Designing for bio-orthogonality and addressability in DNA and RNA origami

Designing for Addressability, Bio-orthogonality and
Abstrac7on Scalability at the Interface of
Compu7ng, Synthe7c Biology and Nanotechnology
Prof. Natalio Krasnogor

Interdisciplinary Compu7ng and Complex Biosystems (ICOS) Research Group
hEp://ico2s.org/

Centre For Synthe7c Biology and the Bioeconomy (CSBB)
hEps://www.ncl.ac.uk/csbb/
Centro Nacional de Biotecnologia – Madrid - 2018
@ico2s
@nkrasnogor

Outline
•  Mo7va7on
•  Designing bio-orthogonality and addressability
for DNA (RNA) origami
•  Designing for physiological condi7ons
•  Designing for abstrac7on scalability
•  Conclusions

Outline
•  Mo1va1on
•  Conclusions

Working the Interfaces
The
Cool
Stuﬀ
Compu1ng
Synthe1c
Biology
Nano
technology

Multiscale Computation in Nature
•  A Research Programme
Programmable algorithmic entry to
the vast world of nanoscale physical,
chemical & biological systems and
processes
ComputerScience
Information & Algorithms
Embedded behavior
Robustness
Uncertainty
Complexity
Tradeoffs
How does “The Logistics of Small Things” look like?
How does “The Decision Making in/with Small Things” take place?
How is “Uncertainty Handled by Small Things” ?
How to stack-up “abstractions” to achieve higher complexities programmatically?

•  2 nm in diameter
•  0.34 nm width of
nt
•  1 turn = 10.5bp
Stability based on:
•  base pairing
•  base stacking
DNA nanotechnology
dates back to ‘80
DNA as nanomaterial

DNA AND RNA ORIGAMI TECHNIQUE: MAIN CONCEPT

Rothemund, Folding DNA to create nanoscale shapes and pa4erns, Nature 2006

Douglas et al.
A Logic-Gated Nanorobot for
Targeted Transport of Molecular
Payloads, Science 2012

Linko and Dietz, The enabled state of DNA nanotechnology, Current Opinion in Biotechnology 2013

Inten7on
•  Develop DNA/RNA origami techniques that are cell-
compa7ble
–  As liEle biological crosstalk as possible
–  Folding buﬀer ≈ cell cytoplasm || colony/bioﬁlm extracellular media
–  Physiological temperatures
–  Addressability
–  Survivability
•  Why?
–  Synthesis of DNA/RNA origami nanostructures in vivo.
•  RNA origami as programmable loci to co-locate bio-processes (e.g.
biosynthesis pathways)
•  New kinds of post-transcrip7on, post-transla7on control devices
–  Cell-instructable origami nanostructures ex vivo.
•  New kinds of inter-cellular communica7on devices, mul7-cellular
organisa7on architects

Outline
•  Mo7va7on
•  Designing bio-orthogonality and
addressability for DNA (RNA) origami
•  Conclusions

Scaﬀold requirements
•  Biological orthogonality
•  Unique addressability


Scaﬀold sequence as a limi7ng
factor:
•  Biological parts

Scaﬀold sequence as a limi7ng
factor:
•  Biological parts
•  Sequence repe77ons
5 6 7 8 9 10 11 12 13
Length
0
100
101
102
103
104
Repeats
pUC19
6 9 12 15 18 21 24 27 30 33 36 39 42
Length
0
100
101
102
103
104 M13
5 6 7 8 9 10 11 12 13 14 15
Length
0
100
101
102
103
104
105 -phage

Superstring problem:
ﬁnd a shortest circular ‘supersequence’ that
contains all possible subsequences of length n
over given alphabet.

Nicolaas Govert de Bruijn

De Bruijn sequence for DNA alphabet
Genera7on
AAACAAGAATACCACGAC
TAGCAGGAGTATCATGAT
TCCCGCCTCGGCGTCTGC
TTGGGTGTTT(AA)

De Bruijn sequence for DNA alphabet
Genera7on
AAACAAGAACACGACTAG
CAGTATCATGATTCCCGCC
TCGTCTGCTTGGGTGTTT(
AA)
Bioparts
Filtering
TAC, GAG, GCG

DNA origami design
pUC19
2.6 kb

De Bruijn
2.6 kb

5.3 x 101409 unique sequences
roughly 1080 of par7cles in observable universe

De Bruijn Sequence

B(4,n): predeﬁned sub-sequences (of n nucleo7des length) that appear just once in the whole main sequence while working
with n long units (or longer).

