Summary

High-speed magnetisch pincet voor nanomechanische metingen aan krachtgevoelige elementen

Published: May 12, 2023
doi:

Summary

Hier beschrijven we een snelle magnetische pincetopstelling die nanomechanische metingen uitvoert op krachtgevoelige biomoleculen met de maximale snelheid van 1,2 kHz. We introduceren de toepassing ervan op DNA-haarspelden en SNARE-complexen als modelsystemen, maar het zal ook van toepassing zijn op andere moleculen die betrokken zijn bij mechanobiologische gebeurtenissen.

Abstract

Magnetisch pincet met één molecuul (MT’s) heeft gediend als krachtig hulpmiddel om biomoleculen, zoals nucleïnezuren en eiwitten, krachtig te ondervragen en is daarom klaar om nuttig te zijn op het gebied van mechanobiologie. Omdat de methode vaak afhankelijk is van beeldgebaseerde tracking van magnetische kralen, heeft de snelheidslimiet bij het opnemen en analyseren van afbeeldingen, evenals de thermische fluctuaties van de kralen, de toepassing ervan lang belemmerd bij het observeren van kleine en snelle structurele veranderingen in doelmoleculen. Dit artikel beschrijft gedetailleerde methoden voor de constructie en werking van een MT-opstelling met hoge resolutie die nanoschaal, millisecondedynamiek van biomoleculen en hun complexen kan oplossen. Als toepassingsvoorbeelden worden experimenten met DNA-haarspelden en SNARE-complexen (membraanfusiemachines) gedemonstreerd, waarbij de nadruk ligt op hoe hun voorbijgaande toestanden en overgangen kunnen worden gedetecteerd in de aanwezigheid van piconewton-schaalkrachten. We verwachten dat snelle MT’s zeer nauwkeurige nanomechanische metingen mogelijk zullen blijven maken op moleculen die krachten in cellen waarnemen, overbrengen en genereren, en daardoor ons begrip van mechanobiologie op moleculair niveau verdiepen.

Introduction

Cellen voelen en reageren actief op mechanische prikkels. Daarbij vertonen veel biomoleculen krachtafhankelijke eigenschappen die dynamische structurele veranderingen mogelijk maken. Bekende voorbeelden zijn mechanosensitieve ionkanalen en cytoskeletelementen die de cellen voorzien van belangrijke mechanische informatie uit hun omgeving.

Daarnaast kunnen moleculen die een unieke krachtdragende aard vertonen ook in bredere zin als mechanosensitief worden beschouwd. Lokale vorming en smelten van nucleïnezuurduplexen, evenals hogere-ordestructuren zoals G-quadruplexen, spelen bijvoorbeeld een cruciale rol bij replicatie, transcriptie, recombinatie en meer recent genoombewerking. Bovendien voeren sommige neuronale eiwitten die betrokken zijn bij synaptische communicatie hun functies uit door fysieke krachten te genereren die de niveaus van typische intermoleculaire interacties overschrijden. Het maakt niet uit welk voorbeeld men bestudeert, het onderzoeken van nanomechanica van de betrokken biomoleculen met hoge spatiotemporale precisie zal zeer nuttig blijken te zijn bij het onthullen van moleculaire mechanismen van de bijbehorende mechanobiologische processen 1,2,3.

