Theory-definition of life A self-sustaining chemical system capable of Darwinian evolution

Carol Cleland Carl Sagan Our adopting this theory-definition tells you that we think that the only way to generate matter having the properties that we value in life is through a process of: (a) replication, (b) where replicates have non-anticipatory imperfections, that (c) are themselves replicable. Natural selection does the rest. What does Darwinism need for an informational polymer? Building blocks must fit Schrödinger’s aperiodic crystal Must keep structure constant with changing information Needs a repeating backbone charge (negative or positive) Must keep properties constant with changing information What does Darwinism need for an informational polymer? Building blocks must fit Schrödinger’s aperiodic crystal Must keep structure constant with changing information Needs a repeating backbone charge (negative or positive) Must keep properties constant with changing information

Schrödinger in 1943 knew nothing about DNA. But he knew that simple binding cannot guarantee fidelity of information transfer needed for biology. For that, Schrödinger needed the physics of phase transitions. For that, exchangeable informational building blocks must all have the same size/shape. They must all fit in an aperiodic crystal structure.

This eliminates some polymer “concepts” For example, many suggest that inter- base hydrogen bonding is not important

Myron Goodman USC

Not an incoherent view • Hydrophilic part of the DNA is the phosphates, on outside. • The nucleobases are the hydrophobic parts. • Bases move into stacked Schrödinger aperiodic “crystal”. • This requires uniform size/shape (= geometry). • Hydrophobic interactions drive duplex formation energy. • Hydrogen bonds incidental; can be dispensed with. Goodman’s view influenced modern synthetic biology Hydrophobic size complementary added base pairs Eric Kool Ichiro Hirao Floyd Romesberg

Hydrophobic pairs replicated by DNA polymerases Floyd got E. coli to replicate a plasmid with a hydrophobic pair. But when not constrained by a polymerase active site...

… the double helix is distorted when size- complementary hydrophobic bases are not joined by hydrogen bonds.

Oily structures intercalate, distort DNA. This defeats the Schrödinger aperiodic crystal structure. The universal informational genetic polymer will not exclusively rely on hydrophobic forces for self-assembly Synthesis lest us test this with genetic systems that completely exploits H-bonds puADD pyADD puDAA pyDAA H H Donor N H O N Acceptor AcceptorO2N O H N Donor N N H N N Donor N H N N Acceptor Acceptor R Donor R N N N Acceptor R O H N Donor Donor R N H O Acceptor G C H V H J pyADA puDAD pyDAD puADA H H Acceptor O H N N Donor Donor N H O Acceptor N N Donor N H N N Acceptor N H N N Donor R Acceptor R N N N R O H N Donor R N H O Acceptor Acceptor Donor T H amA K H X

pyAAD puDDA pyDDA puAAD H H Tag H C O Acceptor 3 H N N Donor Donor O2N N H O Acceptor N N H N N Donor Donor N H N N Acceptor R R Acceptor N N N Donor R N H O Acceptor H N S B Acceptor R O Donor H Z H P Artificially Expanded Genetic Information System (AEGIS) These building blocks are interchangeable in size and shape

(A) Georgiadis host-guest A with reverse transcriptase (cyan/green) bound 16mer hachimoji DNA. (B) Hachimoji DNA PB (green), PC (red), PP (blue) B C D E atop GC DNA. (C) Self-complementary P Z CTTATPBTASZATAAG Z P Z P S B S B Z P (D) Self-complementary B S S B S B P CTTAPCBTASGZTAAG. P Z P Z Z Z P (E) Self-complementary CTTATPPSBZZATAAG

Aperiodic crystal violated most by A:T, not hachimoji pair What does Darwinism need?

