Church Lab: DNA memory IO

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Church Lab DNA memory I/O
DARPA BAA 01-26 BIO-COMP http//arep.med.harvar
d.edu
Technical challenges and risks DNA computing
so far focused on computing (which is slow
relative to silicon computing). This project, in
contrast, explores one of the few working
sub-nanometer input/output, memory
manufacturing processes. The major challenge is
integration with silicon computing while
maintaining the nano-size advantages.
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85 Single DNA molecule detection as amplified
polonies
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Proof of 26 steps of 0.34 nm each
Mean Intensity 58, 0.5 40, 6.5
0.3, 48 0.4, 43
FITC ( C )
CY3 ( T )
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Church Lab DNA memory I/O DoD impact

• Future of computing
• unattended bio-monitoring
• 3D memory arrays
• sub-nm3 per bit (currently 1014 nm3 )
• Input light, chemicals/toxins
• Output nm-scale positioning

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Church Lab DNA memory I/O Deliverables (full
version page 1 of 2)
 1 DNA memory 11 Find SSR upper-limit
oligonucleotides required for reversible,
irreversible, interwoven systems 12 Establish
alternative to Flp involving Lambda-Red
homologous recombination.   13 Design a variant
usable in whole organism cell-lineage
monitoring.  14 We will test an error-prone
polymerase in year two with DNA fluorescent-base
extension as the output. 15 A goal of 2
kilobits of storage at one bit-per-base in a year
3-4 option.   2 A/D Input in vivo allostery, in
vitro light 21 TetR or other sensor inputs will
be studied and integrated into a recombinational
system. 22 Effects of DNA length on Zinc finger
specificity, 5 domains harvested from a
genome. 23 Insert the cAMP-triggered ribozyme
at the 5' end of GFP such cAMP affects
translation. 24 Incorporation of a fluorescent
nucleotide by RNA polymerase under control of a
caged ATP or GTP. 25 Use these to regulate the
incorporation of dNTPs in the format of an
RNAPol-etaDNAPol fusion.     3 D/A Output
polonies micromirrors 31 3D arrays of DNAs
polymerases will use in situ sequencing in gels
chemically cleavable dNTPs. 32 Photocleavable
versions of the above will be tested. 33 A
microfluidic synthesis of 3000 oligomers
(30-mers) will be synthesized using a micromirror
array 34 Bridge a one-cm pattern with nm
precision using a chemi-optical combination.
Controlled polymerase stepper-positioners will
incorporate branching oligonucleotides at nm
scale. 35 Incorporation of electronic or
optical computing components will be attempted
(option)
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Deliverables (page 2 of 2)
  In vitro minigenome 41 A synthetic genome
replication dependent in vitro translation of GFP
or luciferase using E.coli lysates. 42 As
above but showing dependence on added EF-Tu of
measures of replication rate. 43 A highly
purified translation system will be used to
extend the results of 41 or 42. 44 A SSR or
recombination method will be tested for the
transfer of genes for expression in minigenomes.
45 We will develop a rolling circle primer
immobilization method. 46 We will assess the
doubling rates varying number of primers, or
amount of nicking.   Computational 51
Coordination with other DARPA BIO-COMP
participants. 52 The known 3D models will be
adapted to include any sequence changes initially
EF-Tu mutations.  53 Software for inclusion of
microscopic morphology, dynamics, and stochastics
will be developed. 54 As an alternative to the
direct allosteric design, we will develop a model
for signal transduction cascade, which will be
based on two-hybrid and microarray data 55
Metrics for optimality and quality assessment and
outlier identification will be developed and
refined.