Nucleic Acids in Medicine & Biotechnology

CHEM 90628

Spring 2022

Topic #9
DNA Robots and Data Storage

March 28, 2022

Lecture
DNA robots and data storage Outline of subtopics

• DNA nanotechnology
– Introduction
– DNA nanostructures
– Applications (e.g. nanorobots and nanomedicine)
• RNA nanotechnology
– Introduction
– RNA nanostructures
– Applications (e.g. nanomedicine)
• RNP nanotechnology & applications
• DNA data storage
DNA robots and data storage Introduction to nanotechnology

Nucleic acid nanotechnology deals with the design, study and application of
synthetic structures based on DNA

In today’s lecture, we’ll focus on DNA molecules being used as

• Construction material to assemble accurate, biocompatible and functional
nanostructures (e.g. robots)
• A high-capacity information storage

Aim of structural DNA nanotechnology is to create 3D objects with both precise

architecture and function that can be programmed via their sequence

Ideally, nanostructures
• are 1-100 nm
• can self assemble
• can be used for diverse applications in biology, chemistry and physics
DNA robots and data storage Introduction to nanotechnology

DNA is well suited to construct a

wide variety of architectures on the
nanoscale level

• Diameter of dsDNA is ~2 nm
• Structurally repeating unit with
helical pitch of ~3.4-3.6 nm
• Rigid molecule with a
persistence length of ~50 nm
• Watson-Crick base pairing
properties
• ssDNA overhangs (aka ‘sticky
ends’) enable hierarchical
assembly by acting as glue

But, it is an inherently linear

molecule so how is multi-
dimensionality achieved?
DNA robots and data storage Introduction to nanotechnology

Nadrian Seeman was a crystallographer, who

started with the original goal of DNA-assisted
structure determination of difficult-to-crystallize
molecules in the 1980s

Seeman was inspired by Holliday junctions

Four-way junctions can serve as a construction

unit and this marked the beginning of DNA
nanotechnology Nadrian “Ned” Seeman
New York University

Founding Father of Structural

DNA Nanotechnology
DNA robots and data storage DNA nanotechnology

Number of arms around junction could be shortened to 3 or expanded to 5, 6, 8, or

12 arms

Inherent flexibility of multi-way junctions made them suitable for constructing

polyhedra

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

The next advance was “DNA

origami”, which is a method to
build nanostructures by folding
a long, single strand of DNA
using hundreds of short
“staples” made of
complementary DNA strands

Origami method initially used to

create planar structures but
was quickly adapted to make
3D shapes

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

Some notable examples of objects made using DNA origami

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

Alternative methods of large-scale assembly are based on ssDNA tiles containing

4 domains

Adjacent ssDNA tiles connect to each other via base pairing with complementary
domains and continue to form DNA lattices composed of parallel DNA helices

Molecular canvas
strategy is where
selective ssDNA tiles
from a pool of tiles
assemble to form a
specific pattern

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

Alternative methods of large-scale assembly are based on ssDNA tiles containing

4 domains

Adjacent ssDNA tiles connect to each other via base pairing with complementary
domains and continue to form DNA lattices composed of parallel DNA helices

DNA brick strategy

creates lego-like
building blocks

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

Blunt end base stacking and shape complementarity can also be used to
construct nanostructures

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

Few examples of protein-mediated assembly of DNA nanostructures

DNA-protein nanowire, proteins act as ‘staples’

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

Size of DNA origami nanostructures is limited by length of scaffold strand

Most constructed using the M13 ssDNA scaffold (~7,000 nts)

Possible to create scaffolds ranging from ~700-50,000 nts

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage DNA nanotechnology

Assembly of nanostructures is not high tech

Isothermal assembly is possible, especially for protein-mediated assembly

DNA robots and data storage DNA nanotechnology

Various computational tools are available to design DNA nanostructures

DNA robots and data storage Summary: DNA nanotechnology

The field of DNA

nanotechnology is
expansive and has
evolved remarkably
in the past ~35
years

Nanostructures can
be assembled
based on
• Hybridization
• Base
stacking/shape
complementarity
• Protein-DNA
interactions