B(4,3) S = “AAACAATAAGACCACTACGATCATTATGAGCAGTAGGCCCTCCGCTCCTGTGTCGGTTCTGGG”.

No 3-nucleo7de subsequence appears more than once, thus gran7ng the important unique addressability feature.
•  non-biological
•  highly-addressable
•  up to unique
addressability
•  circular

Energe7c op7misa7on
DBS Random

Two aspects of addressability

Staple addressability
188 domains 646 domains 4444 domains
0.0
0.2
0.4
0.6
0.8
1.0
Addressabilitymeasure Scaffold
DBS
natural

Scaﬀold addressability
small medium large
DNA origami size
0.0
0.2
0.4
0.6
0.8
1.0
Probability
origin
DBS
viral

Scaﬀold prepara7on protocols

• M13
• pUC19
• PCR circular
• PCR linear
• RNA/DNA hybrid

De Bruijn DNA scaﬀold prepara7on protocol


AFM imaging (pUC19)

AFM imaging (De Bruijn)

De Bruijn sequence encoded in a
plasmid
T7 RNA Polymerase
De Bruijn RNA scaﬀold prepara7on protocol


Labeling of scaffold DBS using Alexa 488 Ulysis®
Nucleic Acid Labeling kit
Purifica7on with Micro Bio-Spin®
P-30 spin column (BioRad)
Agarose gel electrophoresis imaging using Typhoon laser scanner (Alexa 488: excita7on 488 nm, emission 532 nm; EtBr:
excita7on 510 nm, emission 590 nm)
Agarose gel electrophoresis of Alexa 488
labeled DBS scaffold imaged before (len) and
aner (right) ethidium bromide staining. Lanes.
1: low range ssRNA ladder;
2: Alexa 488 labeled scaffold DBS;
3: not labeled scaffold DBS.
Alexa 488 EtBr
scaffold
De Bruijn scaffold stability in vivo

RNA DBS scaffold electropora7on in NEB 5-alpha electrocompetent E. coli (1.8 kV, 200 Ω, 25 μF)
Cells recovering for 25 or 40 minutes at 37 °C under shaking
Centrifuga7on, washing and cells embedding in agarose plugs
Cells lysis in agarose plugs
Agarose plugs loading into the wells of 1% agarose gel and imaging using Typhoon laser scanner (Alexa 488:
excita7on 488 nm, emission 532 nm; EtBr: excita7on 510 nm, emission 590 nm) .
Alexa 488 EtBr
scaffold
scaffold scaffold scaffold
1-4 no incuba7on; 5-8 10 min preincuba7on; 10 no electropora7on control
scaffold scaffold
De Bruijn scaffold stability in vivo

Outline
•  Mo7va7on
•  Conclusions

Expression in large quan77es

Forma7on of stable interac7ons

Control of gene expression
New tools for the programmable control of gene expression
RNA origami as organel-like structure opera7ng as an ar7ﬁcial
regulatory machine
Designing for Physiological Condi7ons: RNA
Advantages

Prepara7on of Chemically Modiﬁed RNA
Nanostructures

Endo et al., Chem. Eur. J. 2014, 20, 15330-15333, willey

Design and Synthesis of RNA Origami

•  Bio-orthogonality (biological
inert and uniquely
addressable sequences)
•  Physiologically compa7ble
folding at 37 °C
•  Assembly monitoring using a
split light up Broccoli aptamer
system

Broccoli Aptamer (1)

l  Ligand binding RNA sequence

l  Fluorogenic dye: DFHBI-1T, cell permeable with negligible
toxicity in living cells

l  Selec7on using combined SELEX/FACS approach.

(Filonov et al., J. Am. Chem. Soc. 2014, 136, 16299-16308)

Broccoli Aptamer (2)

l  Folding in vivo without the use of a tRNA scaﬀold required by Spinach
aptamer.

l  Lower dependance on magnesium for folding (robust imaging in E. coli
and without the need for addi7onal magnesium in media).

l  In vivo: Broccoli RNA imaging in E. coli.
Scale bar, 2 μm.

