Artificial DNA in Living Cells

Floyd E. Romesberg, Ph.D.

National Academy of Sciences Forum on Synthetic Biology

19 September 2014

Washington, DC

• Efforts to expand the genetic

alphabet by unnatural base pair

design and polymerase evolution

Chemical Biology and Biophysics

• Novel evolution-inspired

approaches to antibiotic

discovery

• Biophysical characterization

of how protein dynamics are

evolved for biological

function

Expansion of the Genetic Alphabet/Code

B

B

B B DNA Polymerase RNA Polymerase

AA*

B

B

AA*

Seminal work of Steve Benner (FAME) Peter Schultz (TSRI)

Predominantly Hydrophobic Nucleotides

(sugar & phosphate not shown)

S

BTp

O

BFr

NH

IN

NN

N

BTz NAP 2MN 3MN DMN 1HN 2HN

OHHO

N

O

O

7OFP

N

O

S

4TFP

N

O

O

4OFP

N

S

O

7OTP

N

S

O

4OTP

N

O

S

7TFP

NN N

N

NN

N

NNNN

NN

N NN

7AI PP ImPyNeb M7AI 4M7AI

NN N

N

NNN

P7AI PM7AI PPP

N

Np 8Q

N ON O

PICS

N O

PIM

N O

N

PNICS ICS

N O

5MICS

N O

MICS

N S

SICS

N O

N

NICS

N O

N

oNICS

N S

N

SNICS

N S

SoNICS

Assays: Thermostability and Kinetics

0.33

0.34

0.35

0.36

0.37

0.38

0.39

0 40 50 60 70 80 90

A 260

T, ºC

5’-GCGTACXCATGCG-3’ 3’-CGCATGYGTACGC-5’

X N

Extension

Incorporation

Structure of a First Generation

Hydrophobic UBP

With D.Wemmer (UC Berkeley) and P. Schultz (TSRI)

Second Generation Hydrophobic Nucleotides

BEN MM1 MM3MM2 DM3 DM5DM4DM2

F

2FB 4FB

F

F

3FB

F

2,3DFB 2M3FB3,4DFB

F

3,5DFB

F

F

F

DM TM2TM

3F4MB TFB

F

F

FF F

F

2Br 3Br 2CN4Br 4CN

N

N

3Py2Py3CN

N

4Py

N

PYR

Br

Br

Br

CN

CN

CN

O N

3MP

N

4MP

N N

4MOP5MP

N N

4FP

N

5FP3FP

N

3MOP

F

F

O

O

O O O

O

O O O

FO

3CB

Cl

2OMB

3OMB MMO24OMB DMOMMO1

O

O

O

O O

O

O

S

MTp

S

ETp

S

MOTp

O

MFr

S

Tp

O

O

DMFr

TMB

SAR Issue

X

dYTP insertion

dNTP (extension dY:dX)

X Y

Efficient extension: Primer terminus dY–minor groove hydrogen-bond acceptor

Template dX–minor groove hydrophobic group

Efficient dYTP Insertion: dYTP–minor groove hydrophobic group

template dX–minor groove hydrophobic group

Two Screens of 3600 Candidate UBPs

Extension Screen

Full Length Screen

X

X

XY

+dNTPs

+dNTPs dYTP

+SYBR

+SYBR

XY

X

SYBR

SYBRSYBR

A Family of UBPs that are Efficiently

Replicated and Transcribed in Vitro

<<

<

Sequencing Analysis of Replication Fidelity

Calibration Curve for

d5SICS-dNaM Replication

1 0 % natu ral

L R

OneTaq PCR with d5SICS-dNaM

DNA Amplification Efficiency (%) Fidelity (%)

ACT b GTG 2.5×1012 97 99.945 ± 0.016

GTC b GGT 1.5×1012 95 99.874 ± 0.035

AGC b CGT 3.5×1012 96 99.930 ± 0.003

CCG b GAA 8.1×1012 >99 99.664 ± 0.037

GTA b TGT 3.1×1012 95 99.987 ± 0.021

AGA b AGT 8.5×1012 >99 >99.98

CCT b AAA 8.4×1012 >99 99.866 ± 0.014

GGT b TCC 2.6×1012 94 99.958 ± 0.012

ACT b b GTG 3.8×1012 96 ≈ 99.5

ACT b A b GTG 2.7×1012 94 ≈ 99.5

ACT b GTGACT b GTG 2.0×1012 93 99.47

NNN b NNN 4.8×1012 97 99.925 ± 0.008

PCR Selection

X = 5SICS

Y = NaM

Single Nucleotide Frequency

dNaM dNaM dNaM dNaM

Nucleotide position relative to dNaM

Single Nucleotide Frequency

24.7%

18.7%

Nucleotide position relative to dNaM

dNaM dNaM dNaM dNaM

Correlation Analysis

dNaM dNaM dNaM dNaM

Nucleotide position relative to dNaM

Dinucleotide Frequency

X X X

X X

Dinucleotide Frequency

3.5%

2.3%

X X

X X

Structural Studies of d5SICS-dNaM

With Tammy Dwyer (U. San Diego)

Structural Studies of d5SICS-dNaM:

Two Questions

With K. Betz & A. Marx

Structural Studies of d5SICS-dNaM

Structural Studies of d5SICS-dNaM

Expansion of the Genetic Alphabet/Code

B

B

B B DNA Polymerase RNA Polymerase

AA*

B

B

AA*

Extracellular Degradation

of dNaM and d5SICS

Activity of Phaeodactylum tricornutum NTT

(PtNTT2) in E. coli (dATP uptake)

Transporter 1

Transporter 2

Transporter 3

Transporter 4

Transporter 5

Transporter 6

Transporter 7

Transporter 8

PtNTT2-Mediated Uptake in E. coli

pACS and pINF (X=NaM, Y=TPT3)

d5SICS-dNaM and dTPT3-dNaM

The Experiment

Transform E. coli with pACS, induce PtNTT2, add dXTPs,

transform with pINF, grow, recover pINF and characterize

Controls: transform with pUC19 instead of pINF

do not induce PtNTT2

do not add dXTPs

Analysis of pINF After Growth in E. coli

(15 h, 22 Doublings, 107-Fold Amplification)

Sequencing Analysis of pINF After Growth in E. coli

(15 h, 22 Doublings, 107-Fold Amplification)

Global Nucleoside Content in pINF and

pUC19 via LC-MS/MS

With Ivan Correa, Nan Dai, and Jeremy Foster (NEB)

Expansion of the Genetic Alphabet/Code

B

B

B B DNA Polymerase RNA Polymerase

AA*

B

B

AA*

Reddit AMA

• 773 comments; score of 3056 (93% upvoted)

• Much of the dialogue was technical, unlike what was generated by the

NYT and NPR stories

Jim Thomas Comments on

Legal/Regulatory Implications

“The arrival of this unprecedented ‘alien’ life

form could in time have far-reaching ethical,

legal and regulatory implications,” Jim Thomas

of the ETC Group, a Canadian advocacy

organization, said in an email. “While synthetic

biologists invent new ways to monkey with the

fundamentals of life, governments haven’t

even been able to cobble together the basics of

oversight, assessment or regulation for this

surging field.”

Comments in Response to

Pollack’s NYT Article

Comments in Response to

Pollack’s NYT Article

Comments in Response NPR Health Blog

Objections to Comparison with Nature

Imagine writing a story with a language with only four

letters. A fifth and sixth letter would let you write more

Interesting stories.

Objections to Comparison with Nature

Imagine writing a story with a language with only four

letters. A fifth and sixth letter would let you write more

Interesting stories.

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