Folding and replication in complex dynamic molecular networks
Liu, Bin
DOI:
10.33612/diss.99784510
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Publication date: 2019
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Liu, B. (2019). Folding and replication in complex dynamic molecular networks. University of Groningen. https://doi.org/10.33612/diss.99784510
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Chapter
5
Self‐replication
Promotes
the
Formation of Complex Folded Structures
The replication of genetic information and its expression into folded functional structures are key biochemical processes in life. However, it is very challenging to construct synthetic chemical systems capturing these complex behaviors. Herein, we show that both processes can emerge from dynamic libraries made from simple building blocks. Specifically, we show that folded structures are promoted by the formation of a replicator. The composition of the resulting libraries is dominated by compounds that form non‐covalent interactions either intramolecularly (giving rise to highly specific complex folded structures, composed of 15 identical subunits), or intermolecularly (resulting in self‐replication driven by self‐assembly). Whether foldamers or replicators emerge is dictated by the ratio of the building blocks. Moreover, transient formation of a self‐replicator was also observed, again, depending on the ratio of the two building blocks.
5.1 Introduction
Supramolecular assembly in complex systems has attracted increasing attention over the past two decades.1,2 It has been used to design functional systems that capture important processes in living organisms.3,4 Self‐replication5‐7 is one of the most important ingredients in the origin of life, and self‐replicating molecules are a promising possible starting point for the de novo synthesis of life.8,9 Since von Kiedrowski demonstrated the first self‐replicating system,10 many such systems have been developed based on synthetic molecules11‐17 or biomolecules such as DNA18,19, RNA20‐26 and peptides.27‐30 However, it remains a huge challenge to develop new self‐replicating systems that adaptively respond to a changing environment. In addition, the construction of dissipative self‐replicating systems, like those found in living systems, is even more challenging.31,32 Recently, dynamic combinatorial chemistry33‐37 (DCC) has been shown to be a promising approach for the fabrication of self‐ replicating systems from complex mixtures of interconverting molecules by self‐selection.38 More importantly, since the structure of the building block does not necessarily predetermine the nature of the self‐replicator, the emergence of replicator from DCLs can be susceptible to environmental changes. The emergence of self‐replicators from DCLs can be affected by templates,39 mechanical agitation,38 solvent environments40 and pre‐existing replicators.41,42 Moreover, dynamic self‐replication systems can exhibit parasitic phenomena similar to biological systems.43
Folding is one of the most important processes in which biomolecules adopt specific three‐ dimensional shapes in order to achieve their special biological properties and functions.44‐47 Scientists have made great efforts to achieve the synthesis of folded structures, foldamers, with different functions and properties.48‐58 However, designing simple motifs that can be folded into a specific shape, such as a peptide or nucleic acid, remains challenging. Most of these synthetic folds only feature secondary structures, such as helices and sheets, rather than more complex tertiary or quaternary arrangements.59 Our recent work shows that a dynamic combinatorial approach can allow access to self‐synthesizing foldamers with complex tertiary structures with remarkable yields and minimal synthetic effort. The dynamic combinatorial approach may also provide us the possibility to self‐sort different assemblies which require different building blocks in complex chemical networks. Self‐ sorting of different assemblies in complex systems may reveal behavior that goes beyond that accessible in individual subsystems.60
Key processes of Darwinian evolution are the replication of genetic information and its expression into functional molecules such as folded proteins. The RNA world hypothesis suggests replication and the expression of genetic information depend on folded RNA enzymes.61 Replication and folding processes play very important roles in biological systems. Yet, there is no report on the relationship between these two processes within chemical systems. Most synthetic systems only capture a single process, and no examples exist of chemical systems that facture both processes simultaneously. The limiting design rules for developing self‐replicating molecules and folded assemblies in synthetic systems makes it challenging to access systems that integrate folding and replication. A primary sequence can, in principle, either replicate or fold, where a delicate balance between inter and intramolecular interactions might favor one over the other.