Single-molecule krachtspectroscopiemethoden hebben gediend als krachtige hulpmiddelen om de mechanische eigenschappen van biomoleculen 2,4,5,6 te onderzoeken. Ze kunnen structurele veranderingen in nucleïnezuren en eiwitten gelijktijdig met de toepassing van kracht volgen, waardoor krachtafhankelijke eigenschappen worden onderzocht. Twee bekende opstellingen zijn optische pincetten en magnetische pincetten (MT’s), die kralen ter grootte van een micron gebruiken om moleculen 5,6,7,8 te manipuleren. In deze platforms worden polystyreen (voor optische pincetten) of magnetische kralen (voor MT’s) vastgebonden aan doelmoleculen (bijv. Nucleïnezuren en eiwitten) via moleculaire “handvatten”, meestal gemaakt van korte fragmenten van dubbelstrengs DNA (dsDNA). De kralen worden vervolgens verplaatst om kracht uit te oefenen en in beeld gebracht om hun locaties te volgen die rapporteren over structurele veranderingen in doelmoleculen. Optische en magnetische pincetten zijn grotendeels uitwisselbaar in hun toepassingen, maar er zijn belangrijke verschillen in hun benaderingen van het regelen van kracht. Optische pincetten zijn intrinsiek positiekleminstrumenten die kralen in positie vangen, waardoor de uitgeoefende kracht fluctueert wanneer een doelconstructie vormveranderingen ondergaat; Extensieverhoging, zoals door het uitvouwen, maakt de tether los en vermindert de spanning, en vice versa. Hoewel actieve feedback kan worden geïmplementeerd om de kracht in optische pincetten te regelen, werken MT’s daarentegen van nature als een krachtklemapparaat, waarbij gebruik wordt gemaakt van stabiele, verafgelegen magnetische krachten door permanente magneten, die ook bestand zijn tegen omgevingsverstoring.

Ondanks hun lange geschiedenis en eenvoudige ontwerp zijn MT’s achtergebleven bij optische pincetten in hun toepassingen voor zeer nauwkeurige metingen, grotendeels vanwege technische uitdagingen bij het volgen van snelle kralen. Onlangs hebben verschillende groepen echter gezamenlijk leiding gegeven aan een veelzijdige verbetering van zowel hardware als software voor MT-instrumenten 2,9,10,11,12,13,14,15,16,17,18,19 . In dit werk introduceren we een voorbeeld van een dergelijke opstelling die op 1,2 kHz draait en beschrijven we hoe deze kan worden gebruikt om nanomechanische metingen uit te voeren op krachtgevoelige biomoleculen. Als modelsystemen gebruiken we DNA-haarspelden en neuronale SNARE-complexen en onderzoeken we hun snelle, structurele veranderingen in het piconewton-regime. DNA-haarspelden vertonen eenvoudige tweetoestandsovergangen in een goed gedefinieerd krachtbereik van20,21 en dienen daarom als speelgoedmodellen om de prestaties van een pincetopstelling te verifiëren. Terwijl de SNARE-eiwitten zich samenvoegen tot een krachtgevoelig complex dat membraanfusie22 aandrijft, zijn ze ook uitgebreid bestudeerd door krachtspectroscopie met één molecuul 14,23,24,25. Standaardbenaderingen voor het analyseren van gegevens en het extraheren van nuttige informatie over thermodynamica en kinetiek worden gepresenteerd. We hopen dat dit artikel de toepassing van zeer nauwkeurige MT’s in mechanobiologische studies kan vergemakkelijken en lezers kan motiveren om hun eigen krachtgevoelige interessesystemen te verkennen.

Protocol

Alle materialen en apparatuur die in dit protocol worden beschreven, worden vermeld in de materiaaltabel. LabVIEW-software om de hieronder beschreven snelle MT-installatie te bedienen, evenals de MATLAB-scripts om voorbeeldgegevens te analyseren, worden gedeponeerd op GitHub (https://github.com/ShonLab/Magnetic-Tweezers) en openbaar beschikbaar. 1. Constructie van apparatuur OPMERKING: Het algemene principe van de high-speed MT-constr…

Representative Results

KrachtkalibratieDe resultaten van de twee krachtmeetmethoden (laterale verplaatsingsvariantie van kralen en vermogensspectrumanalyse) verschilden met 0-2 pN (figuur 2G). Volgens de resultaten in figuur 2F kunnen we betrouwbaar tot 30 pN bereiken met gewone neodymiummagneten. Twee-toestandsovergangen van een 8 bp DNA-haarspeldWe onderzochten eerst de nanomechanica van een korte DNA-haarspeld (<…