Alam et al., ACS Synth. Biol. 2017, 6, 1710-1721.
Split Broccoli for Visualising RNA-RNA Assembly In Vivo

DBS: bio-orthogonal De Bruijn sequence
Split Light-up Broccoli System: Design and oxRNA
Simula7on

F-30 Broccoli
' B i o - o r t h o g o n a l ' s t r u c t u r e
reengineered from ϕ29 three-way
junc7on mo7f
(Filonov et al. Chem. Biol. 2015 22,
649-660)

m-Fold Predicted Structures and Split System Design
l  8 base pairs of F-30 Arm 1
l  Elimina7on of the terminal 4 nt
loop UUCG
l  Elonga7on in 5' and 3' end with
RNA sequences complementary
to a pre-selected DBS

Light-up Split Aptamer: in gel imaging
Broccoli
Split1
Split2
Complementary
Split1/Split2 DBS
Broccoli
Split1
Split2
Compl.
Split1/Split2 DBS
Split1+Split2
Split1+Split2+Compl.
Split1/Split2 DBS
Broccoli
Split1
Split2
Compl.
Split1/Split2 DBS
Split1+Split2
Split1+Split2+Compl.
Split1/Split2 DBS

Fluorescence emission intensity (expressed in arbitrary units, a.u.) in 40
mM HEPES pH 7.4, 100 mM KCl, 1 mM MgCl
2
Split Aptamer System: Spectroﬂuorometer Measurements


Fluorescence emission intensity (expressed in arbitrary units, a.u.) of Split1,
Split2 and complementary Split1/Split2 DBS. Error bars indicate standard
devia7on (n=3). Limit of detec7on: 54 arbitrary units (a.u.)
Split Aptamer System: Spectroﬂuorometer Measurements
at Diﬀerent [c] and at 0.5 mM MgCl2

Fluorescence emission intensity (expressed in arbitrary units, a.u.) of background and
Broccoli aptamer (0.27μM) in aptamer buffer and in folding buffer with decreasing KCl
concentra7on
Influence of Potasium Chloride Concentra7on on Broccoli
Fluorescence Emission Intensity
Aptamer buffer Folding buffer

l  Bio-orthogonality

l  Folding at 37 °C (isothermal condi7ons)

l  Assembly monitoring through the
new light up split aptamer system

Light Up Aptamer Origami Nano-Ribbon

RNA Origami Folding:
Preliminary Screening by Gel Electrophoresis
6% TBE gel electrophoresis of well-folded two dimensional RNA origami
Scaﬀold
Staple strands
RNA origami
Puriﬁed
RNA origami

RNA Origami: AFM Imaging (1)
scale bar 20 nm; b: scale bar 10 nm

Scale bar 20 nm
Dimensions: 25.6 nm ± 2.7 nm, 5.3 nm ± 0.8 nm
RNA Origami: AFM Imaging (2)

Normalized fluorescence value: 0.7 (standard devia7on: ± 0.1, n=3)
Scaffold
Staple strands
Broccoli
Purified
RNA origami
Scaffold
Staple strands
Broccoli
Purified
RNA origami
Light-Up RNA Origami:
In Gel Imaging and Spectrumfluorometer
Measurements

STABILITY OF DBS RNA SCAFFOLD IN VIVO (1)
Agarose gel electrophoresis of A488 labeled DBS, no DBS1 and noDBS2 scaﬀold
sequences.
Ladder
A488 DBS
A488 no DBS1
Ladder
A488 DBS
A488 noDBS2
A488 no DBS1
A488 noDBS2

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
STABILITY OF DBS RNA SCAFFOLD IN VIVO (2)
RNA DBS electropora7on in E.coli
Cells recovery
Cells embedding in agarose plugs and lysis
Agarose plugs loading in wells (1% agarose gel)
A488 no DBS sequences were used as nega7ve control: both sequences do not show a dis7nct
band. Centro Nacional de Biotecnologia – Madrid - 2018

Outline
•  Mo7va7on
•  Conclusions

Living Cells Are Informa7on Processors
LeDuc et al. Towards an in
vivo biologically inspired
nanofactory. Nature (2007)

But What About Memory?
•  DNA has been used to store data.
•  DNA can store very large amount of data.
•  DNA is a durable, eﬃcient and cheap digital substrate.

