Puzzle after genome decoding

1. Proteins coding: 2%

1. Primary transcripts cover 75% of the human

genome, with processed transcripts covering 62%

3. A large portion of the genome has been

transcribed into RNAs

4. What are those RNAs’ functions????????

 Long non-coding RNA
at a glance
The world leader in serving science ~ an emerging research field and its
research resources

Aaron Chen, Ph.D.

Field Application Scientist,
Level Biotechnology Inc.
 Outline
Background
• What are lncRNAs?

Detection of lncRNAs
• Design of qPCR assays

siRNAs to target lncRNAs

• Strategies to improve function and specificity

3
 Outline
Background
• What are lncRNAs?

Detection of lncRNAs
• Design of qPCR assays

siRNAs to target lncRNAs

• Strategies to improve function and specificity

4
 Non-coding RNA (ncRNA)

Protein Coding Non-protein Coding

mRNA
Non-coding RNA

Protein

5
Protein-coding genes vs. Bio-complexity

Number of protein-coding genes

Szymanski and Barciszewski, Beyond the proteome: non-coding regulatory RNAs,

Genome Biology 2002 6
Protein-coding genes vs. Bio-complexity

~ 21,000

Szymanski and Barciszewski, Beyond the proteome: non-coding regulatory

RNAs, Genome Biology 2002 7
Protein-coding genes vs. Bio-complexity

Human C. elegans
3%

% of genome
encoding proteins

non-coding

Szymanski and Barciszewski, Beyond the proteome: non-coding regulatory

RNAs, Genome Biology 2002 8
 Protein-coding genes vs. Bio-complexity

Human C. elegans
3%

% of genome
encoding proteins

non-coding

Can the increased Bio-complexity be explained

by noncoding RNAs?
Szymanski and Barciszewski, Beyond the proteome: non-coding regulatory
RNAs, Genome Biology 2002 9
 Classes of Non-coding RNAs

1. Housekeeping RNA
 tRNA, rRNA, snoRNA, snRNA
 Translation, ribosome mature, RNA splicing…etc

2. Small regulatory RNA

 miRNA (21-24 nt), piRNA (26-31 nt)
 development and disease

3. Long noncoding RNA (lncRNA)

 ≥200 nt
 Most prevalent class of ncRNA
 XIST, HOTAIR…
 development and disease

(1) De Sacco et. al. Int. Journal of Mol. Sci. (2012), 13.
(2) Taft et al., J Pathol 2010; 220: 126–139, Non-coding RNAs: regulators of disease
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Genomic locations of lncRNAs

Derrien et al, 2012, Genome Research

11
Characteristics of lncRNA expression

Compared with mRNAs,

lncRNAs show lower expression in 16 tissues
Derrien et al, 2012, Genome Research
12
Characteristics of lncRNA expression

More tissue-specific expression than

protein-coding genes
Derrien et al, 2012, Genome Research
13
 Roles of lncRNA

e.g. RNA pol III e.g. Evf2

e.g. TLS/CBP/P300 e.g. HOTAIR

(1) De Sacco et. al. (2012) Int. Journal of Mol. Sci

(2) Rinn and Chang, (2012)
14 Annu Rev Biochem
lncRNAs in the nucleus

Transcriptional interference

lncRNA can function in cis (neighbor genes)

lncRNA can function in trans (distant genes)

lncRNA can bind and sequester transcription factors

Batista and Chang, Cell (2013)

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lncRNAs in the cytoplasm

Promote mRNA stability

Bind to 3' UTR and promote mRNA degradation

Bind to 5' mRNA and promote translation

Bind to mRNA and inhibit translation

Batista and Chang, Cell (2013)

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lncRNAs in human development

X-chromosome inactivation
XIST compensates dosage by silencing an X-
chromosome in females

Brown et al., Cell 1992

Regulating imprinting
Air recruits histone methyl-transferase to silence
genes in the Igf2r locus during early development

Sleutels et al., Nature 2002

Epigenetic regulation
Functional demarcation of active and silent
chromatin domains in human HOX loci by
noncoding RNAs [HOTAIR]
Rinn et al., Cell 2007

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lncRNAs in human disease

HIV Replication
NEAT1 Long Noncoding RNA and Paraspeckle
Bodies Modulate HIV-1 Posttranscriptional
Expression
mBio 2013

Alzheimer’s Disease
Knockdown of BACE1-AS Nonprotein-coding
Transcript Modulates Beta-Amyloid-Related
Hippocampal Neurogenesis
Int J Alzheimers Dis. 2011

Melanoma
Melanoma-upregulated SPRY4-IT1 Modulates
Apoptosis and Cell Invasion
Cancer Research 2011

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The rise in the number of lncRNA publications

2013

J Pathol 2010; 220: 126–139

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The rise in the number of lncRNA publications

2013

J Pathol 2010; 220: 126–139

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 General approaches to lncRNA studies

1. Determine lncRNA of interest

 Expression profiling (Normal vs. disease tissue)
 Deep sequencing
 Literature searches
 Protein capture assays
 In situ hybridization
 qPCR

2. Functional screen in characterized cell-based assay

 lncRNA knockdown

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Example – Role of NEAT1 lncRNA in HIV infection

Zhang et al., (2013). mBio. 4:1

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 Tools for investigation of lncRNAs

1. Detection of lncRNA expression

 qPCR assay (Probe system is better)

2. Knockdown of lncRNA transcripts

 lncRNA knockdown (by siRNA)

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 Outline
Background
• What are lncRNAs?

