Liquid Crystal Self-Assembly of dNTPs and rNTPsas Autocatalytic Pathway for Ribozymes Formation

Tommaso P. Fraccia

University of Milan and San Raphael University of Rome

Veli Lošinj - Sept 26th, 2017

How to get long RNA prebiotically?

Joyce 2002, Nature

Even if we assume nucleotides were available on early earth,we still have more than one problem:

Concentration problem Solubility problem Reactivity problem Hydrolysis problem Stability problem

Houston, we have a problem!

How to get long RNA prebiotically?

Singularities - Landmarks on the pathways of life.C. De Duve 2005, Cambr. Univ. Press.

We still lack of a process capable to allow the onset of the RNA world starting from simple building blocks conceivably available on early Earth

"How RNA could possibly have emerged from the clutter withouta "guiding hand" would baffle any chemist. It seems possibleonly by selection, a process that presupposes replication.[...] Theneed seems inescapable for some autocatalytic process such thateach lengthening step favors subsequent lengthening. Only inthis way could the enormous kinetic obstacle to chain elongationbe surmounted. [...] Any invoked catalytic mechanism mustaccommodate the participation of a template, for there can havebeen no emergence of true RNA molecules without replication"

SELF-ASSEMBLY (favors stable aggregates)

PHASE TRANSITION (introduces new symmetries)

PHASE COEXISTENCE (selects molecules by partitioning)

CATALYTIC ENVIRONMENTS (favors chemical reactions)

AND EVERYTHING IS SOFT, MISCIBLE, REVERSIBLE, MARGINALLY STABLE

Soft-matter physics and Origin Of Life:a pathway of self-organization

Liquid mixture Self-assemblyPhase transition and coexistence

Chemical changes by catalytic effects

The guiding hand can be written in RNA chemical - physical properties

end-to-end attraction

lateral repulsion

Interaction between fragments of DNA and RNA double helices

DNA/RNA: a physical point of view

columnar LC phaseDNA/RNA

Livolant, Nature (1986)

Long DNA/RNA and Liquid Crystals

PA

102 – 104 nbcLC < 100 mg/ml

DNA/RNA BECOMES LIQUID CRYSTAL BECAUSE

OF ITS ELONGATED SHAPE AND ITS RIGIDITY

50µm

A Structure for Deoxyribose Nucleic Acid Watson J.D. and Crick F.H.C.Nature 171, 737-738 (1953)

The highly concentrated liquid-crystalline phase of DNA is columnar exagonalLivolant, Levelut, Doucet, BenoitNature, (1989)

DNA & LIQUID CRYSTALS: A RECURRING CONNECTION

X-ray diffraction pattern obtained by M H F Wilkins and R Gosling in late 1950 showing a clear crystalline arrangement.

Short DNA/RNA and Liquid Crystals

hydrofilic

5’-C

GA

TC

G-3’

5’-

CG

AT

CG

-3’

6 to 20 bases

Short DNA/RNA and Liquid Crystals

6 to 20 bases

stacking between

distinct helices

hydrofilic

5’-C

GA

TC

G-3’

5’-

CG

AT

CG

-3’

pairing and stacking

between overhangs

PA

Short DNA/RNA and Liquid Crystals

Nakata et al. Science 2007Zanchetta et al. JACS 2008

6 to 20 bases

stacking between

distinct helices

hydrofilic

5’-C

GA

TC

G-3’

5’-

CG

AT

CG

-3’

SHORT DNA/RNA BECOMES LIQUID CRYSTAL

BECAUSE OF A HIERARCHY OF SELF-ASSEMBLY STEPS

pairing and stacking

between overhangs

LC ordering is a phase transition

ISO

LC

C. De Michele, T.B., F. Sciortino, Macromolecules, 45 (2012) 1090 T. Kouriabova et al. J. Mat. Chem. 20, 10366 (2010)

THE LIQUID CRYSTAL ORDERING STABILIZES THE AGGREGATES

Average aggregate length

DNA/RNA LCs in Molecular MixturesLC formation promotes the entropy driven phase separation

of DNA double helices from single strands or flexible polymers (PEG)