Herein, we show how self‐replication promotes the formation of a foldamer through self‐ sorting in a two building blocks DCL: the emergence of self‐replicators is resulting from intermolecular non‐covalent self‐assembly and the formation of foldamers driven by intramolecular interactions. The foldamer is formed following the emergence of replicators, and the distribution of replicators and foldamers in the DCL is determined by the proportion of the two building blocks. Moreover, the process of simultaneous formation and destruction of self‐replicating molecules was also observed from the same DCL. Such processes may help us to further understand life‐like behavior.31
5.2 Results and discussion
5.2.1 Self‐sorting of a self‐replicator and a foldamer
We have previously reported the emergence of self‐replicating molecules and folded complex structures from DCLs made from dithiol‐functionalized building blocks. Self‐ replicating molecules emerge in DCLs made from building blocks functionalized with short peptide chains composed of alternating hydrophobic and hydrophilic amino acid residues to facilitate the generation of β‐sheets and an aromatic dithiol core for thiol‐disulfide exchange. Building block 1 (Scheme 5.1, left) has a structure similar to those giving rise to the previously reported self‐replicating molecules, except that the amino acid attached to the aromatic core is modified from glycine to β‐alanine. Stirring building block 1 (2.0 mM) in borate buffer (50 mM, pH = 8.0) containing 1.0 M NaCl in the presence of air results in a small DCL containing different macrocyclic disulfides. Initially, cyclic tetramer (14) emerged
verif build the repl hexa relat Build (ade diffe form pH = show hydr Sche the g the r mixe 1221, fy that 14 is ding block 1 formation o icator with amer and o tively hydro ding block 2 enine) attac erent self‐as med by stirrin = 8.0) contai ws that 215 i rogen bonds eme 5.1. Dyna general replic right part sho ed library of b , and foldame s a self‐repli . The results of itself, sug
a relatively ctamer repl ophobic, whi (Scheme 5. hed to the ssembled str ng an aqueo ning 1.0 M N s a cyclic pr s, hydrophob amic combina ation mechan ows the foldin building block er 215 is genera cator by ad s show that t ggesting aut small ring s icators) eme ich facilitate 1, right) feat same arom ructure is fo us solution o NaCl with th oduct forme bic interactio atorial librarie nism for the sh ng for the nuc s 1 and 2, rep ated at the en ding 10 mo the addition tocatalytic b size (compar erged from es the forma
tures an am matic dithiol
ormed in th of building b
e presence o ed by folding ons and π‐π s es made from hort peptide f cleobase equi plicator 14 is f nd. l% of prefor of preforme behavior (Fig ring with th this DCL is ation of sma
ino acid (asp core as bu he library. F lock 2 (2.0 m of air (Figure g of 15 subu stacking. building bloc functionalized ipped building formed first, b rmed 14 into ed 14 significa gure S5.1). e previously because bu all macrocyc partic acid) a uilding block oldamer 215 mM) in borate e 5.1a). X‐ray units throug cks 1 and 2: t d building bloc g block (215 in but then gives
o a fresh DC antly acceler The fact th y reported c uilding block clic replicato and a nucleo k 1. Howeve 5 was select e buffer (50 y crystallogr h intramole the left part s ck (14 in this c n this case); i s way to repli CL of rates hat a cyclic 1 is ors.62 obase er, a tively mM, aphy cular hows case); n the icator
The fold sing dith exch In o bloc pres com cycli mixt sele Figu (c) 1 In o the dom bloc this different n ing make it le chemical iol core whi hange buildin rder to prov ck 1 and 2 ( sence of 1.0 mposed of cy ic trimer 112
ture of repli ctive format re 5.1. UPLC a .0 mM 1 and rder to obta time evolut minated by s cks. After 2 d macrocycle
ature of the is possible t network. M ich ensures ng blocks wit e this conjec ([1] = [2] = 0 M NaCl. T clic trimer 12 22 (Figure 5.1 icator 14 and tion of mixed analysis of DC 1.0 mM 2 in b ain more insi
tion of the small macro days, the con became the e noncovale to investigat Moreover, bo that replica th each othe cture, a smal 1.0 mM) in The library 221 and folda 1c). Instead o d foldamer 2 d cyclic trime CLs made from borate buffer ghts into the library was ocyclic trime ncentration o e dominant p ent interacti te the self‐s oth building ators and fo er by thiol‐di ll DCL of disu n aqueous b reached eq amer 215, acc of narcissist 215), social s er 1221 and fo m (a) 2.0 mM (50 mM, pH = e self‐sorting monitored ers and tetr of the mixed product afte on associate orting of re blocks 1 and lded structu sulfide excha ulfides was g borate buffe uilibrium aft companied b ic self‐sortin self‐sorting o oldamer 215. building block = 8.0, 1.0 M N g process in (Figure 5.2 amers, cont d cyclic trime r 4 days stir