A genetic system able to change encoded information without changing its regular structure (= Schrödinger) Homochirality is a derivative of this A genetic system able to change its encoded information content without changing its physical chemical behavior Such as: its solubility, its molecular recognition, reactivity

Lessons from terran biochemistry In proteins, polysaccharides, most every other class of molecules, including abiological polymers, physical behavior Sickle cell, 1 and reactivity change dramatically even with amino acid small structure changes. change Polyelectrolyte allows DNA to be Darwinian Can change its information without changing its behavior

sugar as scaffold (!?) - charged phosphate backbone (!?) O - O O - O O - P O O P O O P O O O O O O X O P O X O X T/U X C O O N A N R N N G N H N O N N O H N O N H N N N H base pairs H H H N H H N O H H H H N N O N N O N O H H N G A O N N N N H R N N N O N N T/U O O O C

O O O P O O O O O P P P - O O - - - O O O O O O Synthesis again supports this. Made DNA without repeating charges

O O O O N N P NH S NH -O O O N N NH2 N N NH2 O O NH2 NH2 O N O N - P O N S N O O O N N H N N H O O

NH2 NH2 O O O - P N S N O O O N O N O O O Natural Oligosulfone Zhen Huang DNA O Christian Schneider Rule-based molecular evolution fails n > 6 Richert, C., Roughton, A. L., Benner, S. A. (1996) Nonionic analogs of RNA with dimethylene sulfone bridges. J. Am. Chem. Soc. 118, 4518

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ribo-ASO2USO2GSO2GSO2USO2CSO2ASO2U Longer oligosulfones fold (like proteins); Different oligosulfone sequences have very different properties (like proteins). Compare peptides, with repeating dipole

δ+ H R1 R2 R3 + + + + N δ δ δ δ C − δ− δ− δ− δ δ− O + H R6 R5 δ δ+ δ+ δ+ N R4 C − − − δ δ δ δ− O (above) A backbone with a repeating dipole easily folds (below) A backbone with a repeating charge extends to template R1 R2 R3 R4 R5 ------

Polyelectrolyte backbone slows folding, allows templating. = polyelectrolytes are soluble in water. Sulfones fold, no Watson Crick rules

O USO2G N H O R N N R O H N

O Do not overlook interstrand repulsion

Coulomb’s law

S P S P S P S P predicts that the backbones repel. Duplex is more C A T stable in high salt. G base coulombic charge:charge Drives strand- pairs A repulsion T G C strand inter- actions as far

S P S P S P S P from backbone as possible. sugar as scaffold charged phosphate backbone

This is what gives Watson-Crick rules Darwinian informational biopolymers molecules must be charged

O 1. Keeps the biopolymer dissolved (in water). O P N NH 2. Backbone-backbone coulombic repulsions -O O N N NH2 force strand-strand contacts to Watson-Crick O edges of nucleobases (= rules). NH O 2 3. Polyanion discourages folding. - P O N N O O N N H O neutral polymer Radius = length (1/2) NH2 O O P N - - O ------O N O ------O ------polyanionic polymer O Radius = length >>(1/2) Synthesis lets us test this with genetic systems that completely exploits H-bonds puADD pyADD puDAA pyDAA H H Donor N H O N Acceptor AcceptorO2N O H N Donor N N H N N Donor N H N N Acceptor Acceptor R Donor R N N N Acceptor R O H N Donor Donor R N H O Acceptor G C H V H J pyADA puDAD pyDAD puADA H H Acceptor O H N N Donor Donor N H O Acceptor N N Donor N H N N Acceptor N H N N Donor R Acceptor R N N N R O H N Donor R N H O Acceptor Acceptor Donor T H amA K H X

pyAAD puDDA pyDDA puAAD H H Tag H C O Acceptor 3 H N N Donor Donor O2N N H O Acceptor N N H N N Donor Donor N H N N Acceptor R R Acceptor N N N Donor R N H O Acceptor H N S B Acceptor R O Donor H Z H P Artificially Expanded Genetic Information System (AEGIS) Thermodynamic parameters for 8- letter hachimoji DNA s predict pairing as well as in 4-letter GACT DNA pyAAD puDDA pyDDA puAAD H H Acceptor H3C O H N N Donor Donor O2N N H O Acceptor N N N H N N Donor Donor N H N N Acceptor R R Acceptor C N C N Donor H O Acceptor H N S R N B Acceptor R O Donor H Z H P