LC phase coexistence -> SEGREGATION-> selection of double strands

Phase separation and liquid crystallization of complementary sequences in mixtures of nanoDNA oligomers.G. Zanchetta, et al. PNAS, 105, 1111 (2008)

PA FLUO

ISO

LC

200µm

Random-sequence DNA oligomersA pool of oligomers with random sequences is the most likely

outcome of a hypothetic random chemical ligation of mononucleotidesthat could have taken place in early Earth

Liquid Crystal Self-Assembly of Random-Sequence DNA Oligomers.T. Bellini, G. Zanchetta, T.P. Fraccia, R. Cerbino, E. Tsai, G.P. Smith, M.J. Moran, D.M. Walba, N.A. Clark PNAS 109, 1110 (2012)

PA

1012 different strands

LC ordering -> SELECTION OF STRUCTURES-> segregation of linear from non linear polymers

20mer: 5’-NNNNNNNNNNNNNNNNNNNN-3’50µm

Can it have a role in the prebiotic synthesis of long RNA?

Very short DNA oligomers: 4 base long

5’- GCCGp - 3’

5’-GCCGp-3’ - -

5’- ATTAp - 3’

5’-ATTAp-3’ - -

Fraccia et al. ACS Nano, 2016

Columnar (COL)

n

Nematic (N*)

or

Isotropic (ISO)

=

5’- GCTAp - 3’

5’-GCTAp-3’ - -

5’- GTACp - 3’

5’-GTACp-3’ - - - -

DUPLEX FORMATION, LINEAR AGGREGATION & LC FORMATION

ALL AT THE SAME TIME

Even the shortest: LC ordering of dATP - dTTP

under submission

Starting solution:50 mM dATP-dTTPpH 7.5No added saltT = 5°C

500µm500µm

500µm

Even the shortest: LC ordering of dATP - dTTP

under submission

Starting solution:50 mM dATP-dTTPpH 7.5No added saltT = 5°C

500µm500µm

Even the shortest: LC ordering of dATP - dTTP

under submission

500µm

Starting solution:50 mM dATP-dTTPpH 7.5No added saltT = 5°C

adenine thymine

cytosine guanine

guanine quadruplexes

Liquid Crystals ordering of dNTPs

under submission

LCs seem to know about Watson – Crick pairing…

cDNA (mg/ml)cDNA (mg/ml)cDNA (mg/ml)

T (°

C)

T (°

C)

T (°

C)

dTTP + dATPdCTP + dGTP dNTPs mix

LC

GROWING STABILITY

GUANOSINE QUADRUPLEXES

under submission

Liquid Crystals ordering of dNTPs

Which is the arrangement of dNTPs?

larger qSTACKING PEAK

smaller qCOLUMNAR PEAK

LC of single basesdATP + dTTP 1:1

T = 5 °C

LC of DNA 12mersT = 20 °C

Intercolumnar distance = 2.3 nm

Base stacking distance = 0.334 nm

under submission

column of dimersinter-columnar distance

lines computed from concentration for columns formed by stacks of 2, 3, and 4 bases

Still duplexed DNA even without backbone

under submission

Liquid Crystals ordering of NTPs (almost)

GTP

CG CTP

dATP ATP

dTTP

dUTP

UTP

TTPto be published

NO LC

LC

UTP

dTTP

PA

Message:

• DNA and RNA order in LC phases in a very broad range of lengths

• As length increases LCs become more stable

1bp

4bp

12bp

100bp

LC order as a template for ligationliquid crystal order keeps the terminals of the oligomers in continuous physical contact

Can this proximity favor the formation of chemical bonds?