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage Applications

DNA
nanostructures
used to
assemble
fluorescent
materials (e.g.
quantum dots)

DNA can be
conjugated to
fluorophore,
which will be
spatially
distributed in
known locations
with high
precision
• fluorescent
nanoscale
rulers
• sensors
Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001
DNA robots and data storage Applications

DNA
nanostructures
have been used to
study biophysical
properties of other
molecules

High-precision
positioning of
molecules
achieved with DNA
origami hinge

Hinged DNA
origami used to
study nucleosome
unwrapping,
stability, and the
forces between
nucleosomes

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage Applications

DNA nanostructures and scaffolds have been used for macromolecular structure
determination (NMR and cryoEM) by facilitating protein binding, controlling
position and orientation

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage Applications
DNA robots and data storage Applications

DNA nanotechnology has also produced DNA-based machines, which can be

used as tools to control intra- or intercellular functions

DNA robotics are physically active molecular devices composed of DNA

Walking is driven by repetition of energy cascades via

• Strand hybridization
• Enzymatic reactions
• Environment-induced conformational changes
DNA robots and data storage Applications

DNA walker known as a

molecular spider is
directed across surface
DNA robots and data storage Applications

Multiple DNA robots perform parallel sorting task on DNA origami surface

DNA robots
independently execute
operations via
hybridization reactions
• cargo pickup
• random movement
to adjacent stepping
stones
• cargo drop off

Each robot has

• foot domain for
walking
• Arm/hand domain
for picking up and
dropping off cargo
DNA robots and data storage Applications
DNA robots and data storage Applications

DNA nanorobot is a
hexagonal barrel that
consists of hinged DNA
origami with protruding
ssDNA to anchor the
molecular payload (antibody
or gold nanoparticles)

DNA origami held together by

DNA duplexes to form a
clam-shaped nanostructure,
which encloses payload

DNA duplexes are aptamer

sequences that act as ‘locks’
DNA robots and data storage Applications

The ‘keys’ are cell-generated

biomarkers (PDGF)

Formation of aptamer-
substrate complex unlocks
origami-based shell

Released payload stimulates

intracellular function
DNA robots and data storage Applications

DNA nanorobots are able to

• carry molecular loads
• transport chemical to cells
• activate intracellular transformations
DNA robots and data storage Summary: DNA nanotechnology applications

Clever manipulation
of DNA based on
well-known
properties

DNA
nanostructures can
be dynamic, such
as robots

Reading
assignment will
focus on a DNA
nanorobot used for
cancer

Xavier and Chandrasekaran (2018) Nanotechnology 29: 062001

DNA robots and data storage Outline of subtopics

• DNA nanotechnology
– Introduction
– DNA nanostructures
– Applications (e.g. nanorobots and nanomedicine)
• RNA nanotechnology
– Introduction
– RNA nanostructures
– Applications (e.g. nanomedicine)
• RNP nanotechnology & applications
• DNA data storage
DNA robots and data storage RNA nanotechnology

RNA architectonics is the scientific study of the principles of RNA architecture

with the aim of constructing RNA nanostructures of any arbitrary size and shape

Additional terminology commonly used in the field

DNA robots and data storage RNA nanotechnology

DNA and RNA share general features along with unique chemical and structural
properties

Distinct approaches to nanostructure design and assembly

DNA sequence
design to satisfy
domain
complementarity
combined with
Holliday junction
motifs

RNA exploits
conserved,
naturally evolved
motifs with
predictable
tertiary
structures
DNA robots and data storage RNA nanotechnology

Compare chemical, structural and supramolecular modularity of DNA and RNA

Jaeger & Chworos (2006) Curr Opin Struct Biol 16: 531-543
DNA robots and data storage RNA nanotechnology

Study fueled by increase in RNA structural information

• ribosome
• RNase P
• group I and
group II introns
• spliceosome

RNA tertiary
folding and
assembly
principles are
significantly
different from
those of DNA and
offer new
possibilities for
the rational design
of complex
nanoarchitectures

