Download Nucline RNA and Its Uses

Nucline RNA
SunPillar, LLC
©C. Allen Black; SunPillar, LLC 2002
What is Nucline?
• Nucline is a full length mRNA molecule.
• It is conditionally translated into a protein.
• Protein is only expressed when researcher-defined
gene profiles are present in the cell.
• It can be used to modify, tag, and even destroy
cells that express the gene profile.
• It is not siRNA (methylates target DNA) or
antisense (blocks mRNA translation.) These only
“turn off”/”turn down” protein expression.
©C. Allen Black; SunPillar, LLC
mRNA translation
• 30s ribosome subunit binds the 5’ cap.
• It scans through the mRNA sequence.
• It stalls at the AUG start condon, binds the 50s subunit and
beings translating the protein.
• The 5’ Untranslated Region (UTR) upstream of the start
codon regulates scanning and initiation.
translation begins
30s
5'
scan
UTR
50s
30s
AUG
protein sequence
©C. Allen Black; SunPillar, LLC
3'
mRNA can be “blocked” by
5’ UTR secondary structure
• The ribosome requires energy to scan through the UTR.
• A mRNA with strong secondary structure prevents the
ribosome from ever reaching the AUG codon. The
ribosome cannot melt the structure.
• E.g. Antisense, stem-loops, and hairpins impede the
ribosome and prevent translation.
scan
30s
5'
stal l
AUG
protein sequence
©C. Allen Black; SunPillar, LLC
3'
Nucline: designing a “drug” by
example
• Pick a disease: B cell lymphoma
• Pick what gene you wish the target cell to express:
pertussis toxin (suicide gene)
• Pick sequences (normal or mutant) that are only
expressed in B cell lymphoma (mutant c-myc and
IgM)
• Insert construct into an mRNA transcription
vector, runoff mRNA, and ligate repressors.
• Deliver mRNA via liposomes, virosomes, ballistic
penetration, electroporation etc.
©C. Allen Black; SunPillar, LLC
Step 1: the protein
• Construct suicide gene.
• AUG placed upstream of pertussis toxin.
• All transfected cells will die. mRNA permanently
“on.” No secondary structure.
50s
translation
30s
5'
AUG
Pertussis Toxin
©C. Allen Black; SunPillar, LLC
3'
Step 2: the 5’UTR
• Place sense sequences (100bp) corresponding to cmyc and IgM in the 5’UTR.
• All transfected cells will die. mRNA permanently
“on.” No secondary structure present.
50s
30s
5'
scan
IgM
30s
c-myc
AUG
translation
pertussis toxin
©C. Allen Black; SunPillar, LLC
3'
Step 3: Ligate antisense
• Add anti- sense sequences (100 bp)
complementary to IgM and c-myc in the 5’UTR.
IgM
5'
IgM
c-myc
c-myc
AUG
pertussis toxin
©C. Allen Black; SunPillar, LLC
3'
Nucline in normal cells
In IgM-, c-myc- cells, antisense stays “stuck” to
Nucline. The antisense has only Nucline coded
sense sequences to bind. No translation in normal
cells.
30s
stall
5'
IgM
IgM
c-myc
c-myc
AUG
©C. Allen Black; SunPillar, LLC
pertussis toxin
3'
Nucline in normal B cells.
• Anti-sense will hybridize to its complements.
• In normal B cells (non-c-myc cells), IgM wt mRNA
competes off the antisense. The antisense “choses”
between binding Nucline or wt IgM RNA. Both have
sense sequences so competitive hybridization occurs.
• The c-myc antisense stays “stuck” to Nucline. Still, no
translation. Half of secondary structure still present.
IgM
stall
30s
5'
IgM
wild type IgM mRNA
IgM
AUG
c-myc
c-myc
AUG
pertussis toxin
©C. Allen Black; SunPillar, LLC
3'
Nucline in B cell lymphoma.
• Anti-sense will hybridize to its complements.
• IgM wt mRNA competes off the IgM antisense.
• c-myc wt RNA competes off the c-myc antisense.
TRANSLATION. All of secondary structure removed.
