By Yuqiong Xia2014

Oligonucleotides

Pharmaceutical Biotechnology

Keywords

2

Oligonucleotide, ribonucleotide, deoxy-ribonucleotides, macrophage

Transcription factor, decoy, repressor, activator Ribozyme, DNAzyme, aptamer, riboswitch

生物制药工程 夏玉琼 西安电子科技大学

Outline

3

Introduction to oligonucleotides Oligonucleotides with different functions Other Application

生物制药工程 夏玉琼 西安电子科技大学

Introduction to oligonucleotides

4

Oligonucleotides (ONs) (Short) chains of (chemically modified) ribo- or

deoxyribonucleotides

http://www.livescience.com/37247-dna.html

25-mer: 25nt

生物制药工程 夏玉琼 西安电子科技大学

Why ONs can be used as a drug

5

Specificity A sequence of 15-17 bases occurs only once in the

human genome

生物制药工程 夏玉琼 西安电子科技大学

Why ONs can be used as a drug

6

Activity Bind to transcriptional factors (TFs) and interfere with

the function of biomolecules Bind to specific receptors of cells, which will trigger

therapeutic immune response

https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQhAU5jNJdWHAJJHcdHvpmQRIr8vnT_j9bZNE21l8QfvUfe7sjrWw

生物制药工程 夏玉琼 西安电子科技大学

Limitations of Ons 1/2

7

Off-target effects Partial sequence homology may affect expression

other than targeted gene

生物制药工程 夏玉琼 西安电子科技大学

Limitations of ONs 2/2

8

Short circulation time Rapid uptake by macrophages and excretion by

kidneys ONs are sensitive to nucleases

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/aviruses/macrophage-antg2656.JPGhttp://www.sigmaaldrich.com/content/dam/sigma-aldrich/articles/life-science-innovations/issue25/tarfigure1.gif

生物制药工程 夏玉琼 西安电子科技大学

9

The structure of ONs： 1st generation

PS-ONs, more stable in serum Longer circulation half life 40-60

h (DNA < 10 min) More binding to proteins, being

toxic

硫代磷酸 DNA

生物制药工程 夏玉琼 西安电子科技大学

10

The structure of ONs： 2nd generation

Less toxic than PS-ONs Long circulation half life (30 days) But poor substrate for RNase H

生物制药工程 夏玉琼 西安电子科技大学

11

The structure of ONs： 3rd generation

Superior stability and RNA binding properties lack RNase H activationBut poor substrate for RNase H

生物制药工程 夏玉琼 西安电子科技大学

Outline

12

Introduction to oligonucleotides Oligonucleotides with different functions Triple helix-forming oligonucleotides (TFO) Transcription factor decoys Ribozyme/DNAzyme Antisense RNA/miRNA/siRNA Aptamer/riboswitch

Other Application

生物制药工程 夏玉琼 西安电子科技大学

13

Triple helix-forming oligonucleotides (TFO)生物制药工程 夏玉琼 西安电子科技大学

Triple helix-forming oligonucleotides (TFO)

14

TFO binds to polypurine strands in the major groove of dsDNA

Prevent transcription initiation or elongation by binding promoter, gene or regulatory DNA regions

生物制药工程 夏玉琼 西安电子科技大学

Limitations and solutions of TFO

15

Limitations Difficult to form stable triple helix, due to electrostatic

repulsion

Solutions Use PNA (peptide nucleic acid) instead of DNA or

RNA

生物制药工程 夏玉琼 西安电子科技大学

Transcription factor

16

Transcription factor is a protein that binds to specific DNA sequences, thereby controlling the transcription of DNA to mRNA

Transcription factors can promote (as an activator), or block (as a repressor) the recruitment of RNA polymerase to specific genes.

生物制药工程 夏玉琼 西安电子科技大学

17

Transcription factor decoys 生物制药工程 夏玉琼 西安电子科技大学

Ribozyme

18

Discovered in 1982 Ribozyme can catalyze specific biochemical reactions,

similar to protein enzymes

http://en.wikipedia.org/wiki/File:Ribozyme.jpg

生物制药工程 夏玉琼 西安电子科技大学

DNAzyme

19

Discovered in 1994 DNAzymes are DNA

molecules that can perform a chemical reaction, such as catalytic action

DNAzymes probably exist only in the laboratory

http://onlinelibrary.wiley.com/doi/10.1002/cbic.v11:7/issuetochttp://en.wikipedia.org/wiki/Deoxyribozyme

生物制药工程 夏玉琼 西安电子科技大学

Antisense RNA

20

Definition A single-stranded RNA that is complementary to a

mRNA

Application Use transgenic plants to express antisense RNA,

leading to gene silencing

Limitation Lack effective design, biological activity, and efficient

route of administration

http://en.wikipedia.org/wiki/Antisense_RNA

生物制药工程 夏玉琼 西安电子科技大学

Antisense RNA

21 http://biowiki.ucdavis.edu/@api/deki/files/46/07_antisense.GIF?revision=1

Antisense RNA

生物制药工程 夏玉琼 西安电子科技大学

siRNA

22

Small interfering RNA (siRNA) is a class of ds RNA, 20-25 base pairs in length

Interfere with the expression of specific genes with complementary nucleotide sequences.