Jaeger & Chworos (2006) Curr Opin Struct Biol 16: 531-543
DNA robots and data storage RNA nanotechnology

Extensive analysis of RNA and RNP structures has yielded various RNA parts
useful for RNA architectonics
• Secondary: junctions involve points of contact where multiple helices converge
• Tertiary shows interacting motifs

Grabow & Jaeger (2014) Acc Chem Res 47: 1871-1880

 DNA robots and data storage RNA nanotechnology

Structural and interacting motifs (see previous slide) can be combined

Grabow & Jaeger (2014) Acc Chem Res 47: 1871-1880

 DNA robots and data storage RNA nanotechnology

Heart
shapes
require
complex
collection
of
different
bends
and
coaxial
stacks

Geary et al. (2017) Nano Letters 17: 7095-7101

DNA robots and data storage RNA nanotechnology

What assembly
strategies are
used for RNA
nanostructures?

Grabow & Jaeger (2014) Acc Chem Res 47: 1871-1880

DNA robots and data storage RNA nanotechnology

(a) Single-step self assembly is where all molecules are mixed together and
annealed via slow cooling (most common for DNA)
(b) Stepwise hierarchical self assembly is where specific sets of molecules are
pre-assembled before mixing to form final architecture
(c) Scaffolded self
assembly (or
scaffolded RNA
origami) is where
long ssRNA is
folded into
arbitrary shape
with small oligos
acting as ‘staples’

Jaeger & Chworos (2006) Curr Opin Struct Biol 16: 531-543
 DNA robots and data storage RNA nanotechnology

Scaffolds are size

controllable

Length of “line region”

can be
shortened/extended
by one pitch of dsRNA
(A-form)

Grabow & Jaeger (2014) Acc Chem Res

47: 1871-1880
DNA robots and data storage RNA nanotechnology

Lee et al. (2015) Nano Today 10: 631-655

DNA robots and data storage Applications

RNA nanoparticles can be developed into targeted drug delivery systems

Fusion constructs
• RNA-based therapeutics (e.g. siRNA, miRNA)
• Targeting ligands (e.g. aptamers)

Conjugated to 5’/3’ ends

• Chemical ligands (e.g. folate, galactose)
• Chemotherapeutic drugs

Fine tune pharmacological profile in vivo by adjusting size, shape, and other
properties of RNA nanoparticle
DNA robots and data storage Applications

pRNA-3WJ nanoparticles
• Folate as targeting ligand
• Alexa 647 for imaging
• siRNA targeting BRCAA1 (breast cancer-associated antigen 1), which is
overexpressed in gastric cancer

RNA nanoparticles internalized into cells via folate receptor-mediated endocytosis

RISC processes BRCAA1 siRNA region

Hague et al. (2018) WIREs RNA 9: e1452

DNA robots and data storage Applications

pRNA-3WJ nanoparticles
• HER2 as targeting aptamer
• siRNAs targeting MED1 (ER coactivator Mediator Subunit 1), which is a key
tissue-specific mediator of breast cancer metastasis and therapeutic resistance

RNA nanoparticles deliver siRNA to HER2-overexpressing human breast cancer

cells

Hague et al. (2018) WIREs RNA 9: e1452

DNA robots and data storage Applications
 DNA robots and data storage Applications

Assembly of RNA nanoparticles in cells or in vivo has been challenging due to

• Instability of RNA after expression
• RNA misfolding

pRNA-3WJ motif is a robust scaffold that undergoes spontaneous assembly

Hague et al. (2018) WIREs RNA 9: e1452

DNA robots and data storage Applications

RNA origami in real time

RNA synthesized as continuous strands and

can fold to form modular units

Stages of RNA cotranscriptional folding

for an RNA tile
1. First hairpin stem-loop folds upon
synthesis (gray box)
2. More hairpin loops form
3. First domain of tile (i.e. modular building
block) is complete to interact with a
second domain once it is synthesized
4. Tertiary contacts (e.g. kissing loop, teal
box) between two domains