AUG
c-myc
30s
5'
scan
IgM
c-myc
50s
wild type c-myc
mRNA
translation
30s
c-myc
AUG
pertussis toxin
©C. Allen Black; SunPillar, LLC
3'
But this is biology,
will it really work?
©C. Allen Black; SunPillar, LLC
Detecting Estrogen
Receptor Overexpression
•
•
•
•
•
•
Using the plasmid provided the GFP along with the ER regions was amplified using the
two primers Nucln FP and Nucln RP.
RNA was synthesized using this PCR DNA and Ribomax kit from Promega.
MCF-7 cells were purchased from ATCC and were grown in RPMI medium with 10%
fetal calf serum.
For transfection 1 ug RNA was mixed 1ug of Nucline and boiled for 10 min and allowed
to hybridize. To this 2 ul of Tfx-50 reagent (Promega) was added and incubated for 10
min with 300 ul serum free medium. This was added to MCF-7 cells, after washing the
monolayer with RPMI without serum. The cell density was about 60 to 70%.
Transfection was allowed for 30 min after which the medium was removed and
replaced by serum containing medium. The cells were observed under microscope after
24 hrs post-transfection
ER Target Sequence: 5’ ggtcacgtggtaactatttggtcacgtggtaactattt
Special Thanks to Dr. Augustine Rajakumar (University of Pittsburgh) and Dr. Yunus
Luqmani (Kuwait University) for providing these Estrogen Receptor data.
©C. Allen Black; SunPillar, LLC
MCF-7 Cells (Bright Field)
©C. Allen Black; SunPillar, LLC
MCF-7 Positive Control
GFP Plasmid
©C. Allen Black; SunPillar, LLC
Nucline Transfection
Specific for Estrogen Receptor Overexpression
©C. Allen Black; SunPillar, LLC
Repeated Again
©C. Allen Black; SunPillar, LLC
What about in vivo?
• Mouse solid tumor model (MM2MT breast
carcinoma). DeFatta, RJ, Li, Y., De Bendetti,
Cancer Gene Therapy 9:503 (2002).
• Introduced RNA with impenetrable 5’ UTR
(hairpins). RNA coded suicide gene.
• DNA coding the RNA was lipofected into
animals.
• eIF4E (cap binding protein) over expressed in
tumor, not regular cells. It “melts” RNA structure
in cancer but not normal cells. 120 day survival:
0/10 control; 7/10 experimental. 70% cure rate.
©C. Allen Black; SunPillar, LLC
Curing Cancer (in mice)
• DeFatta et al. used “protein assisted” melting.
• Nucline uses “sequence assisted” competitive
hybridization to melt secondary structure.
• Nucline is sequence specific, eIF4E melts any
secondary structure.
• Mode of melting irrelevant for biological effect
• RNA with de-repressible secondary structure
(confering specificity) cures cancer in mice.
Nucline design increases the utility and specificity.
©C. Allen Black; SunPillar, LLC
Uses
• FACS (deliver fluorescent protein)
• (+)/(-) cell selection (suicide/proliferation
inducing genes)
• Disease models (suicide genes, repair genes)
• Wound repair/gene defects (deliver proliferation
inducing genes or new “correct” gene)
• Detect mRNA/(s)tructural RNA profiles,
mRNA/sRNA concentration, mRNA/sRNA
sequences.
©C. Allen Black; SunPillar, LLC
Nucline Logic
• It is a logic system where A,B are cellular mRNA
sequences and Y is a protein to be expressed in those cells
alone.
• IF A is present, THEN release Y. (One antisense blocking
translation)
• IF A and B are present, THEN release Y. (different
antisense targets on same Nucline RNA, e.g. lymphoma
Nucline)
• IF A or B is present, THEN release Y. (Combine two
different kinds of Nucline molecules)
• IF A is present > 100 copies, THEN release Y. (Nucline
with 101 redundant antisense stuck to it)
• IF A but not B present, THEN release Y. (advancing)
• IF A present < 100 copies, THEN release Y. (advancing)
©C. Allen Black; SunPillar, LLC
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©C. Allen Black; SunPillar, LLC