Fraccia, T. P.; Smith, G. P.; Zanchetta, G.; Paraboschi, E.; Yi, Y.; Walba, D. M.; Dieci, G.; Clark, N. A & Bellini, T. Abiotic Ligation of DNA Oligomers Templated by Their Liquid Crystal Ordering. Nat. Commun. 6, 6424 (2015)

Fraccia, T. P.; Zanchetta, G.; Rimoldi, V.; Clark, N. A. & Bellini, T. Evidence of Liquid Crystal–Assisted Abiotic Ligation of Nucleic Acids. Orig. Life Evol. Biosph. 45, 51–68 (2015)

Non-enzymatic ligation reaction

+ HO

O-

O

O

P

=

O

OHO

O

P

=

O

Shabarova et al. 1981-1991, Taran et al. 2010

longer DNA duplex

1st Step:Terminal Phosphate activation

2st Step:Nucleophilic substitution

3’P DNA ligation in LC phase

DNA - PEG mixed DNA – PEG domainsCf DNA = = 80 – 110 mM= 300 - 400 mg/ml

Increasing PEG (osmotic pressure)

PA

FLUO

200µm

D1p(12mer)

- - - - - - - -- - - -5’-CGCGAATTCGCG p-3’

5’-CGCGAATTCGCG p -3’

C0 DNA = 5mM = 19 mg/ml CPEG = 0 – 400 g/l

PEG 8 kDa

LC forming sequences

cPEG (100 g/l)0 1 2 3 4

12

24

36

48

12084

D1p/PEG mixture D1p LC domains

DNA/PEG mixture <N> < 2DNA LC domains <N> ≈ 9

Increasing Temperature

How to compare ISO and LC phases segregated PEG?D1p/PEG phase diagram is temperature dependent…

D1p LC D1p ISO

25°C 65°C

25 µm25 µm

...and PEG osmotic pressure controls LC stability:high PEG -> higher melting T, lower PEG -> lower melting T

LC melting

ISO vs LC domains

ISO vs LC domains

12

24

36

48

120

30 40cPEG (w%)

[PEG]/[DNA]=50Reaction Time = 24hTemp = 65°C

D1p LC

D1p ISO

<N>ISO ≈ 3<N>LC ≈ 9LADDER

Not just concentration dependence

5mM

10mM

50mM

5’P RNA ligation catalyzed by LCs

yield 72%

yield 31%

Flu

ore

scen

ce In

ten

sity

Polymer SizePolymer Size

5’-p CGCGAAUUCGCG -3’ 5’-p CGCGAAUUCGCG-3’

Circular/linear productsdecreases in LC phase

ISO LC

Flu

ore

scen

ce In

ten

sity

to be published

Reaction conditions:

10 mM RNA 5’P10 mM HEPES pH 7.560 mM MgCl2300 mM EDC0 – 30 %W PEG 8 kDa24 - 48 hT = 25 – 40°C

5’-p GAUCGC -3’5’-p GAUCGC-3’

Features of Liquid Crystals Autocatalysis Self-assembly and ordering from

monomers to long DNA/RNA

Self-templating Non–Enzymatic ligation, protection from hydrolysis

Complementarity and structural selection through phase separation

Everything is soft, miscible, reversible and marginally stable

100µm

LC autocatalysis conjecture

LC

c, L

T

L

P(L)

LC

c, L

T

Growing length by non-equilibrium cycling Structural selection

L

P(L)

LC autocatalysis conjecture

Folding polymers

solubility in water

acc

iden

tal

chem

istr

yeq

uili

bri

um

& n

on

-eq

uili

bri

um

sta

tist

ica

l phy

sics

evo

luti

on

LIQUID CRYSTAL

AUTOCATALYSIS

RNA WORLD

irreversible collapse of immiscible molecules

isotropic dispersion of water-soluble

molecules

breaking of chains recycles them through

the LCA

LC-assisted chain lengthening feeds the RNA world

boundary ofmarginal solubility

NA fragments, nucleotides,amphiphilic molecules

folding RNA strands, ribozymes

LC autocatalysis conjecture

Giuliano Zanchetta

Noel Clark

Greg Smith

Marco Todisco

Tommaso Bellini

and thanks to you for listening!S e e d G r a n t

Emily Hayden Joe Theis