rnai blast blast smith water man

8/7/2019 RNAi Blast BLAST Smith Water Man

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SmithSmith--Waterman vs Blast inWaterman vs Blast in

siRNA Oligonucleotide DesignsiRNA Oligonucleotide Designand Selectionand Selection

Christine LeeChristine Lee

Dr. Cecilie Boysen, Ph.D.Dr. Cecilie Boysen, Ph.D.

Paracel,Paracel, Applied High Performance ComputingApplied High Performance Computing

Southern California Bioinformatics InstituteSouthern California Bioinformatics Institute

Summer 2004Summer 2004

Funded by the National Science Foundation and National Institute of HealthFunded by the National Science Foundation and National Institute of Health



OutlineOutline

History of RNAiHistory of RNAi

Small interfering RNA (siRNA) MechanismSmall interfering RNA (siRNA) Mechanism

siRNA design and selectionsiRNA design and selection

Blast vs SmithBlast vs Smith--WatermanWaterman

Project Objectives and ResultsProject Objectives and Results

Conclusions & Future WorkConclusions & Future Work



History of RNAiHistory of RNAi

Discovered in 1998 by Andrew Fire, CraigDiscovered in 1998 by Andrew Fire, Craig

Mello, and colleaguesMello, and colleagues

RNAiRNAi silencing of gene expression bysilencing of gene expression bydsRNA moleculesdsRNA molecules

Organism used: Caenorhabditis elegansOrganism used: Caenorhabditis elegans



Short interfering RNA (siRNA)Short interfering RNA (siRNA)

MechanismMechanism

http://www.bioteach.ubc.ca/MolecularBiology/AntisenseRNA/siRNA.gif



siRNA Selection & Design:siRNA Selection & Design:

Avoiding CrossAvoiding Cross--HybridizationHybridization Important to guard against strong crossImportant to guard against strong cross--

hybridization to other geneshybridization to other genes

CrossCross--hybridization with nonhybridization with non--specific targetsspecific targetsresults in wasted lab time and materials, as wellresults in wasted lab time and materials, as wellas inaccurate conclusionsas inaccurate conclusions

Preliminary sequence analysis allows verificationPreliminary sequence analysis allows verificationof candidate oligos to protect against crossof candidate oligos to protect against cross--hybridizationhybridization



siRNA Selection & DesignsiRNA Selection & Design

Hybridization concerns:Hybridization concerns: siRNA mismatch tolerancesiRNA mismatch tolerance

Insertion/deletion vs mismatchInsertion/deletion vs mismatch

Query: 1 GAACTTATCTTCCTTCTTC 19Query: 1 GAACTTATCTTCCTTCTTC 19

||||||||||||||||||||||||||||||||||||||

Sbjct: 3783 GAACTTATCTTCCTTCTTC 3801Sbjct: 3783 GAACTTATCTTCCTTCTTC 3801

Query: 19 GAAGAAGGAAGATAAGTTC 1

||||||||| || ||||||

Sbjct: 778 GAAGAAGGATGAGAAGTTC 796



Blast vs SmithBlast vs Smith--WatermanWaterman

Blast may potentially miss relevant alignmentsBlast may potentially miss relevant alignments

Using word size seven, nearly 6% of allUsing word size seven, nearly 6% of allpossible alignments with three mismatchespossible alignments with three mismatchesbetween 21between 21--mers will be missedmers will be missed

Increasing word size or allowing moreIncreasing word size or allowing moremismatches contribute to higher rate of mismatches contribute to higher rate of

missed hitsmissed hits SmithSmith--Waterman is said to have higherWaterman is said to have higher

sensitivity, so why not use it?sensitivity, so why not use it?



Project ObjectivesProject Objectives

Test set: 10,000 19Test set: 10,000 19--mer oligos/siRNAsmer oligos/siRNAs

Test database: RefSeqTest database: RefSeq

Comparison study between Blast andComparison study between Blast andSmith WatermanSmith Waterman

15/1915/19 --> Percent Identity threshold set to> Percent Identity threshold set to

78% e78% e--value adjustment from default of value adjustment from default of 10. E10. E--value 500 usedvalue 500 used



A Closer Look at SmithA Closer Look at Smith--WatermanWaterman

& Blast Parameters& Blast Parameters

AlgorithmAlgorithm AlignmentAlignment Score/Score/

(ID)(ID)

ParamParam MatchMatch MisMis--

matchmatch

GO/GO/

GEGE

GapGap

TotalTotal

SmithSmith

Water manWater man

Query: 19 TCACCGTAGATGCTCTTTC 1Query: 19 TCACCGTAGATGCTCTTTC 1

|| |||| ||||||||||||| |||| |||||||||||

Sbjct: 2376 TCSbjct: 2376 TC--CCGTGGATGCTCTTTC 2393CCGTGGATGCTCTTTC 2393

2929

17/1917/19

(89%)(89%)

defaultdefault +2+2 --22 --33

BlastBlast Query: 1 gaaagagcatctacgg 16Query: 1 gaaagagcatctacgg 16

||||||||||| ||||||||||||||| ||||

Sbjct: 2393 gaaagagcatccacgg 2378Sbjct: 2393 gaaagagcatccacgg 2378

1212

15/1615/16

(93%)(93%)