ed with self plicators and d 2 have the res are in p ange. generated by r (50 mM, p ter 7 days by a small am g60 (which s occurred,60 g k 1, (b) 2.0 mM aCl) after stirr the two buil a). Initially, taining one er 1221 rapid ring. Foldam f‐replication d foldamers e same arom principle, ab y stirring bui pH = 8.0) in and was m mount of ano should result giving rise to M building blo ring for 10 day lding blocks the library or two bui dly increased mer 215 starte and in a matic le to lding n the ainly other t in a o the ock 2, ys. DCL, was lding d and ed to
form of c was conc cont form form form iden bloc obse rem had requ form fold Figu with 5.2.2 In o obse furth tran m after all of yclic trimer performed centration o taining build mation of 122 mation of th mation in a ntical condit ck 1 through erved before aining build reached co uires the em m the folded ed complex re 5.2. Time e 6 mol% 1221 2 Characteri order to und
erved previo her charact nsmission ele f building blo 1221 and fol d in order f 1221 increa ding blocks 21, which pro
e folded com separate li ions. The pr h thiol‐disulfi e all of build ing block 2 mpletion. Th mergence of structure. T structures in evolution of D in borate buff ization of ne derstand wh ously for othe erized by c ectron micro ock 1 was co damer 215. A to assess w ased rapidly u 1 (1.0 mM) oves that the mplex struct brary which reformed fo ide exchange ding block 1 was convert hese results self‐replicat Thus, the em n this two co DCLs made fro fer (50 mM, p ew formed se hether the m er peptides‐b circular dich oscopy (TEM onsumed an A seeding e whether it upon adding and 2 (0.50 e small cyclic ture in the a h only cont oldamer 215 w
e (Figure S5 was consum ted to folda
suggest tha tor to consu ergence of s omponent dy m (a) 1.0 mM pH = 8.0, 1.0 M elf‐replicato mechanism based replica hroism (CD), M). The CD s d the library xperiment f is a self‐re g 6 mol% pre 0 mM) in th c trimer 1221 above library ains buildin was disassem .5). Note als med. Afterwa mer after th at the forma ume those b self‐replicato ynamic chem M 1 and 1.0 mM M NaCl). ors of self‐repl ation, the ne , thioflavin spectra of re y ended up c ocused on c plicator (Fig eformed 1221
he ratio bias
is a replicato
y is delayed g block 2 u mbled upon so that, no f ards it rapidly he process o
tion of folda building bloc or promotes mical network M 2, (b) 1.0 m ication is th ewly formed T fluoresce eplicator 14 consisting m cyclic trimer gure 5.2b). 1 into a fresh sed towards or. Note tha compared t under other adding bui foldamer 215 y formed an of self‐replica amer in this cks which ca the formatio k.
mM 1 and 0.5 m
he same as replicators w ence assays showed pos ainly 1221 The h DCL s the t the to its rwise lding 5 was nd all ation s DCL nnot on of mM 2 that were and sitive
helic stru inte pres TEM fibro form beha Figu spec mM, 5.2.3 We libra stro build city at 206 n cture62 (Fig nsities were sence of β‐s M micrograp ous nanostr med self‐repl avior of prev re 5.3. (a) CD ctra, (c) and (d , pH = 8.0, 1.0 3 Ratio effec then investi ary. The res ngly on the ding blocks, m and negat ure 5.3a). S relatively w sheet structu hs of the li uctures (Fig licators were vious replicat spectra (reco d) TEM microg 0 M NaCl) that cts igated how t ults describ e ratio of th
while the fo
tive helicity a Similar helic weak. Thioflav ures for rep
braries dom gures 3c an e driven by t tors.62 orded at ident graphs of libra t were domina
the ratio of ed above al he two build
ormation of around 220 cities were vin T fluores plicators 14 a minated by r d 3d). Thes he formation tical concentra aries made fro ated by 14 and the two bu lready indica ding blocks, folded mac nm, indicativ observed f cence measu nd 1221 (Fig replicators 1 se results d n of β‐sheet, ations), (b) th om building b d 1221, respec ilding blocks ated that th because re crocycles 215 ve of the pre or replicato urements als ure 5.3b). N 14 and 1221 s emonstrate , which is co ioflavin T fluo locks 1 and 2 tively. s affects the he library be eplicator 122 requires the esence of β‐s or 1221, but so confirmed Negative sta showed bun that the n nsistent with orescence emi 2 (borate buffe outcome of ehavior dep 21 contains e self‐replica sheet t the d the ining ndled ewly h the ission er, 50 f the ends both ating
mol fold diffe und whe diffe build mol repl max libra prod build conc inco afte Figu conc (50 m ecules to co amers. A se erent ratios
er the same en they had erent produ ding blocks e fraction o icator 1221 in ximum at 0. aries when t ducts of the ding block 2 centration o orporated int r all of build re 5.4. Summ centration of t mM, pH = 8.0, onsume all o eries of libra ([1]/[2] = 90 e conditions reached eq ct distributi 1 and 2. The f building b ncreased wit 6 mM of bu the concentr e libraries w in the librar of replicator to a replicat ing block 1 h
mary of the the two build , 1.0 M NaCl).
