• uses siRNAs encapsulated in lipid nanoparticle

miRNA biogenesis

RNA-guided gene silencing in eukaryotes

RNA interference (RNAi)

• genome encoded

• regulate gene expression

siRNAs generated from invading viral dsRNA

• transposable elements in the germ line

• genome stability

• artificial miRNAs and siRNAs

• their precursors

• their antagonists

• upregulation

• downregulation

• mutation of regulator/regulon

• global events, linked to silencing pathways

• selective events, linked to miRNA

• overexpression (improved RISC loading)

• increased target complementarity

• miRNA gene knockouts

• anti-miR

No, as it is very moderate

difficult to detect under lab-controlled conditions

• stress signal mediation

• stress signal modulation

• negative feedback: signal resolution

• positive feedback: phenotypic switching

• buffering: signal stability

because cancer is a stress condition

• either miRNA dysregulation leads to disease

• or disease leads to miRNA dysregulation

• more stable

• signatures are less complex

Yes

• miRNA: stemloop primer-based PCR for small RNA detection

• RNA: microarrays or RNA sequencing

• shorter

• stable ss or ts

• protection by associated proteins

• other class: circRNA, as they have no 5’ or 3’ ends

stress, eg. cancer

• amplified: in genomic regions

• deleted: in chromosomes

• lncRNA

• circRNA

because it exhibits selectavle phenotypes, used in SELEX

• class of oligonucleotides for diagnostic/therapeutic use

• high affinity and specificity

• single stranded RNA or DNA

• can be used in SELEX or naturally in riboswitches

• RNA molecules that catalyte reactions, eg.:

  • RNA cleavage

  • ligation

  • phosphorylation

  • oligomerisation

• can be used for SELEX

• PCR

• transcription in vitro

• selection for target binding

• elution

• reverse transcription

• PCR

• transcription in vitro

• PCR

• strand separation

• selection for target binding

• elution

• PCR (other one than in RNA)

• strand separation

• random sequence space: sum of all different sequences at given length

• hamming distance: number of different bases between two sequences of equal length

• structure space: sum of all structures formed by the sequences of given sequence space

• 1 zu n, n zu 1

• goals: high diversity, effective amplification, effective cloning

• variables: priming sites, number of random positions, length

• DNA sequencing

• Mass spectronometry

1. amplify antisense strand and selectively bind it to column

2. denature instead of remove as strand and re-anneal

• cDNA amplification

• RNA transcription

• selection with diversified pool

• principle: species with superior fitness are most likely to be found in near neighborhood

• because all molecules are compromises and need to be active and compatible

1. SELEX with modified nucleotides during transcription/PCR

   1. Important: 2’ o methyl, 2’ amino, 2’ fluoro

2. other moieties can be linked to selected aptamer scaffolds

• selection of aptamer that binds ribozyme substrate

• selection of catalytic ribozyme core

• specific phenotype is enriched in population in range of 100-1000 fold per cycle

by a strong phenotype

• thermodynamic selection of stable-binding RNA molecules

• Keq = kon/koff

• monoLEX / single cycle aptamer SELEX (keq, kon)

• spiegelmers/ mutliple or single cycle antisense SELEX (kon)

with the cell SELEX strategy:

• selection cycle 1, then counter selection, then further selection cycles

• based on differences of target protein expression between two cell types

• biostability and biocompatibility

• extracellular transport

• organ and cell targeting

• in vitro

• ex vivo

• in vivo

• diversity of initial pool of variants

With mutagenetic PCR

• small size

• amendable

• longer shelf life and no cooling necessary

• easy large scale production

• low immunogenicity

• can be used for non-immunogenic targets

• problems: difficult for flexible molecules, expensive but SELEX much cheaper and quicker

• inhibits angiogenesis in tumors in mice

• reduces blood supply to tumors

Usually, they just bind cell surface markers, but some also get internalized by specific cells (eg. for siRNA delivery)

fast binding

kon (speed of binding)

also, length is important , as the longer, the better it binds as they have larger surfaces

• because of long free end and joints

• leads to faster target binding

• in vitro

• computationally

g quartets

tRNA, as three stemloops can be observed at the high plateaus

Option B is correct:

It occours mainly in the cytoplasm and is highly abundant

Option D is correct:

complementarity to a guide RNA and PAM

Option A is correct:

regulation of gene expression

(because piRNA combats transposon expansion, viral defence is only on lower eukaryotes, RNAi doesnt interfere witth genomic DNA)

option D is correct:

dicer and drosha (dicer in cytoplasm and drosha in nucleus processing)

option C is correct:

miRNA duplex structure

option D is correct:

RISC formation, as no drosha nor nuclear export is required. the transcripts are also not capped nor polydenylated

25 ug

Ago1 RISCs, as too few are perfectly paired for Ago2 RISC and 3 and 4 are not fully understood

structure of the effector duplex

option C is correct:

transposons

option D is correct:

one miRNA can regulate multiple genes AND

one gene can be regulated by multiple miRNAs

… has consequences under stress conditions

… the diversity of the initial pool of variants

• T7 RNA polymerase

• Reverse transcriptase

• Taq DNA polymerase

the mfe structure prediction is very reliable and alternative suboptimal folds do not exist/ are unimportant

option D is correct:

asRNA SELEX is a kinetic SELEX and is a selection for high kon

smaller size and ability to target non-immunogenic targets