Discussion

In dit werk introduceerden we een single-molecule krachtspectroscopie-opstelling die structurele veranderingen van biomoleculen met hoge spatiotemporale precisie kan waarnemen. De gebruikte high-speed CMOS-camera verkrijgt 1.200 frames s−1 met een resolutie van 1.280 x 1.024, waardoor 1,2 kHz kralentracking mogelijk is. De snelheid van metingen wordt momenteel echter beperkt door de kraalvolgsoftware, dus de ROI wordt meestal teruggebracht tot kleinere gebieden bij metingen met hoge snelheid. Het hoge vermog…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

Dit werk werd ondersteund door de National Research Foundation of Korea (NRF) subsidie gefinancierd door de Koreaanse overheid (MSIT) (NRF-2022R1C1C1012176, NRF-2021R1A4A1031754 en NRF- 2021R1A6A1A10042944). S.-H.R. werd ondersteund door de NRF-subsidie (2021R1C1C2009717).

Materials

Materials for construct synthesis
Agarose gel electrophoresis system Advance Mupid-2plus
DNA ladder Bioneer D-1037
nTaq polymerase Enzynomics P050A
PCR purification kit LaboPass CMR0112
PEGylated SMCC crosslinker / SM(PEG)2 ThermoFisher Scientific 22102 For SNARE–DNA coupling
Primer B Bioneer 5'-Biotin/TCGCCACCATCATTTCCA-3' For 5-kbp force calibration construct and DNA handles
Primer B_hp IDT 5'-Biotin/TTTTTTTTTTGTTCTCTATTT
TTTTAGAGAAC /AP site/ /AP site/ TCGCCACCATCATTTCCA-3'
For hairpin construct
Primer N Bioneer 5'-C6Amine/CATGTGGGTGACGCGAAA-3' For DNA handles
Primer Z Bioneer 5'-Azide/TCGCCACCATCATTTCCA-3' For DNA handles
Primer Z_5k Bioneer 5'-Azide/TTAGAGAGTATGGGTATATGACA
TCG-3'
For 5-kbp force calibration construct
Primer Z_hp Bioneer 5'-Azide/GTGGCAGCATGACACC-3' For hairpin construct
SYBR Safe DNA Gel Stain ThermoFisher Scientific S33102
λ-DNA Bioneer D-2510 Template strand for PCR
DNA sequences for SNARE proteins
6×His-tagged SNAP-25b (2-206; capitalized) in pET28a homemade tggcgaatgggacgcgccctgtagcggcgca
ttaagcgcggcgggtgtggtggttacgcgca
gcgtgaccgctacacttgccagcgccctagc
gcccgctcctttcgctttcttcccttccttt
ctcgccacgttcgccggctttccccgtcaag
ctctaaatcgggggctccctttagggttccg
atttagtgctttacggcacctcgaccccaaa
aaacttgattagggtgatggttcacgtagtg
ggccatcgccctgatagacggtttttcgccc
tttgacgttggagtccacgttctttaatagt
ggactcttgttccaaactggaacaacactca
accctatctcggtctattcttttgatttata
agggattttgccgatttcggcctattggtta
aaaaatgagctgatttaacaaaaatttaacg
cgaattttaacaaaatattaacgtttacaat
ttcaggtggcacttttcggggaaatgtgcgc
ggaacccctatttgtttatttttctaaatac