of the other aries were s 0/10, 80/20, as the desc quilibrium a ons were o e libraries w lock 2 is be th the increa uilding block ration of bui were replicat ries was raise 1221 decre or (either 14 had been inte experiments ing blocks is 2 building blo set up at in
, 70/30, 60/ cribed above as observed bserved fro were domina elow than 30 ase of buildi k 2. Replic ilding block tor 1221 and ed further, m ased. Note 4 or 1221), an egrated into s at different 2.0 mM and a ocks that ar a total con /40, 50/50, 4 e. The librari
by UPLC/M m the libra ated by repli 0%. In this r ng block 2 in ator tetram 2 was more d foldamer 2 more foldam
that in all nd building replicators. t ratios of b ll of the librar e incapable ncentration 40/60, 30/70 es were stir MS. As show ries with dif
cators 14 an range, the co n the librarie er 14 disapp than 0.8 m 215. As the c er 215 was ge cases, build block 2 form uilding block ries were set u
of giving ris of 2.0 mM 0, 20/80, 10 rred for 10 d wn in Figure fferent ratio d 1221 when oncentration es and reach peared from mM and the concentratio enerated and ing block 1 med foldame ks 1 and 2. up in borate b se to with 0/90) days, 5.4, os of n the ns of hed a m the main on of d the was er 215 Total buffer
5.2.4 Inte build the whe unti To repl show mol% and rapi decr the was the diffe repl repl not for t build Figu 10 m 4 Transient s restingly, tr ding block 2 emergence ereas, once r l all of buildi investigate icator 1221 a wn in Figure % preformed 1.0 mM bu dly became reased and e library after no emergen same librar erent in thes icator 1221 a ication can c the final pro the formatio ding blocks i re 5.5. Time e mol% 1221 in b self‐replicat ransient self is below 30 of replicato replicator 12 ing block 2 w whether tra nd foldamer e 5.5a, the c d replicators uilding block the domina essentially d r all of buildi nce of replica ry (Figure 5 se two expe nd foldamer change the p oduct distribu on of folded ncapable of evolution of D borate buffer ion f‐replication 0 mol% in th ors 14 was 221 emerged was consume ansient self‐ r 215, cross‐s concentratio s 14 was seed k 2. Then re ant species. isappeared f ing block 1 w ator 14 when 5.5b). Althou riments, bot r 215. These pathway for ution. In add macrocycles giving rise to DCLs made fro (50 mM, pH = of 14 was e libraries. I observed b d, replicator ed. ‐replication eeding expe on of replicat
ded into the eplicator 122 Meanwhile, from the lib was transfer n 10 mol% p ugh the pat th gave the s results indic r the formati dition, cross s: self‐replica o foldamers. om 1.0 mM 1 = 8.0, 1.0 M N observed w n those libra efore the fo 14 was tran of 14 can eriments wer tor 14 rapidly DCL made f 21 emerged , the concen rary. Finally, rred to replic reformed re thways to f same produc cate that see ion of the co catalysis did ating molecu 1 and 1.0 mM aCl). when the c aries (Figure ormation of sformed into affect self‐s re performed y increased rom 1.0 mM after 70 ho ntration of s foldamer 2 cator 1221. In plicator 1221
orm the fin ct distributio eding‐induce ompounds in not change ules must seq 2 with (a) 10 concentratio e S5.4a, b an f replicator o replicator sorting betw d (Figure 5.5
at first whe M building blo ours stirring, self‐replicato 15 emerged n contrast, t 1 was seeded nal products on dominate ed transient n the library the requirem quester all o 0 mol%l 14 an n of nd c), 1221, 1221 ween 5). As en 10 ock 1 and or 14 from there d into s are ed by self‐ , but ment f the nd (b)
