A Web-based Software Tool for Designing Small Interface RNA

1
A Web-based Software Tool for Designing Small
Interface RNA

• Blake Adams
• Graduate Student
• Department of Computer Science
• Advisor Dr. Muhammad A. Rahman

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Overview

• Central Dogma of Molecular Biology
• Gene Silencing
• siRNA
• P2X3
• Rational Design
• Thermodynamics
• Web-based Interface Demonstration
• QA

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DNA and RNA

• DNA
• Double Helix
• adenine ,guanine, cytosine,thymine
• Sugar is deoxyribose.
• Mainly acts as a set of directions
• RNA
• Single Strand
• Uracil replaces thymine
• Sugar is ribose
• Can take on several different roles

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The Central Dogma of Molecular Biology

• DNA contains the complete genetic information
  that defines the structure and function of an
  organism
• Proteins are formed using the genetic code of the
  DNA
• Different processes are responsible for the
  inheritance of genetic information and for its
  conversion from one form to another
• Replication a double stranded nucleic acid is
  duplicated to give identical copies. This process
  perpetuates the genetic information.
• Transcription a DNA segment that constitutes a
  gene is read and transcribed into a single
  stranded sequence of RNA. The RNA moves from the
  nucleus into the cytoplasm.
• Translation the RNA sequence is translated into
  a sequence of amino acids as the protein is
  formed. During translation, the ribosome reads
  three bases (a codon) at a time from the RNA and
  translates them into one amino acid

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• Genetic Disorders
• A genetic disorder is a disease caused by
  abnormalities in an individuals genetic material
  (genome). There are four different types of
  genetic disorders
• single-gene - cystic fibrosis, sickle cell
  anemia, Huntingtons disease.
• Multifactorial - heart disease, high blood
  pressure, arthritis, diabetes, cancer, and
  obesity.
• Chromosomal - Down syndrome
• Mitochondrial Alzheimers disease

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Gene Silencing

• Targeting or interfering with a specific gene and
  preventing its expression (in other words,
  preventing it from leading to a protein.)
• One method for silencing these genes is RNA
  interference

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What is RNA Interference?

• A phenomenon in which the introduction of
  double-stranded RNA into a cell causes
  degradation of the complementary mRNA. Since the
  mRNA becomes degraded, it is no longer capable of
  translating the data recorded from DNA, thus the
  gene is no longer producing its unique protein,
  and its effect is silenced.

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siRNA Mechanism

• 2 Step Process
• Initiation
• Generation of siRNA snippets
• Effector
• Degradation of target mRNA

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Initiation Step
Double Stranded RNA
DICER
siRNA
10
Effector Step
siRNA
RNA Induced Silencing Complex (RISC)
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History of siRNA

• 1995 Gou Kemphues notice gene silencing
  effect during an experiment with Caenorhabditis
  elegans. They do not know why it is occurring
  but make note of the phenomenon
• For the next 3 years The basis of the sense
  effect is under investigation
• 1998 Fire Mello verify silencing of C.
  elegans a few dsRNA molecules are sufficient to
  silence the homologous genes expression.

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My Interest in gene silencing

• Bioinformatics Fall 2004
• siRNA sequences are collected from relevant
  papers.
• A scoring algorithm is developed based on
  prevailing design standards and techniques.
• The scoring system is incorporated into a perl
  program designed to score siRNAs.

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P2X3 A gene silencing example

• siRNA relieves chronic neuropathic pain
• Gabriele Dorn et al, Nucleic Acids Research,
  March 2004.
• P2X3 is a gene present in the Rattus norvegicus
  genome (rat) that plays a role in neuropathic
  pain (e.g. Arthritis).
• Development and injection of an effective siRNA
  caused subjects suffering from Arthritis
  (identified by frequency of paw-licking) to lick
  paws less often and urinate with less frequency
  leading the researchers to conclude that
  silencing P2X3 has not only a significant effect
  on Arthritis but could also be used as a
  treatment for incontinence.