attcaaatatgtatccgctcatgaattaatt
cttagaaaaactcatcgagcatcaaatgaaa
ctgcaatttattcatatcaggattatcaata
ccatatttttgaaaaagccgtttctgtaatg
aaggagaaaactcaccgaggcagttccatag
gatggcaagatcctggtatcggtctgcgatt
ccgactcgtccaacatcaatacaacctatta
atttcccctcgtcaaaaataaggttatcaag
tgagaaatcaccatgagtgacgactgaatcc
ggtgagaatggcaaaagtttatgcatttctt
tccagacttgttcaacaggccagccattacg
ctcgtcatcaaaatcactcgcatcaaccaaa
ccgttattcattcgtgattgcgcctgagcga
gacgaaatacgcgatcgctgttaaaaggaca
attacaaacaggaatcgaatgcaaccggcgc
aggaacactgccagcgcatcaacaatatttt
cacctgaatcaggatattcttctaatacctg
gaatgctgttttcccggggatcgcagtggtg
agtaaccatgcatcatcaggagtacggataa
aatgcttgatggtcggaagaggcataaattc
cgtcagccagtttagtctgaccatctcatct
gtaacatcattggcaacgctacctttgccat
gtttcagaaacaactctggcgcatcgggctt
cccatacaatcgatagattgtcgcacctgat
tgcccgacattatcgcgagcccatttatacc
catataaatcagcatccatgttggaatttaa
tcgcggcctagagcaagacgtttcccgttga
atatggctcataacaccccttgtattactgt
ttatgtaagcagacagttttattgttcatga
ccaaaatcccttaacgtgagttttcgttcca
ctgagcgtcagaccccgtagaaaagatcaaa
ggatcttcttgagatcctttttttctgcgcg
taatctgctgcttgcaaacaaaaaaaccacc
gctaccagcggtggtttgtttgccggatcaa
gagctaccaactctttttccgaaggtaactg
gcttcagcagagcgcagataccaaatactgt
ccttctagtgtagccgtagttaggccaccac
ttcaagaactctgtagcaccgcctacatacc
tcgctctgctaatcctgttaccagtggctgc
tgccagtggcgataagtcgtgtcttaccggg
ttggactcaagacgatagttaccggataagg
cgcagcggtcgggctgaacggggggttcgtg
cacacagcccagcttggagcgaacgacctac
accgaactgagatacctacagcgtgagctat
gagaaagcgccacgcttcccgaagggagaaa
ggcggacaggtatccggtaagcggcagggtc
ggaacaggagagcgcacgagggagcttcca
gggggaaacgcctggtatctttatagtcctgt
cgggtttcgccacctctgacttgagcgtcga
tttttgtgatgctcgtcaggggggcggagcc
tatggaaaaacgccagcaacgcggccttttt
acggttcctggccttttgctggccttttgct
cacatgttctttcctgcgttatcccctgatt
ctgtggataaccgtattaccgcctttgagtg
agctgataccgctcgccgcagccgaacgacc
gagcgcagcgagtcagtgagcgaggaagcgg
aagagcgcctgatgcggtattttctccttac
gcatctgtgcggtatttcacaccgcatatat
ggtgcactctcagtacaatctgctctgatgc
cgcatagttaagccagtatacactccgctat
cgctacgtgactgggtcatggctgcgccccg
acacccgccaacacccgctgacgcgccctga
cgggcttgtctgctcccggcatccgcttaca
gacaagctgtgaccgtctccgggagctgcat
gtgtcagaggttttcaccgtcatcaccgaaa
cgcgcgaggcagctgcggtaaagctcatcag
cgtggtcgtgaagcgattcacagatgtctgc
ctgttcatccgcgtccagctcgttgagtttc
tccagaagcgttaatgtctggcttctgataa
agcgggccatgttaagggcggttttttcctg
tttggtcactgatgcctccgtgtaaggggga
tttctgttcatgggggtaatgataccgatga
aacgagagaggatgctcacgatacgggttac
tgatgatgaacatgcccggttactggaacgt