siRNA functions by causing mRNA to be broken down after transcription, resulting in no translation

http://en.wikipedia.org/wiki/SiRNA

生物制药工程 夏玉琼 西安电子科技大学

siRNA

23

siRNA can be produced from dsRNA or enter cells by transfection

http://upload.wikimedia.org/wikipedia/commons/6/6c/RNAi.jpg

20-25 bp

Dicer enzyme

RISC

生物制药工程 夏玉琼 西安电子科技大学

miRNA (definition)

24

Discovered in 1990s A microRNA (abbreviated miRNA) is a small non-

coding RNA molecule (22 nt) found in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression.

http://en.wikipedia.org/wiki/MiRNA

生物制药工程 夏玉琼 西安电子科技大学

How does miRNA function

25

miRNAs function via base-pairing with complementary sequences within mRNA molecules.

As a result, these mRNA strands are silenced.

生物制药工程 夏玉琼 西安电子科技大学

26

How does miRNA function

http://www.nature.com/nbt/journal/v25/n6/fig_tab/nbt0607-631_F2.html

~22 bpmiRNA

生物制药工程 夏玉琼 西安电子科技大学

27

Antisense/Ribozymes/siRNA

停顿

生物制药工程 夏玉琼 西安电子科技大学

Aptamers (适体)

28

Aptamers (from the Latin aptus - fit, and Greek meros - part) are oligonucleic acid or peptide molecules that bind to a specific target molecule.

Classification Nucleic acid aptamers Peptide aptamers

http://en.wikipedia.org/wiki/Aptamer

生物制药工程 夏玉琼 西安电子科技大学

Nucleic acid aptamers

29

Single-stranded DNA or RNA (60 nt)， 3D structure Bind to various molecular targets such as small

molecules, proteins, nucleic acids, and even cells, tissues and organisms.

Offer molecular recognition properties that rival that of antibodies.

http://en.wikipedia.org/wiki/Aptamer

生物制药工程 夏玉琼 西安电子科技大学

30

SELEX-based selection of aptamers生物制药工程 夏玉琼 西安电子科技大学

Riboswitches （核糖开关）

31

Single-stranded DNA or RNA (60 nt)， 3D structure

A riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA

Mostly occur in bacteria, but functional riboswitches of one type (the TPP riboswitch) have been discovered in plants and certain fungi.

http://en.wikipedia.org/wiki/Riboswitches

生物制药工程 夏玉琼 西安电子科技大学

Summary of ONs 1/2

32

Triple helix-forming oligonucleotides (TFO) Form triple-helix and interfere gene expression

Transcription factor decoys Catch transcription factor and interfere gene expression

Ribozymes / DNAzyme Bind to mRNA and cut mRNA molecules

Antisense RNA Bind to mRNA, leading to gene silencing

siRNA/miRNA cut mRNA molecules, form RISC, and cut mRNA

All of the above ONs interfere with gene expression

生物制药工程 夏玉琼 西安电子科技大学

Summary of ONs 2/2

33

Atpamer/riboswitch Bind to small molecules, generally non-nucleotide

molecules

生物制药工程 夏玉琼 西安电子科技大学

Outline

34

Introduction to oligonucleotides Oligonucleotides with different functions Other Application Gene repair Stimulate immune response

生物制药工程 夏玉琼 西安电子科技大学

Triplex helix-forming oligonucleotides

35

Used for site-directed mutagenesis Used for homologous-site-specific recombination

生物制药工程 夏玉琼 西安电子科技大学

Antisense-induced exon skipping

36

Artificially remove one or more exons

http://www.discoverymedicine.com/Qi-L-Lu/files/2009/08/lu_19_fig1.jpg

生物制药工程 夏玉琼 西安电子科技大学

Antisense-induced ribonucleoprotein inhibition

37

Antisense ONs inhibit the functions of ribonucleoproteins by binding to the RNA part of the ribonucleoprotein

生物制药工程 夏玉琼 西安电子科技大学

Outline

38

Introduction to oligonucleotides Oligonucleotides with different functions Other Application Gene repair Stimulate immune response

生物制药工程 夏玉琼 西安电子科技大学

Stimulate immune response

39

Foundation Specific receptors exist that recognize pathogenic

DNA or RNA that subsequently activate a series of genetic programs.

生物制药工程 夏玉琼 西安电子科技大学

Stimulate immune response: an example

40

Principle Prokaryotic DNA contains many CpG dinucleotide

sequences, while mammalian DNA has very few, which are usually methylated.

Design and Result Synthetic ONs containing CpG motifs can mimic

prokaryotic DNA, and induce immune responses.

生物制药工程 夏玉琼 西安电子科技大学

Summary

41

Oligonucleotides Different ONs: TFO, Transcription factor decoys,

Ribozymes/DNAzymes, Antisense RNA /siRNA /miRNA

Aptamer/riboswitch Other Application: Gene repair and stimulate

immune response

生物制药工程 夏玉琼 西安电子科技大学

Homework 6

42

Tell the difference between transcription factor and transcription factor decoy.

Tell the similarity and difference between antisense RNA and siRNA.

生物制药工程 夏玉琼 西安电子科技大学