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siRNA Good Design

• Any given gene can be thousands of nucleotides in
  length.
• What segment should be targeted?
• Cost of purchasing siRNA to use in wet lab after
  design is quite high, a single sample can cost as
  much at 1000.
• Time investment by scientists is increased for
  each unsuccessful siRNA used.

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Rational Design

• Reynolds, A. et al.(2004) Nature Biotechnology
  22, 326-330
• Lays out 8 key factors that significantly improve
  the probability of selecting functional siRNA
  sequences

• 30 - 52 G/C Content
• Three or more A/Us at Positions 15-19 (sense)
• Absence of internal repeats
• A at position 19 of the sense strand

• A at position 3 of the sense
• U at position 10 of the sense
• No G/C at position 19 of the sense
• No G at position 13 of the sense.

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Thermodynamics

• Recent experimental evidence indicates that the
  two strands in the siRNA duplex do not enter into
  the RISC equally
• The less stable 5 end (of the sense OR
  antisense) in the siRNA duplex directs the strand
  to enter into the RISC complex.
• Two recently published papers by Schwartz, et al.
  (Asymmetry in the assembly of the RNAi enzyme
  complex) and Khvorova et at (Functional siRNAs
  and miRNAs exhibit strand bias) demonstrate that
  it is the antisense strand of the siRNA that must
  bind to the RISC complex AND that energy
  differences between the sense and antisense
  (sense energy antisense energy) tend to favor
  the sense when the outcome is positive. It is
  therefore favorable to select a sequence with a
  negative differential.

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Calculating a Thermodynamic Score

• Thermodynamic parameters have been established
  and reported Xia et al. (Biochemistry 1998,
  Thermodynamic Parameters for an Expanded
  Nearest-Neighbor Model for Formation of RNA
  Duplexes with Watson-Crick Base Pairs)
• For a given set of Neighbors in a sequence such
  as AG, there is an established energy value
  (-2.08). Each possible combination of AGTC (20
  in total) has a fixed energy value. Dangling
  nucleotides also have fixed energy values.
• A scoring system developed by Yuan et al.
  (Nucleic Acids Research 2004, siRNA Selection
  Server an automated siRNA oligonucleotide
  prediction server) establishes a system based on
  these values to determine the Energy score of the
  sense and antisense strand of a given siRNA strand

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Calculating a Thermodynamic Score (continued)

• Develop 2 models based on a given target strand
• 5 AA AGTCACCGTCAAGGTGTAT TT 3
• 3 TT TCAGTGGCAGTTCCACATA AA 5
• The 2 models will represent the sense and the
  antisense of the siRNA.
• The first model combines the first 5 nucleotides
  of the 5 end of the sense with the last 5
  nucleotides of the 3 end of the antisense and
  incorporates a 3 dangling nucleotide from the
  antisense. The second model repeats this process
  for the 5 end of the antisense and 3 end of the
  sense. Given the above example
• SENSE
• 5 AGUCA3
• 3U UCAGU5
• ANTISENSE
• 5 AUACA3
• 3UUAUGU5

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Calculating a Thermodynamic Score (continued)

• SENSE
• 5 AGUCA3
• 3U UCAGU5
• ANTISENSE
• 5 AUACA3
• 3UUAUGU5
• The thermodynamic score of the 4 nearest
  neighbors (based on Watson-Crick Nearest Neighbor
  method) are added to the thermodynamic score of
  the Dangling 3 overhang.
• SENSE AG -2.08, GU -2.24, UC-2.35, CA-2.11,
  _A -.10
• (-2.08) (-2.24) (-2.35) (-2.11) (-0.1)
  -8.88
• ANTISENSE AU-1.10, UA -1.33, AC-2.24,
  CA-2.11, _A -.10
• (-1.10) (-1.13) (-2.24) (-2.11) (-0.1)
  -6.88
• Thermodynamic Score is the difference of the
  energy of the 5 sense and the 5 antisense.
• -8.88 (-6.88) -2.00

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Demonstration

• Web-based siRNA Design Tool

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Acknowledgements

• Dr Muhammand A Rahman Assistant Professor,
  University of West Georgia
• Bingbing Yuan MIT, Whitehead Biocomputing Group
  
• Michael Duong McMaster University