Artificial DNA in Living Cells

Floyd E. Romesberg, Ph.D. National Academy of Sciences Forum on 19 September 2014 Washington, DC Chemical Biology and Biophysics

• Efforts to expand the genetic alphabet by unnatural design and polymerase evolution • 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 DNA Polymerase B RNA Polymerase B B

AA* AA*

B B

Seminal work of Steve Benner (FAME) Peter Schultz (TSRI) Predominantly Hydrophobic (sugar & phosphate not shown)

S O NH N N N HO OH

BTp BFr IN BTz NAP 2MN 3MN DMN 1HN 2HN

O O S N O S O

N N N N N N N O N S N O N O N O N S P7AI PM7AI PPP 7OFP 4TFP 4OFP 7OTP 4OTP 7TFP

N N N N N N N N N N N N N N N N N

Neb M7AI 4M7AI 7AI PP ImPy Np 8Q

N

N O N O N O N O N O N O

PICS PIM PNICS ICS 5MICS MICS

N N N

N O N O N S N S N S

NICS oNICS SNICS SoNICS SICS Assays: Thermostability and Kinetics

5’-GCGTACXCATGCG-3’ 3’-CGCATGYGTACGC-5’ 0.39 Incorporation 0.38 Extension A 0.37 260 0.36

0.35

0.34 X N 0.33 0 40 50 60 70 80 90 T, ºC Structure of a First Generation Hydrophobic UBP

With D.Wemmer (UC Berkeley) and P. Schultz (TSRI) Second Generation Hydrophobic Nucleotides

BEN MM1 MM2 MM3 DM2 DM3 DM4 DM5 DM TM TM2 F F F F F F F F F F F

F F F

TMB 2FB 3FB 4FB 3,5DFB 2,3DFB 3,4DFB 2M3FB 3F4MB TFB Br CN Br CN N N N Br CN N O N O

2Br 3Br 4Br 2CN 3CN 4CN 2Py 3Py 4Py PYR 3MP O F O F F Cl

N O N O N O N O N O N O N O O

4MP 5MP 3MOP 4MOP 3FP 4FP 5FP 3CB 2OMB O O O O O

S S S S O O O O

3OMB 4OMB MMO1 MMO2 DMO Tp MTp ETp MOTp MFr DMFr SAR Issue

dYTP insertion

X Efficient dYTP Insertion: dYTP–minor groove hydrophobic group template dX–minor groove hydrophobic group

dNTP (extension dY:dX) Y X Efficient extension: Primer terminus dY–minor groove hydrogen-bond acceptor Template dX–minor groove hydrophobic group Two Screens of 3600 Candidate UBPs

Extension Screen

Full Length Screen SYBR +dNTPs +SYBR X X

X +dNTPs dYTP Y +SYBR Y X X SYBR 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 Frequency

dNaM dNaM dNaM dNaM Nucleotide position relative to dNaM Single Nucleotide Frequency

24.7%

18.7%

dNaM dNaM dNaM dNaM Nucleotide position relative to dNaM Correlation Analysis

dNaM dNaM dNaM dNaM

Nucleotide position relative to dNaM Dinucleotide Frequency

X XX

X X Dinucleotide Frequency

X X

2.3%

3.5%

X X Structural Studies of d5SICS-dNaM

With Tammy Dwyer (U. San Diego) Structural Studies of d5SICS-dNaM: Two Questions Structural Studies of d5SICS-dNaM

With K. Betz & A. Marx Structural Studies of d5SICS-dNaM Expansion of the Genetic Alphabet/Code

B DNA Polymerase B RNA Polymerase B B

AA* AA*

B B 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 DNA Polymerase B RNA Polymerase B B

AA* AA*

B B 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.

play