W 7W 7

e 500e 500

DefaultDefault

+1+1 --33 GG --55

EE --22

--77

BlastBlastQuery: 1 gaaagagcatctacggtga 19Query: 1 gaaagagcatctacggtga 19

||||||||||| |||| ||||||||||||| |||| ||

Sbjct: 2393 gaaagagcatccacggSbjct: 2393 gaaagagcatccacgg--ga 2376ga 2376

2929

17/1917/19

(89%)(89%)

W7W7

e 500e 500

G 1G 1

q 2 r 2q 2 r 2

+2+2 --22 GG --11

EE --22

--33



SmithSmith--Waterman vs. Blast ResultsWaterman vs. Blast Results

Percent Identity: 89% ,GATA3 gene

> gi|4503928|ref|NM_002051.1| Homo sapiensGATA binding protein 3

(GATA3), mRNA

Length = 2365

Score = 31.7 bits (38), Expect = 0.041

Identities = 19/19 (100%)

Strand = Plus / Plus

Query: 1 ctttttaacatcgacggtc 19

|||||||||||||||||||

Sbjct: 299 ctttttaacatcgacggtc 317

SWN hit-4 bin Blast hit-1 bin W7 G1 r2 q2 e500 E2

Original Query Sequence: CTTTTTAACATCGACGGTC



SmithSmith--Waterman vs. Blast ResultsWaterman vs. Blast Results

> gi|4503928|ref|NM_002051.1| Homo sapiens GATA binding protein 3

(GATA3), mRNA

Length = 2365





|||||||||||||||||||


> gi|4557424|ref|NM_001248.1| Homo sapiens ectonucleosidetriphosphate

diphosphohydrolase 3 (ENTPD3), mRNA

Length = 2797


Identities = 17/19 (89%), Gaps = 1/19 (5%)

Strand = Plus / Minus

Query: 1 aa-aatactgagagaggga 18

|| ||||||||| ||||||

Sbjct: 2044 aagaatactgagggaggga 2026

> gi|4503928|ref|NM_002051.1| Homo sapiensGATA binding protein 3

(GATA3), mRNA

Length = 2365





|||||||||||||||||||


SWN hit-1 bin

Blast hit-4 bin

Original Query Sequence: AAAATACTGAGAGAGGGAG



Conclusions and Future WorkConclusions and Future Work

Produce more conclusive statistics for occurrences of Produce more conclusive statistics for occurrences of more accurate Smithmore accurate Smith--Waterman resultsWaterman results

No consensus exists as to which hits are consideredNo consensus exists as to which hits are considereddangerous or significant for crossdangerous or significant for cross--hybridizationhybridization

Creation of a positionCreation of a position--specific matrixspecific matrix

Mutation tolerance on the 5 endMutation tolerance on the 5 end

Low tolerance on the 3 endLow tolerance on the 3 end GU wobbleGU wobble



ReferencesReferences Novina, C and Sharp, P.Novina, C and Sharp, P. The RNAi revolution.The RNAi revolution.

Nature. 2004 Jul 8;430(6996):161Nature. 2004 Jul 8;430(6996):161--4.4. Dorsett, Y and Tuschl, T.Dorsett, Y and Tuschl, T. siRNAs: applications insiRNAs: applications in

functional genomics and potential asfunctional genomics and potential astherapeutics.therapeutics. Nat Rev Drug Discov. 2004Nat Rev Drug Discov. 2004

Apr;3(4):318Apr;3(4):318--29.29. Snove, O Jr. and Holen, T.Snove, O Jr. and Holen, T. Many commonlyMany commonly

used siRNAs risk off used siRNAs risk off--target activity.target activity. BiochemBiochemBiophys Res Commun. 2004 Jun 18;319(1):256Biophys Res Commun. 2004 Jun 18;319(1):256--63.63.

Paroo, Z and Corey, DR.Paroo, Z and Corey, DR. Challenges for RNAi inChallenges for RNAi invivo.vivo. Trends Biotechnol. 2004 Aug;22(8):390Trends Biotechnol. 2004 Aug;22(8):390--4.4.

Amarzguioui, M. et al.Amarzguioui, M. et al. Tolerance for mutationsTolerance for mutationsand chemical modifications in siRNAand chemical modifications in siRNA. Nucl Acids. Nucl AcidsResearch. 2003; 31(2)589Research. 2003; 31(2)589--595.595.