•  DNA data structures for informa7on processing
•  Biological data with programma7c API
Figure 2: Data structures, and common operations to store and retrieve ordered information
A StackA Stack
B QueueB Queue
C TreeC Tree
Last In
First Out
push(data)
pop()
enqueue(data)
dequeue()
First In
First Out
data4
data3
data4
data2
data1
data1
data6
34
2
5
Sub-Tree
Root
Parent Node Child Node
Siblings
Leaf Node
data1
data2
data3
data4
data5
data6
data7
data8
data9
data10
graft(subtree)
prune(subtree)
add(data, parent)
search(data)
getparent(data)

A stack is a data register, two opera7ons: push and pop. You can push
values on the stack, and pop them from the stack. This happens in
LIFO (last in, ﬁrst out) order.

SIGNAL (X)
SIGNAL (Y)
SIGNAL (Y)
SIGNAL (X)
PUSHING POPPING
Last in First out
Stack Data Structure

Image by Ouldridge et al.
The rate constant of the strand-displacement reac7on varies over a factor of 106, from 1 M−1 s−1 to 6 × 106 M−1 s−1.
DNA hybridiza7on and DNA strand displacement

DNA “Bricks”
Smallest brick: 22nt Largest brick: 137nt
l* A
(S) Start
(P) Push
(Q) Pop
(R) Read
(X) Write_x
(TX) Report_x
(TY) Report_y
(Y) Write_y
B* C* A
w*
Y
w
i h
B C A*
A* B
d
C
d*
e
A B* C*
d*
e*
d
B* C* A
v*
X
v
g f
(Z) Releaser
m* l*
r X*
r Y*
(L) Linker
m l
DNAStrands

Stack Recorder - Single Molecule Opera7on
start
Last In
First Out
Recording 4 signals on stack
in order X(1), Y(1), X(2), Y(2)

start
push
Last In
First Out

Last In
First Out

signal X(1)
X(1)
Last In
First Out

X(1)
Last In
First Out
X(1)

push
X(1)
Last In
First Out
X(1)

signal Y(1)
X(1)
Last In
First Out
X(1)
Y(1)

X(1)
Y(1)
Last In
First Out
X(1)
Y(1)

Aner 4 signals pushed to stack:
X(1)
Y(1)
X(2)
Y(2)
Last In
First Out X(1)
Y(1)
X(2)
Y(2)

S
+ P
+ X
Desired Mul7-Molecule Scenario
All stack complexes in solu7on would have iden7cal state

G0
of binding
(kcal/mol)
DNA Sequence Op7misa7on
Domains on the DNA bricks
had their nucleo7de sequences op7mised
so that these mul7ple objec7ves were sa7sﬁed:
Single DNA strands folded into the
correct local topology
Pairs of DNA strands co-folded into the
correct stack topology
Desired reac7ons resulted in irreversible transforma7ons
Undesired reac7ons were minimised
Jerzy Kozyra, Harold Fellermann, Ben Shirt-Ediss, Annunziata
Lopiccolo, and Natalio Krasnogor. 2017. Op1mizing nucleic acid
sequences for a molecular data recorder. Proceedings of the GenePc
and EvoluPonary ComputaPon Conference (GECCO '17). ACM, New
York, NY, USA, 1145-1152. DOI: hEps://doi.org/
10.1145/3071178.3071345

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
Single Tube Experimental Results

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SP

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPX
1 signal

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPXP

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPXPX
2 signals

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPXPXP

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPXPXPX
3 signals

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPXPXPXP

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPXPXPXPX
4 signals

S
P
S
P
X
S
P
X
P
S
P
X
P
X
S
P
X
P
X
P
S
P
X
P
X
P
X
S
P
X
P
X
P
X
P
S
P
X
P
X
P
X
P
X
SPXPXPXPXP
…and so on

stepwise assembled tape with signals X-Y-X-Y-X

bio7nylated report strands with gold nanobeads bind to signal domains
signal loop backbone nanobeads reporter
16nm
3.4nm
3.7nm
5nm 10nm
Transmission electron microscope