tgtgagggtaaacaactggcggtatggatgc
ggcgggaccagagaaaaatcactcagggtc
aatgccagcgcttcgttaatacagatgtaggt
gttccacagggtagccagcagcatcctgcga
tgcagatccggaacataatggtgcagggcgc
tgacttccgcgtttccagactttacgaaaca
cggaaaccgaagaccattcatgttgttgctc
aggtcgcagacgttttgcagcagcagtcgct
tcacgttcgctcgcgtatcggtgattcattc
tgctaaccagtaaggcaaccccgccagccta
gccgggtcctcaacgacaggagcacgatcat
gcgcacccgtggggccgccatgccggcgata
atggcctgcttctcgccgaaacgtttggtgg
cgggaccagtgacgaaggcttgagcgagggc
gtgcaagattccgaataccgcaagcgacagg
ccgatcatcgtcgcgctccagcgaaagcggt
cctcgccgaaaatgacccagagcgctgccgg
cacctgtcctacgagttgcatgataaagaag
acagtcataagtgcggcgacgatagtcatgc
cccgcgcccaccggaaggagctgactgggtt
gaaggctctcaagggcatcggtcgagatccc
ggtgcctaatgagtgagctaacttacattaa
ttgcgttgcgctcactgcccgctttccagtc
gggaaacctgtcgtgccagctgcattaatga
atcggccaacgcgcggggagaggcggtttgc
gtattgggcgccagggtggtttttcttttca
ccagtgagacgggcaacagctgattgccctt
caccgcctggccctgagagagttgcagcaag
cggtccacgctggtttgccccagcaggcgaa
aatcctgtttgatggtggttaacggcgggat
ataacatgagctgtcttcggtatcgtcgtat
cccactaccgagatatccgcaccaacgcgca
gcccggactcggtaatggcgcgcattgcgcc
cagcgccatctgatcgttggcaaccagcatc
gcagtgggaacgatgccctcattcagcattt
gcatggtttgttgaaaaccggacatggcact
ccagtcgccttcccgttccgctatcggctga
atttgattgcgagtgagatatttatgccagc
cagccagacgcagacgcgccgagacagaa
cttaatgggcccgctaacagcgcgatttgctgg
tgacccaatgcgaccagatgctccacgccca
gtcgcgtaccgtcttcatgggagaaaataat
actgttgatgggtgtctggtcagagacatca
agaaataacgccggaacattagtgcaggcag
cttccacagcaatggcatcctggtcatccag
cggatagttaatgatcagcccactgacgcgt
tgcgcgagaagattgtgcaccgccgctttac
aggcttcgacgccgcttcgttctaccatcga
caccaccacgctggcacccagttgatcggcg
cgagatttaatcgccgcgacaatttgcgacg
gcgcgtgcagggccagactggaggtggcaac
gccaatcagcaacgactgtttgcccgccagt
tgttgtgccacgcggttgggaatgtaattca
gctccgccatcgccgcttccactttttcccg
cgttttcgcagaaacgtggctggcctggttc
accacgcgggaaacggtctgataagagacac
cggcatactctgcgacatcgtataacgttac
tggtttcacattcaccaccctgaattgactc
tcttccgggcgctatcatgccataccgcgaa
aggttttgcgccattcgatggtgtccgggat
ctcgacgctctcccttatgcgactcctgcat
taggaagcagcccagtagtaggttgaggccg
ttgagcaccgccgccgcaaggaatggtgcat
gcaaggagatggcgcccaacagtcccccggc
cacggggcctgccaccatacccacgccgaaa
caagcgctcatgagcccgaagtggcgagccc
gatcttccccatcggtgatgtcggcgatata
ggcgccagcaaccgcacctgtggcgccggtg
atgccggccacgatgcgtccggcgtagagga
tcgagatctcgatcccgcgaaattaatacga
ctcactataggggaattgtgagcggataaca
attcccctctagaaataattttgtttaactt
taagaaggagatataccATGGGCAGC
AGCCATCATCATCATCATCACA
GCAGCGGCCTGGTGCCGCGC
GGCAGCCATACTAGCGGAGAT