We with mM only 1221 build repl conc rapi by s rapi to th at th and Figu mM dom simu Thes repl whic 5.5b resu build then explore h different bu ) and 2 (0.3 y replicators 1. Since the ding block icator 1221. centration o dly grew wh elf‐replicato dly destroye hat of the lib he start of th similar resu re 5.6. Altern 1 and 0.3 mM minated by rep ultaneously. se results in icators in re ch building b b. Again, sim ults, combine ding blocks ed the comp uilding block mM) under 14 emerged amount of b 1 into repli After the of building b hile part of r or 14. Then 15 ed and conve brary formed he experimen lts were obta nating additio M 2 in borate plicators 14 a ndicate this sponse to th blocks 1 and multaneous c ed with the DCLs is the petition betw ks as ‘‘food’’. at the same from the lib building bloc cator 1221, library reac locks 1 and replicator 12 5 mol% build erted into re d by mixing t nt. A second ained (Figure ns of portion buffer (50 mM and 1221. (a) A system is a he addition o 2 were adde creation and previous ob ermodynami ween replicat . We prepare e conditions a brary which w ck 2 in this
the final lib ched equilib 2) of buildi 21 was disas ding block 2 eplicator 122 he correspo round of bu e 5.6a). ns of building M, pH = 8.0, 1 Addition of 1
ble to simu of building bl ed at the sam destruction bservations, ically less st tors 14 and 1 ed a DCL con as described was subsequ library was brary contai brium, 85 m ng block 1 w sembled and was added a 1, reaching a nding amoun uilding block blocks 1 and M NaCl), resu 1 and 2, sepa ltaneously c locks. We als me time. The n of self‐repl suggest tha table, even 1221 when th ntaining build above. As e ently conver insufficient ined both r mol% (relati was added. S d the library and most of a product dis nts of buildin addition was
2 to a mixtu ults in switchi arately, (b) ad create and d so conducted e results are icators was at replicator if it is the
ese are prov ding blocks 1 expected, ini rted to replic to convert a replicator 14
ive to the Self‐replicato y was domin replicator 14 stribution sim ng blocks 1 a s also perfor ure made from ing between s ddition of 1 a disintegrate d experimen e shown in Fi observed. T r 14 in these thermodyn vided 1 (1.7 tially cator all of 4 and total or 14 nated 4 was milar and 2 rmed m 1.7 states and 2 self‐ nts in igure These two amic
product in the library which only contains building block 1. In the presence of a sufficient amount of building block 2, all of the species made from building block 1, including replicator 14, will convert to replicator 1221. Yet replicator 14 was able to grow transiently,
even in the presence of competition replicator 1221. Thus, replicator 14 appears to be a more
proficient replicator.
5.3 Conclusion
In conclusion, we were able to witness two of the most important processes of living systems, self‐replication and folding in a single fully synthetic chemical system. The most critical aspect of this system is the self‐sorting between self‐replicators and foldamers. Self‐ replication is the result of intermolecular self‐assembly while the formation of foldamer is driven by intramolecular non‐covalent interactions. The emergence of self‐replicators consumes all of the non‐foldable building blocks (whose existence will destroy any foldamer) and result in a stable fibrous structure. The remaining foldable building blocks then react to give rise to a complex folded structure. The competition between self‐replicators and foldamer is controllable and the emergence of self‐replicators or foldamer can be directed by adjusting the ratio of the building blocks. We also observed the transient formation of a self‐replicator, the formation of which can be induced by adding the respective starting building block to the library. The metastable self‐replicator is eventually consumed and transformed into a stable self‐replicator. The proportion of metastable self‐replicator and stable self‐replicator in the library can be controlled by the ratio of building blocks.
Our results provide a way for non‐templated self‐sorting of different structures in covalent disulfide based dynamic combinatorial libraries, which paves the way for designing complex dynamic systems with multiple coupled functions in the future, such as catalysis for self‐ replicators and binding for foldamers.