In fact, the outlier is A:T, with just 2 hydrogen bonds Why genetic systems in water must have a polyelectrolyte backbone

O O 1. Keeps DNA soluble in water O O - P N NH S N NH 2. Backbone-backbone coulombic O O O N N NH2 N N NH2 O O interactions force strand-strand NH NH O 2 O 2 - P O N N S N N contacts to Watson-Crick edges O O O N N H N N H of the nucleobases (= rules) O O

NH2 NH2 3. Repeating charges discourages O O O P N S N - O O folding; “excluded volume” effect O N O N O O strand-strand O 4. Repeating charge dominates the interactions can molecule's properties, allowing O be anywhere mutation to occur without changing properties of molecule (= evolution) neutral polymer (1/2) Benner, S. A., Hutter, D. (2002) Phosphates, Radius = length DNA, and the search for nonterrean life. A ------second generation model for genetic molecules. ------Bioorg. Chem. 30, 62-80 ------polyanionic polymer Polyelectrolyte Theory of the Gene Radius = length >>(1/2) Using these features necessary for Darwinism and inaccessible without Darwinism to search for life? Building blocks must fit Schrödinger’s aperiodic crystal Must keep structure constant with changing information Needs a repeating backbone charge (negative or positive) Must keep properties constant with changing information Polyelectrolytes easy to concentrate from dilute solution, even in presence of anions containing single charges. = HPO4 = ClO4 = SO4 Br- Cl- biopolymer with repeating charges ------regularly spaced + + + + + + + + + charges on surface DETECTOR Universal life detection in water Regardless of origins, Darwinism requires a genetic polymer with a repeating backbone charge and regular building blocks. Benner, S. A. (2017) Detecting Darwinism from molecules in the Enceladus plumes, Jupiter's moons, and other planetary water lagoons. Astrobiology 17, 840 Polyelectrolytes concentrated coulombically from dilute solution Plate 1 binds polycationic Darwinian biopolymers light out polarized rotated light in due to homochiral Darwinian biopolymer

P R Q P S Flow of sample

A B A C C

light out rotated polarized due to light in homochiral Plate 2 binds polyanionic Darwinian biopolymers AA Darwinian biopolymer How to detect concentrated polyelectrolytes

Refractive index change in total internal reflection system (Biacore)

R1 R2 R3 R4 R5 ------

Detector

Sensitivity is low, long path length, differential refractive index is uncertain with unknown ions being displaced What detection architecture is most sensitive?

Ultraviolet absorption of the side chains

R1 R2 R3 R4 R5 ------

Detector Sensitivity better, long path length, UV spectra of side chains unknown; spectral measurements would be informative? What detection architecture is most sensitive? Macrochirality. Rotation of polarized light greater for a polymer of homochiral building blocks than rotation of the monomers collectively

R1 R2 R3 R4 R5 ------

Detector Applying the Schrödinger criterion to material isolated by exploiting the polyelectrolyte theory of the gene. This does not preclude downstream analysis

Displacement of adsorbed polyelectrolyte à Mass spec

R1 R2 R3 R4 R5 ------ESI MS MALDI MS

Detector

We could get some information about the detailed structure of the polyelectrolyte A complete architecture Martian polar ice •Must find the sample optional Mars ice caps survey accessible surface No caching, correct guesses about locales withdraw for microscopic • Must thaw the sample pressurizing ice inspection Energy from all steps does this “for free” zone melt zone withdraw for • Must disrupt cell compartments microscopic Abrasive dust in Mars ice does this “for free” sonication cell detection • Must separate minerals zone pressure driven pressure Sharpless centrifuge ruptures cells and removes chiral minerals that might interfere cyclonic filtration withdraw for • Pass liquid through capture zone mineralogic and organic Polylelectrolyte capture analysis. • Complete Mars analysis package polyanion capture Can inspect sample microscopically polycation capture Mineralogical sample of planet (borate …) waste Can seek metabolites in evaporated waste visual signal Downstream analysis of polyelectrolytes indicating capture, • No possibility of false positives evaporation pan homochirality