Potentially useful tool for synthetic biologists

DNA robots and data storage Applications

RNA origami in real time … another schematic

DNA robots and data storage RNA nanotechnology

Cotranscriptional folding is an approach specific for RNA

DNA robots and data storage RNA nanotechnology

Comparison of methodologies used in DNA vs RNA nanostructures

DNA robots and data storage RNA nanotechnology

Challenges remaining in the field

• RNA nanoparticles can get trapped in endosomes, which can diminish
therapeutic efficacy

• User-friendly software to design RNA nanoparticles/nanostructures

• RNA motif databases

• Chemical synthesis of RNA strands (large-scale methods, modified RNA and >80
bases)

• Large-scale purification methods

Hague et al. (2018) WIREs RNA 9: e1452

DNA robots and data storage Summary: RNA nanotechnology

Workflow of RNA tectonics

Jaeger & Chworos (2006) Curr Opin Struct Biol 16: 531-543
DNA robots and data storage Outline of subtopics

• DNA nanotechnology
– Introduction
– DNA nanostructures
– Applications (e.g. nanorobots and nanomedicine)
• RNA nanotechnology
– Introduction
– RNA nanostructures
– Applications (e.g. nanomedicine)
• RNP nanotechnology & applications
• DNA data storage
 DNA robots and data storage RNP nanostructures

So we’ve seen what nanostructures can form using DNA and RNA--what about RNPs?

Ohno & Saito (2016) Prog Mol Biol Trans Sci 139: 165
DNA robots and data storage RNP nanostructures

Ribosome is a rich source

for RNP modules

L7-box C/D triangle

• L7Ae protein
• Box C/D kink-turn RNA
motif

L1-rRNA square
L1 ribosomal protein
induces RNA to bend at a
right angle
• Unilateral: proteins on
same side
• Flip-flop: two proteins
on each face

Stability improved with PS

linkages and 2’-dF

Ohno & Saito (2016) Prog Mol Biol Trans Sci 139: 165
DNA robots and data storage RNP nanostructures

RNP nanostructure could be used as a carrier for any functional molecule

RNA can be attached directly onto RNA region of nanostructure

L7-triangle used as an siRNA carrier

Knockdown efficiency of GFP similar to traditional shRNA

Ohno & Saito (2016) Prog Mol Biol Trans Sci 139: 165
DNA robots and data storage RNP nanostructures

Malachite green (MG) aptamer was added to L1-square

MG aptamer binds to a fluorescent dye

In theory, could also add a functional RNA (e.g. ribozyme) or ssRNA to hybridize
with DNA

Ohno & Saito (2016) Prog Mol Biol Trans Sci 139: 165
DNA robots and data storage RNP nanostructures

Proteins could also be loaded

onto RNP nanostructure

Incorporation via fusion proteins

has been tried using the L7-
triangle

GFP, Protein G, or HER2 affibody

fused to L7Ae protein

SKBR3 is an HER2-positive
breast cancer cell line

Ohno & Saito (2016) Prog Mol Biol Trans Sci 139: 165
DNA robots and data storage Outline of subtopics

• DNA nanotechnology
– Introduction
– DNA nanostructures
– Applications (e.g. nanorobots and nanomedicine)
• RNA nanotechnology
– Introduction
– RNA nanostructures
– Applications (e.g. nanomedicine)
• RNP nanotechnology & applications
• DNA data storage
DNA robots and data storage The problem

Currently using magnetic storage technology with petabyte (1 PB = 1 million GB)

and exabyte (1 EB = 1 billion GB) capacities

Growing gap between volume of digital data being created and available storage
capacities

Current storage limited by

• storage density
• energy consumption
• footprint
• lifetime

Optical data storage first

emerged in 1980s
DNA robots and data storage Why DNA?

DNA is an attractive storage medium

Idea introduced early in 1960s

DNA has potential because

• It is incredibly stable (hundreds of thousands of years)
• It has a proven track record (life)
• It is volumetric rather than planar
• It is incredibly dense: store one bit per base and one base is a few atoms
DNA robots and data storage Outline of subtopics

The first word of Richard Feynman’s suggested message to future civilizations

rendered in 3 different DNA formats

Storage density depends on encoding schemes

Theoretically, DNA can encode two bits/nucleotide = 455 EB/g of ssDNA

Theoretical limit of volumetric density is above 10 EB/mm3

Current methods have only been able to use a fraction of theoretical maximum
DNA robots and data storage Why DNA?
DNA robots and data storage Fountain

Fountain approaches the theoretical maximum information stored per nucleotide

DNA robots and data storage Random Access - Organick et al.