5.4. Experimental section
5.4.1 General methods
All chemicals, unless otherwise stated, were purchased from Sigma‐Aldrich and used as received. Acetonitrile (ULC‐MS grade), water (ULC‐MS grade) and trifluoroacetic acid (HPLC grade) were purchased from Biosolve BV. Peptide building block 1 was purchased from Cambridge Peptides Ltd. (Birmingham, UK) with 95% purity. The synthesis of building block 2 was described in Chapter 2.
Buffer preparation
Borate buffer (50 mM, pH = 8.0) was prepared by dissolving sodium tetraborate (3.84 g, Na2B4O7.10H2O) in 200 mL doubly distilled water. Then the pH was adjusted to 8.0 by
addition of concentrated HCl. Library preparation
Building blocks were dissolved in borate buffer (50 mM, pH 8.2) in the presence of 1.0 M NaCl. All libraries were set up in an HPLC vial (12×32 mm) with a Teflon‐coated screw cap. All HPLC vials were equipped with a cylindrical stirrer bar (2×5 mm, Teflon coated, purchased from VWR) and were stirred at 1200 rpm using an IKA RCT basic hot plate stirrer. All experiments were performed at ambient conditions.
UPLC and UPLC‐MS analysis
UPLC analyses were performed on a Waters Acquity H‐class system equipped with a PDA detector, using a detection wavelength of 254 nm. Samples were injected on an Phenomenex Aeris Peptides 1.7 μm (150 × 2.1 mm) column, purchased from Phenomenex, using ULC‐MS grade water (eluent A) and ULC‐MS grade acetonitrile (eluent B), containing 0.1 V/V % TFA as modifier. A flow rate of 0.3 mL/min and a column temperature of 35 °C were applied.
UPLC‐MS analyses were performed using a Waters Acquity UPLC H‐class system coupled to a Waters Xevo‐G2 TOF. The mass spectrometer was operated in positive electrospray ionization mode with the following ionization parameters: capillary voltage: 3 kV; sampling cone voltage: 20 V; extraction cone voltage: 4 V; source gas temperature: 120 °C; desolvation gas temperature: 450 °C; cone gas flow (nitrogen): 1 L/h; desolvation gas flow (nitrogen): 800 L/h.
Circular Dichroism
Spectra were recorded at room temperature using a JASCO J715 spectrophotometer and HELMA quartz cuvettes with a path length of 1 mm. All spectra were recorded at room temperature from 190 nm to 300 nm, with 2 nm step interval and 3 scans with a speed of 200 nm/min. Solvent spectra were subtracted from all spectra. Samples were diluted to a concentration of 0.15 mM with respect to the concentration of building block. Thioflavin T (ThT) Fluorescence Assay Fluorescence measurements were performed on a JASCO FP 6200 fluorimeter using quartz cuvettes with 1 cm path length. For the ThT measurements, a freshly prepared solution of thioflavin T (dissolved to 2.2 mM in 50 mM borate buffer at pH = 8.0 and filtered through a 0.2 µm syringe filter) was diluted to 22 µM with the same buffer. An aliquot of 450 µL of this diluted solution was transferred to the cuvette, followed by the addition of 80 µL of sample (diluted with borate buffer to 80 µM total building block concentration just prior to the measurement). Spectra were recorded after an incubation time of 2 minutes. The excitation wavelength was set at 440 nm and spectra were recorded in the range of 480‐700 nm with a slit width of 5 nm, using a cutoff filter at 480 nm. The blank spectrum (i.e. fluorescence of the