Kim Lennox, MScResearch Scientist, Integrated DNA Technologies

Methods to knock down lncRNAs

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• Lower organisms: mRNA > lncRNA• Higher organisms: lncRNA >>

mRNA• Added regulatory functions of

lncRNAs:– Correlate with an increased

ability of multicellular organisms to differentiate into many different cells types

– Allow an organism to achieve greater diversity from the same number of protein coding genes

Importance of lncRNAs in mammals

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Target LocationmRNA cytoplasm + nucleussplice nucleus onlymiRNA cytoplasm > nucleus

lncRNAcytoplasm > nucleuscytoplasm = nucleuscytoplasm < nucleus

Does cellular localization matter when choosing a silencing reagent?

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Rinn study: RNA-FISH on 61 lncRNAs

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Cabili et al. (2015) Localization and abundance of human lncRNAs at single-cell and single molecule resolution. Genome Biol, 16:20.

Cell lines: HeLa, hlF, hFF

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RISC

Target RNA

RNA cleavage

siRNA

RNA cleavage

Target RNA

RNA interference (RNAi)

Target RNA

RNase H1

ASO (DNA)

Antisense oligonucleotides (ASOs)

> cytoplasm > nucleus

Study design• lncRNA targets

– 2 nuclear– 3 cytoplasmic– 3 nuclear + cytoplasmic

• Knockdown reagents– Antisense oligonucleotides (ASOs)

• 12 DNA-PS (20mers)• 12 2′OMe-PS 5-10-5 chimeras (same sequence as DNA-PS)• 6 LNA™ longRNA GapmeRs (sites selected by Exiqon)

– siRNA• 12 Dicer-substrate siRNAs (DsiRNAs) (27mer, IDT design)• 12 siRNAs (21mer, Thermo Fisher design)• 4 Silencer® Select siRNAs (21mer, Life Technologies design)

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The knockdown reagents

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Methods• Transfection (ASOs and siRNAs):

– HeLa cells (Huh7 for NRON); all data replicated in HCT116 cells – Biological triplicates – 96-well format – Lipofectamine® 2000– Chemistry-matched negative control sequences

• RNA was prepared 24 hours post-transfection• RT-qPCR:

– Two qPCR assays for each target (one towards the 5′ end and one towards the 3′ end)

– Triplicate qPCRs – Quantification standard curves on each 384-well plate – Results normalized against internal control HPRT and SFRS9 gene expression levels

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Targets

Nuclear Cytoplasmic Nuclear + Cytoplasmic

Target 1: MALAT1 (8 kb)

Target 3: NRON (2.7 kb)

Target 6: TUG1 (7.5 kb)

Target 2: NEAT1 (3.7 kb)

Target 4: DANCR (0.9 kb)

Target 7: CasC7 (9.3 kb)

Target 5: OIP5-AS1 (1.9 kb)

Target 8: HOTAIR (2.3 kb)

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In situ hybridization: MALAT1 (nuclear)

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In situ hybridization: NEAT1 (nuclear)

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In situ hybridization: CasC7 (nuclear + cytoplasmic)

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MALAT1 knockdown in HeLa cells: nuclear

For mRNA knockdown, we usually expect >80% success rate for the DsiRNA algorithm.

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NEAT1 knockdown in HeLa cells: nuclear

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NRON knockdown in Huh-7 cells: cytoplasmic

NRON function involves tight binding of multiple protein species → no knockdown.Therefore, needed to move to a different cytoplasmic target. 15

DANCR knockdown in HeLa cells: cytoplasmic

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OIP5-AS1 knockdown in HeLa cells: cytoplasmic

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TUG1 knockdown in HeLa cells: nuclear + cytoplasmic

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CasC7 knockdown in HeLa cells: nuclear + cytoplasmic

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HOTAIR knockdown in HeLa cells: nuclear + cytoplasmic

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Comparison summary

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2′OMe-PS ASOLNA-PS ASO

LNA-siRNA

DsiRNAsiRNA

Combinatorial approach can have additive effects for lncRNAs localized in both the nucleus and cytoplasm

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ASO DsiRNA ASO + DsiRNA

ASO DsiRNA ASO + DsiRNA

ASO DsiRNA ASO + DsiRNA

0

20

40

60

80

100

120

Combinatorial Knockdown10 nM5 nM2.5 nM1 nM

% R

emai

ning

RNA

MALAT1Nuclear

OIP5-AS1Cytoplasmic

CasC7Both

Comparison of ASO chemistry/design potency at the same 6 sites in MALAT1

2nd generation “Gapmer” ASOs are more potent than DNA-PS 23

Summary• Overall performance of ASOs vs. siRNAs varies with the

dominant cellular localization of the lncRNAs that were targeted:– ASOs were more often effective at knocking down nuclear

lncRNAs.– siRNAs were more often effective at knocking down cytoplasmic

lncRNAs.– Better knockdown can be achieved by combining RNAi reagents

with ASOs. • Characterizing the localization of a targeted lncRNA and

selecting the appropriate knockdown method is important for improving the success of the knockdown experiment.

• If lncRNA localization is unknown, trying both methods may be prudent.

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Articles from IDT scientists:

• www.idtdna.com/decoded (Small RNAs/Functional Genomics section)– A New Renaissance for Antisense in the Era of lncRNA– Using Antisense Technologies to Modulate Noncoding RNA

Function• www.idtdna.com (Search for “antisense

oligonucleotide”)– Antisense Oligonucleotides: Strategies and Applications

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Online tools:

• www.idtdna.com/scitools (Gene Regulation and Knockdown section)– Predesigned DsiRNA Selection ToolSelects DsiRNA Duplexes and

TriFECTa® Screening Kits for your sequence– RNAi Design ToolGenerates duplex siRNA sequences for RNAi

applications

• For additional ASO design assistance, please email ApplicationSupport@idtdna.com

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