AcknowledgementsAcknowledgements

Dr. Cecilie BoysenDr. Cecilie Boysen (advisor) Parcel Scientific Staff (advisor) Parcel Scientific Staff

David MeyerDavid Meyer Paracel Software EngineerParacel Software Engineer

Stephanie PaoStephanie Pao Paracel Technical Sales EngineerParacel Technical Sales Engineer

Frances TongFrances Tong Paracel InternParacel Intern

William WhiteWilliam White Paracel Technical WriterParacel Technical Writer

Southern Ca lifornia Bioinformat ics Inst it ut e 2004 Fa cult ySout hern Ca lifornia Bioinformat ics Inst it ut e 2004 Fa cult ya nd Staff :a nd Staff :Dr. Ja mil Moma nd, Dr. Na ncy Wa rt erDr. Ja mil Moma nd, Dr. Na ncy Wa rt er--Perez,Perez,Dr. Sa ndra Sha rp & Dr. Wendie Johnst on,Dr. Sa ndra Sha rp & Dr. Wendie Johnst on,& Ja ckie Leung& Ja ckie Leung

Fellow int ernsFellow int erns NIH & NSFNIH & NSF



Short interfering RNA

Mechanism

Post-transcriptional gene

silencing.

Novina, C and Sharp, P. The RNAi

revolution. Nature Vol 430. July 8,

2004.



Dorsett, Y and Tuschl, T.

siRNAs: applications in

functional genomics and

potential as therapeutics. Nat

Rev Drug Discov. 2004

Apr;3(4):318-29.

�Reverse genetic approaches ± expensive and time consuming

�siRNAmay be chemically synthesized or expressed from DNA vectors



MicroRNAsMicroRNAs

Short RNAs 19Short RNAs 19--25 nucleotides25 nucleotides

Abundant, single stranded RNAs encoded inAbundant, single stranded RNAs encoded ingenomes of most multicellular organisms: from fewgenomes of most multicellular organisms: from fewthousand to 40,000 molecules per cellthousand to 40,000 molecules per cell

Some evolutionarily conserved andSome evolutionarily conserved anddevelopmentally regulateddevelopmentally regulated

Translational silencing.

Picture from:

Novina, C and Sharp, P. The RNAi

revolution. Nature Vol 430. July 8,

2004.



Differences between siRNA andDifferences between siRNA and

miRNAmiRNAsiRNAsiRNA

Promote the cleavagePromote the cleavageor degradation of or degradation of

mRNAsmRNAs Sense strand hasSense strand has

exactly the sameexactly the samesequence as the target sequence as the target

strandstrand Target genes orTarget genes or

genetic elements fromgenetic elements fromwhich they originatedwhich they originated

miRNAmiRNA

Regulate theRegulate theexpression of mRNAs;expression of mRNAs;

transcription is not transcription is not impeded and mRNAsimpeded and mRNAsnot destroyednot destroyed

Imperfect baseImperfect base--pairingpairing

between mRNA targetsbetween mRNA targetsand miRNAand miRNA

Regulate separateRegulate separategenesgenes



Interchangeability of siRNAs andInterchangeability of siRNAs and

miRNAsmiRNAs miRNA may act like siRNAmiRNA may act like siRNA

* perfect or near* perfect or near--perfect complementarity toperfect complementarity to

cellular mRNAscellular mRNAs Could siRNA also work like miRNA?Could siRNA also work like miRNA?

* synthetic siRNA partially complementary to* synthetic siRNA partially complementary to

reporter gene inhibited its expressionreporter gene inhibited its expression Distinction between single site with almost Distinction between single site with almost

exact complementarity and numerous partiallyexact complementarity and numerous partiallycomplementary binding sitescomplementary binding sites



Laboratory and ClinicalLaboratory and Clinical

Applications of siRNAApplications of siRNA In C. elegans, simple experiment: inject dsRNA,In C. elegans, simple experiment: inject dsRNA,

soak in dsRNA solution, or feed with bacteriasoak in dsRNA solution, or feed with bacteria

expressing dsRNAexpressing dsRNA In worms, screening for obesity and ageingIn worms, screening for obesity and ageing

In fruitflies, purified long dsRNA used to identifyIn fruitflies, purified long dsRNA used to identifyroles of genes in cholesterol metabolism androles of genes in cholesterol metabolism andheart formationheart formation

Therapeutic potential of siRNAs for humansTherapeutic potential of siRNAs for humans



FileFile TypeType BasesBases SequencesSequences # of Oligos# of Oligos

BRCA1BRCA1 fastafasta 32433243 11 32553255

GATA3GATA3 fastafasta 30703070 11 30703070

HLAHLA--moleculemolecule fastafasta 29182918 11 29182918

InsulinInsulin--likelike--

growthgrowth--factor factor

fastafasta 49894989 11 49714971

InterleukinInterleukin--receptor receptor

fastafasta 14511451 11 14331433

NFKB1NFKB1 fastafasta 41044104 11 41864186

Serine kinaseSerine kinase fastafasta 35063506 11 34883488

SerotoninSerotonin--

receptor receptor

fastafasta 19271927 11 19091909

TNF2TNF2 fastafasta 16691669 11 16511651

VinculinVinculin fastafasta 56475647 11 56295629

TotalTotal 3255432554 1010



Paroo, Z and Corey, DR. C hallenges for RNAi in vivo.

Trends Biotechnol. 2004 Aug;22(8):390-4.



Blast vs SmithBlast vs Smith--Waterman SpeedWaterman Speed

Test ResultsTest Results

11.35

205.69

46.7

346.08

0

50

100

150

200

250

300

350

Defau e500

SWN

Bla

Time in

minutes

rnai blast blast smith water man

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