corresponding dye in solvent only) was subtracted from each spectrum. Negative‐staining Transmission Electron Microscopy A small drop (5 µL) of sample was deposited on a 400 mesh copper grid covered with a thin carbon film (supplied by Van Loenen instruments). After 30 seconds, the droplet was blotted on filter paper. The sample was then stained twice with a solution of 2% uranyl acetate (5 µL) deposited on the grid, and blotted on filter paper after 30 seconds. The grids were observed in a Philips CM12 electron microscope operating at 120 kV. Images were recorded on a slow scan CCD camera. UPLC methods: Method for the analysis of DCLs made from building blocks 1 and 2: t / min % B 0 10 10 40 12 90 13 90 14.5 10 17 10
5.4.2 Kine Figu (2.0 Seed Figu mM, prefo 2 Appendix etic profiles f re S5.1. Kinet mM) in borat ding experim re S5.2. Grow , pH = 8.0, 1 ormed 14, dem for self‐repli tic study of lib te buffer (50 m ment for self wth of 14 with 1.0 M NaCl) w monstrating t icator 14 and braries made f mM, pH = 8.0) f‐replicator 1 time in a DCL without seed he autocataly d foldamer 2 from (a) build ) with 1.0 M N 14 L made from ding and upo ytic nature of t 215 ding block 1 (2 NaCl. 1.0 mM build n seeding wi the formation 2.0 mM) and ( ding block 1 in th 10 mol % n of 14. (b) building bl n borate buffe % (in terms o ock 2 er (50 f [1])
Effe 1 an Figu = 8.0 40; ( ct of the rat nd 2 re S5.3. UPLC 0, 1.0 M NaCl) (f) 50; (g) 60; ( tio of buildin C analysis of li ) at a total co (h) 70; (i) 80; ( ng blocks on braries made ncentration o (j) 90; (k) 100 the product from building of 2.0 mM at d mol % of buil t distributio g blocks 1 and different ratio ding block 2. n of the libra d 2 in borate b s: (a) 0; (b) 10 aries made f buffer (50 mM 0; (c) 20; (d) 3 from M, pH 0; (e)
Kine Figu pH = 40; ( etic profiles o re S5.4. Kinet = 8.0, 1.0 M N (e) 50; (f) 60; ( of libraries m tic analysis of NaCl) at a tota (g) 70; (h) 80; made from b f libraries mad al concentrati (i) 90 mol % o building bloc de from build ion of 2.0 mM of building blo cks 1 and 2 w ding blocks 1 a M at different ock 2. with differen and 2 in bora ratios: (a) 10 nt ratios ate buffer (50 0; (b) 20; (c) 3 mM, 0; (d)
Buil Figu build disas Tota Figu bloc of th ding block 1 re S5.5. Kinet ding block 1 ssembling into al peak area re S5.6. Tota k 2 (2.0 mM); he building blo 1 triggered fo tic analysis of (1.0 mM) in o monomer 2 of the librar l UPLC peak a ; (c) building b ocks are essen oldamer disa a library mad borate buffe by building b ry made from area of librari blocks 1 (1.0 m ntially indepen assembly de from folda er (50 mM, p block 1 and tra m building b ies made from mM) and 2 (1 ndent of the m mer (1.0 mM pH = 8.0, 1 M ansforming int blocks 1 and m (a) building 1.0 mM), show macrocycles. , in terms of M NaCl), show to self‐replica 2 g block 1 (2.0 wing that the monomer [2] wing foldame ator 1221. mM); (b) bu molar absorp ) and er 215 ilding ptivity
5.4.2 Figu pH = Figu anal 1722 1378 2 UPLC and re S5.7. UPLC = 8.0, 1.0 M Na re S5.8. Mas
ysis of a DC 2.55 [M+4H] 8.37 [M+5H]5 UPLC‐MS An C analysis of th aCl) after stirr ss spectrum L made from ]4+, 1378.24 5+. nalyses he DCL made f ring for 36 ho of 215 (rete m 1 (1.6 mM [M+5H]5+; o from 1 (1.6 m urs. ntion time 3 ) and 2 (0.4 observed m/ mM) and 2 (0.4 3.46 min in mM). Calcu /z: 2296.84 4 mM) in bora Figure S5.7) lated m/z: 2 [M+3H]3+, 17 ate buffer (50 from the LC 296.40 [M+3 722.81 [M+4 mM, C‐MS 3H]3+, 4H]4+,