Store 35 files (>200 MB) in >13,000,000 DNA oligos

A comparison to prior work shows that coding scheme has similar logical
redundancy but requires lower sequencing coverage to recover files
DNA robots and data storage Random Access - Organick et al.

Primer marking enables faster retrieval

DNA robots and data storage
DNA robots and data storage

Lifetime is the period of time for which technology can reliably store and
retrieve data

Lifetime is an important consideration for various reasons

• frequency of investment into new storage
• Operational cost
• Maintenance cost
• Big data centers prefer longer lifetimes

DNA half-life is 500 y in harsh

conditions (increased by
protecting DNA in spores,
Deinococcus radiodurans)

Most energy consumed

depends on DNA synthesizers
and sequencers
DNA robots and data storage

What are the drawbacks?

DNA synthesis and

sequencing
• Is vulnerable to various
errors (substitutions,
insertions, deletions)
• Is time consuming and
lowers throughput

Cost of DNA synthesis and

sequencing are declining so
it is believed that DNA
storage will be cost effective
in coming years

Transfer rates and access

times are slow

Use PCR to read only

selected region of interest
DNA robots and data storage CRISPR and CAMERA

Biologists studying cells would like to have a data

recorder to better understand processes, such as
cancer, aging, environment, and embryonic
development (akin to airplane flight recorder)

CRISPR introduces random errors, which can be used

as markers or barcodes to track how cells
“differentiate” from one state to another

CAMERA = CRISPR-mediated analog multi-event

recording apparatus

CAMERA eliminates stochastic mixture and also

records whether a cell experiences a stimulus, by how
much and for how long

CAMERA can record exposure to light, antibiotics and

viral infection
DNA robots and data storage CRISPR and CAMERA

CAMERA = CRISPR-mediated analog multi-event

recording apparatus

Two forms of CAMERA

CAMERA1
• In bacteria, use two “recorder” plasmids (R1 and
R2) that settle at a stable ratio
• Another plasmid with Cas9 and gRNA
• CRISPR machinery will target R1 for destruction
when bacterial cell experiences a specific stimulus
(e.g. antibiotic)
• After stimuli, sequence plasmids and measure
R1:R2 ratio to determine how long cells had been
exposed to drug
DNA robots and data storage CRISPR and CAMERA

CAMERA = CRISPR-mediated analog multi-event

recording apparatus

Two forms of CAMERA

CAMERA2
• dCas9 (catalytically dead mutant) linked to cytidine
deaminase, which converts C to T
• To record an event, gRNA shuttles cytidine
deaminase to a “safe harbor” gene, whose DNA
can be altered without harming the cell
• CAMERA turned on in presence of Wnt activity (the
stimulus), inscribing a record of signals in the safe
harbor gene

This study used as few as 10 cells, ideally want to be

single cell

CAMERA can also erase recorded information

Next, need to show it can work in animal model

DNA robots and data storage CRISPR and movies
DNA robots and data storage Summary: data storage

DNA holds much potential as a

medium to store data

In your lifetime, do you expect

DNA to become the storage
medium of choice? Why or why
not?

If yes, about what year would you

predict technology to be ready?

If no, what will keep the

technology from the mainstream?
DNA robots and data storage

Assignment for Friday (4/1)

Read the following research articles:

1. A DNA nanorobot functions as a

cancer therapeutic in response
to a molecular trigger in vivo
(2018) Nature Biotechnology

2. Smart cancer therapy with DNA

origami (2018) Nature
Biotechnology (News and Views)

Trailer:
https://www.youtube.com/watch?v=y Reminders
h304Z88AW8 • April 8th: Third draft of mini-grant
proposal is due
• April 11th: Start student
presentations