Rule-Based Kine7c Model of DNA Chemistry
pop
push
read
X …stack
stack… push
stack… X …stack
stack… X push
stack… push
stack… push stack…
…stack
start push
X
read
pop
start
…stack
push
X …stack
+
+
+
(1)
(2)
(3)
(4) +
…stack + X
…stack +
…stack + pop push
Recording Signals
Popping Signals
(5)
All reac7ons
considered
irreversible
inert dsDNA product
inert dsDNA product
Two hybridisa7on
rate constants:
Two strand-
displacement
rate constants:

Recording Signals:
Model Parameter Space Explora:on
≠

Recording Signals:
Model Parameter Space Explora:on
≈

Recording: Approximate Kine7c Model Rate Constants
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
SP SPXP SPXPXP
Experiment Model

Actual Mul7-Molecule Scenario
S + P + X
— Complexes can have several isoforms
— Unintended side reac7ons take place
— DNA complexes have ﬁnite diﬀusion and reac7on rates
— Finite wait 7mes mean that chemistry is s7ll under kine7c control

You Can Build To Compute
Tetra-Pyridyl Porphyrin (TPyP) on Au(111)
Structural unit
to func1onalise
(~ 1nm)
With G. Terrazas, P.
Moriarty and N.
Chapness But: the smaller the
processor you build the
dumber it is!

Backbone
Self-assembly coun7ng
process
•  Blue porphyrin-7les act as counters1,2 “seeded” via
red porphyrin-7les
•  Backbones are spa7al limits controlling blue-
porphyrin-7les assembly
1 Q. Cheng et al. Op7mal self-assembly of counters at temperature two. In FoundaPons of Nanosciense, 2004.
2 P. Moisset. Computer aided search for op7mal self-assembly systems. In N. Krasnogor et al. (Eds.), Systems Self-Assembly MulPdisciplinary Snapshots, 2008.
m1
m2
Embedded Discrete Process of Computa1on (I)
Backbone
Es = 0.50
E11 = 1.00
E22 = 0.20
E12 = 0.20
Es = 0.60
E11 = 0.40
E22 = 0.20
E12 = 0.10

Embedded Discrete Process of Computa1on (II)
Checkers paEern
(spa7al interac7ons)
•  Highly ordered self-assembled structure
•  Spontaneous internal arrangements
•  Globally complex shape with locally
simple organisa7on
λ (y)
λ (y)
(x)
(ε) (ε)
(x)
q1
q2
ε, x, y Є [0, 1]
ε + x + y = 1
x >> ε >> y
Computed by a ﬁnite state
machine-like process
ε: probability of mistaking symbol
λ: new diagonal begins

Es = 0.50 E11 = E22 = 0.10 E12 = 0.40
Es = 0.50 E11 = E22 = 0.10 E12 = 0.30
Es = 0.50 E11 = E22 = 0.30 E12 = 0.40
Es = 0.50 E11 = E22 = E12 = 0.30
Diﬀerently programmed spa7al
interac7ons generate:
•  micro level features (order/
disorder)
•  macro level features (regular/
irregular shape)

Construc7on as a Mean of Informa7on Processing
l  DNA & RNA origami based on:
l  bio-orthogonal De Bruijn Sequence
l  unique addressability
l  RNA origami isothermal folding at 37 °C
•  The no toxicity of DFHBI-1T, its ability to assemble at 37°C
without ini7al thermal denatura7on and reduced magnesium
dependance è light-up split aptamer a poten7al tool for
protein-free ﬂuorescence detec7on of endogenous RNA in live
bacterial cells
l  DNA (& RNA) Data Structures may lead to a “full-stack” for in
vivo informa7on processing

Thank you
•  Victor for the invita7on
•  The brilliant PhD students and postdocs
•  UK’s EPSRC for funding
•  U. S7mming, U., J.Y. Gu - School of Chemistry, Newcastle University
•  K. Voitchovsky, L. Piantanida -School of Physics, Durham University
99 IPMU 2018 – Cadiz, Spain
hEps://portabolomics.ico2s.org/

Designing for bio-orthogonality and addressability in DNA and RNA origami

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