Figu DCL [M+1 Figu of a obse re S5.9. Mass made from 1 1H]+. re S5.10. Mas DCL made fro erved m/z: 10 s spectrum of 1 (1.6 mM) an ss spectrum o om 1 (1.6 mM 75.06 [M+2H] 21 (retention nd 2 (0.4 mM of 1123 (retent M) and 2 (0.4 m ]2+, 717.20 [M time 4.48 mi ). Calculated tion time 6.20 mM). Calculat M+3H]3+. n in Figure S5 m/z: 462.10 0 min in Figur ted m/z: 1075 .7) from the L [M+1H]+; obse e S5.7) from t .30 [M+2H]2+, LC‐MS analysi erved m/z: 46 the LC‐MS an , 717.20 [M+3 s of a 62.23 alysis 3H]3+;
Figu of a obse Figu a DC obse re S5.11. Mas DCL made fro erved m/z: 12 re S5.12. Mas CL made from erved m/z: 689 ss spectrum o om 1 (1.6 mM 31.58 [M+2H] ss spectrum o m 1 (1.6 mM) 9.64 [M+2H]2 of 1222 (retent M) and 2 (0.4 m ]2+, 821.23 [M of 23 (retentio and 2 (0.4 m + , 460.22 [M+ tion time 7.41 mM). Calculat M+3H]3+. n time 7.73 m mM). Calculat +3H]3+. 1 min in Figur ted m/z: 1231 min in Figure S ted m/z: 689. e S5.7) from t .43 [M+2H]2+, S5.7) from the 63 [M+2H]2+, the LC‐MS an , 821.29 [M+3 e LC‐MS analy 460.09 [M+3 alysis 3H]3+; ysis of 3H]3+;
Figu a DC obse Figu of a obse re S5.13. Mas CL made from erved m/z: 774 re S5.14. Mas DCL made fro erved m/z: 84 ss spectrum o m 1 (1.6 mM) 4.37 [M+1H]+ ss spectrum o om 1 (1.6 mM 5.68 [M+2H]2 of 11 (retentio ) and 2 (0.4 m , 387.87 [M+2 of 1122 (retent M) and 2 (0.4 + , 564.26 [M+ n time 8.16 m mM). Calculat 2H]2+. tion time 8.29 mM). Calcula +3H]3+. min in Figure S ted m/z: 774 9 min in Figur ted m/z: 845 S5.7) from the .37 [M+1H]+, e S5.7) from t .76 [M+2H]2+, e LC‐MS analy 387.18 [M+2 the LC‐MS an , 564.17 [M+3 ysis of 2H]2+; alysis 3H]3+;
Figu of a obse Figu a DC 772. re S5.15. Mas DCL made fro erved m/z: 118 re S5.16. Mas CL made from 35 [M+4H]4+; ss spectrum o om 1 (1.6 mM 82.52 [M+3H] ss spectrum o m 1 (1.6 mM) a observed m/z of 1421 (retent M) and 2 (0.4 m ]3+, 887.27 [M of 14 (retentio and 2 (0.4 mM z: 1543.99 [M tion time 8.49 mM). Calculat M+4H]4+. n time 8.74 m M). Calculated M+2H]2+, 1029. 9 min in Figur ted m/z: 1182 min in Figure S d m/z: 1543.7 .52 [M+3H]3+, e S5.7) from t .49 [M+3H]3+, S5.7) from the 70 [M+2H]2+, 1 772.51 [M+4 the LC‐MS an , 887.12 [M+4 e LC‐MS analy 1029.47 [M+3 H]4+. alysis 4H]4+; ysis of 3H]3+,
Figu a DC 965. [M+4 Figu of a 668. [M+3 re S5.17. Mas CL made from 19 [M+4H]4+, 4H]4+, 772.53 re S5.18. Mas DCL made fro 26 [M+3H]3+, 3H]3+, 501.56 ss spectrum o m 1 (1.6 mM) a , 772.35 [M+ [M+5H]5+. ss spectrum o om 1 (1.6 mM , 501.45 [M+ [M+4H]4+. of 15 (retentio and 2 (0.4 mM +5H]5+; observ of 1221 (retent ) and 2 (0.4 m +4H]4+; obser n time 9.07 m M). Calculated ved m/z: 19 tion time 9.59 mM). Calculate rved m/z: 20 min in Figure S d m/z: 1929.3 29.49 [M+2H 9 min in Figur ed m/z: 2002 002.84 [M+1H S5.7) from the 37 [M+2H]2+, 1 H]2+, 1286.54 e S5.7) from t .78 [M+1H]+, H]+, 1001.71 e LC‐MS analy 1286.58 [M+3 [M+3H]3+, 96 the LC‐MS an 1001.89 [M+2 [M+2H]2+, 66 ysis of 3H]3+, 65.29 alysis 2H]2+, 68.30
Figu of a 579.
5.5
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) re S5.19. Mas DCL made fro 51 [M+4H]4+;References
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