Detection of lncRNAs
• Design of qPCR assays

siRNAs to target lncRNAs

• Strategies to improve function and specificity

24
 Sources for design of qPCR assays

1. Source: RefSeq (NCBI)

2. Gene Type: ncRNA (but excluding microRNAs, tRNA … etc.)

3. Transcript accession: NR_ or XR_

25
 Considerations for design of qPCR assays

• Assay designed to span exons

 Preventing gDNA amplification
• Primers/probe set BLASTed
 Preventing non-specific amplification
 Among 10 primer pairs for lncRNAs, 2-3 primers detect to protein coding genes
• Stringent design parameters
 GC content, sequence length, Tm, etc.
• Detecting all known splice variants
Splice Variant 1

Splice Variant 2

Splice Variant 3

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 Ideal qPCR lncRNA expression assays
• Probe and primer sets increase specificity

• Design algorithm results in high specificity

No cross-targeting to protein coding transcripts

• Sequence information provided for primers and probes

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Examination of lncRNA Expression

High Cq =
Low expression

Low Cq =
High expression

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 Outline
Background
• What are lncRNAs?

Detection of lncRNAs
• Design of qPCR assays

siRNAs to target lncRNAs

• Strategies to improve function and specificity

29
 Non-coding RNA (ncRNA)

Protein Coding Non-protein Coding

5’-p RISC
p-5’

mRNA
Non-coding RNA

RISC p-5’

Protein

30
 Challenges for lncRNA knockdown

(1) lncRNA is a functional molecule, not an

intermediate

(2) siRNA recognition sites may be RISC

in-accessible

(3) lncRNA could be predominantly

localized to the nucleus

(4) siRNA may off-target to overlapping

protein coding genes

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 Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown

1. siRNA design algorithm for effective silencing

2. siRNA pool format to reduce off-target and

enhance function

3. Chemical modification to improve specificity

32
 Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown

1. siRNA design algorithm for effective silencing

2. siRNA pool format to reduce off-target and

enhance function

3. Chemical modification to improve specificity

33
 siRNA design algorithm

(1) BLAST analysis for both strands of all coding and

non-coding genes

(2) Target consensus regions of all isoforms

(3) Avoid targeting protein-coding transcripts (NM_)

(4) Filter out highly conserved microRNA seeds

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Ex. siRNA-mediated Knockdown of 15 lncRNAs

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 Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown

1. siRNA design algorithm for effective silencing

2. siRNA pool format to reduce off-target and

enhance function

3. Chemical modification to improve specificity

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 Use of siRNA pool

How to ensure the siRNA target region

is accessible to loaded RISC?

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 Use of siRNA pool

Combining 4 efficient siRNA pool

 Improve possibility of lncRNA degradation !

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 Effective knockdown of lncRNAs

CDKN2B-AS1 in HeLa cells BDNF-AS1 in hNDF cells

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 Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown

1. siRNA design algorithm for effective silencing

2. siRNA pool format to reduce off-target and

enhance function

3. Chemical modification to improve specificity

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 Chemical modification select RISC-loading strand

S AS siRNA
S BDNF RNA
AS BDNF-AS1 RNA

RISC
AS

S BDNF RNA

RISC or
S

AS BDNF-AS1 RNA

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Dual-strand modifications to improve specificity

Chemical modified siRNA

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 Chemical modifications reduce off-targets

S AS siRNA
S BDNF RNA
AS BDNF-AS1 RNA

RISC
AS

S BDNF RNA

RISC or
S

AS BDNF-AS1 RNA

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 Chemical modifications reduce off-targets

S AS siRNA
S BDNF RNA
AS BDNF-AS1 RNA

RISC
AS

S BDNF RNA

RISC or
S

AS BDNF-AS1 RNA

44
 Chemical modifications reduce off-targets

RISC
S

AS BDNF-AS1 RNA

45
 Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown

1. siRNA design algorithm for effective silencing

2. siRNA pool format to reduce off-target and

enhance function

3. Chemical modification to improve specificity

46
 Discussion and Questions

Thank You for Your Attention

Aaron Chen, Ph.D.

Field Application Scientist,
Level Biotechnology Inc.
aaronchen@mail.level.com.tw

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 FAQs

I want to silence lncRNAs that you don’t have products for.

What should I do?

http://www.thermoscientificbio.com/design-center/

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Q & A siRNA siDESIGN Center

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Q & A siRNA siDESIGN Center

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Q & A siRNA siDESIGN Center

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