Pharmaceutical Biotechnology - Xidianweb.xidian.edu.cn/yqxia/files/20140429_231446.pdfSummary of ONs 1/2 32 Triple helix-forming oligonucleotides (TFO) Form triple-helix and interfere

By Yuqiong Xia2014

Oligonucleotides

Pharmaceutical Biotechnology

Keywords

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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

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Introduction to oligonucleotides Oligonucleotides with different functions Other Application


Introduction to oligonucleotides

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

deoxyribonucleotides

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

25-mer: 25nt


Why ONs can be used as a drug

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

human genome


Why ONs can be used as a drug

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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


Limitations of Ons 1/2

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Off-target effects Partial sequence homology may affect expression

other than targeted gene


Limitations of ONs 2/2

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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


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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


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The structure of ONs： 2nd generation

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


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The structure of ONs： 3rd generation

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


Outline

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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


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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)

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TFO binds to polypurine strands in the major groove of dsDNA

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


Limitations and solutions of TFO

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Limitations Difficult to form stable triple helix, due to electrostatic

repulsion

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

RNA


Transcription factor

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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.


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

Ribozyme

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Discovered in 1982 Ribozyme can catalyze specific biochemical reactions,

similar to protein enzymes

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


DNAzyme

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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


Antisense RNA

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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


Antisense RNA

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

Antisense RNA


siRNA

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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


siRNA

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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


miRNA (definition)

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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


How does miRNA function

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

As a result, these mRNA strands are silenced.


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How does miRNA function

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

~22 bpmiRNA


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Antisense/Ribozymes/siRNA

停顿


Aptamers (适体)

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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


Nucleic acid aptamers

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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


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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）

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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


Summary of ONs 1/2

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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


Summary of ONs 2/2

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

molecules


Outline

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


Triplex helix-forming oligonucleotides

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


Antisense-induced exon skipping

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Artificially remove one or more exons

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


Antisense-induced ribonucleoprotein inhibition

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


Outline

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


Stimulate immune response

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Foundation Specific receptors exist that recognize pathogenic

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


Stimulate immune response: an example

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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.


Summary

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Oligonucleotides Different ONs: TFO, Transcription factor decoys,

Ribozymes/DNAzymes, Antisense RNA /siRNA /miRNA

Aptamer/riboswitch Other Application: Gene repair and stimulate

immune response


Homework 6

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Tell the difference between transcription factor and transcription factor decoy.

Tell the similarity and difference between antisense RNA and siRNA.


Documents

Pharmaceutical Biotechnology - Xidianweb.xidian.edu.cn/yqxia/files/20140429_231446.pdfSummary of ONs 1/2 32 Triple helix-forming oligonucleotides (TFO) Form triple-helix and interfere