ATCGCCGAGGACGCAGACAT
GCGCAATGAGCTGGAGGAGA
TGCAGAGGAGGGCTGACCAG
CTGGCTGATGAGTCCCTGGA
AAGCACCCGTCGCATGCTGC
AGCTGGTTGAAGAGAGTAAA
GATGCTGGCATCAGGACTTT
GGTTATGTTGGATGAGCAAG
GCGAACAACTGGAACGCATT
GAGGAAGGGATGGACCAAAT
CAATAAGGACATGAAAGAAG
CAGAAAAGAATTTGACGGAC
CTAGGAAAATTCGCCGGCCT
TGCCGTGGCCCCCGCCAAC
AAGCTTAAATCCAGTGATGC
TTACAAAAAAGCCTGGGGC
AATAATCAGGATGGAGTAGT
GGCCAGCCAGCCTGCCCG
TGTGGTGGATGAACGGGAG
CAGATGGCCATCAGTGGTG
GCTTCATCCGCAGGGTAAC
AAATGATGCCCGGGAAAAT
GAGATGGATGAGAACCTG
GAGCAGGTGAGCGGCATC
ATCGGAAACCTCCGCCAC
ATGGCTCTAGACATGGGCA
ATGAGATTGACACCCAGA
ATCGCCAGATCGACAGGA
TCATGGAGAAGGCTGATT
CCAACAAAACCAGAATTG
ATGAAGCCAACCAACGTG
CAACAAAGATGCTGGGAA
GTGGTTAAggatccgaattcgag
ctccgtcgacaagcttgcggccgcactc
gagcaccaccaccaccaccactgagat
ccggctgctaacaaagcccgaaagga
agctgagttggctgctgccaccgctgag
caataactagcataaccccttggggcct
ctaaacgggtcttgaggggttttttgctga
aaggaggaactatatccggat
6×His-tagged VAMP2 (2-97, L32C/I97C; capitalized) in pET28a homemade tggcgaatgggacgcgccctgtagcggcgca
ttaagcgcggcgggtgtggtggttacgcgca
gcgtgaccgctacacttgccagcgccctagc
gcccgctcctttcgctttcttcccttccttt
ctcgccacgttcgccggctttccccgtcaag
ctctaaatcgggggctccctttagggttccg
atttagtgctttacggcacctcgaccccaaa
aaacttgattagggtgatggttcacgtagtg
ggccatcgccctgatagacggtttttcgccc
tttgacgttggagtccacgttctttaatagt
ggactcttgttccaaactggaacaacactca
accctatctcggtctattcttttgatttata
agggattttgccgatttcggcctattggtta
aaaaatgagctgatttaacaaaaatttaacg
cgaattttaacaaaatattaacgtttacaat
ttcaggtggcacttttcggggaaatgtgcgc
ggaacccctatttgtttatttttctaaatac
attcaaatatgtatccgctcatgaattaatt
cttagaaaaactcatcgagcatcaaatgaaa
ctgcaatttattcatatcaggattatcaata
ccatatttttgaaaaagccgtttctgtaatg
aaggagaaaactcaccgaggcagttccatag
gatggcaagatcctggtatcggtctgcgatt
ccgactcgtccaacatcaatacaacctatta
atttcccctcgtcaaaaataaggttatcaag
tgagaaatcaccatgagtgacgactgaatcc
ggtgagaatggcaaaagtttatgcatttctt
tccagacttgttcaacaggccagccattacg
ctcgtcatcaaaatcactcgcatcaaccaaa
ccgttattcattcgtgattgcgcctgagcga
gacgaaatacgcgatcgctgttaaaaggaca
attacaaacaggaatcgaatgcaaccggcgc
aggaacactgccagcgcatcaacaatatttt
cacctgaatcaggatattcttctaatacctg
gaatgctgttttcccggggatcgcagtggtg
agtaaccatgcatcatcaggagtacggataa
aatgcttgatggtcggaagaggcataaattc
cgtcagccagtttagtctgaccatctcatct
gtaacatcattggcaacgctacctttgccat
gtttcagaaacaactctggcgcatcgggctt
cccatacaatcgatagattgtcgcacctgat
tgcccgacattatcgcgagcccatttatacc
catataaatcagcatccatgttggaatttaa
tcgcggcctagagcaagacgtttcccgttga
atatggctcataacaccccttgtattactgt
ttatgtaagcagacagttttattgttcatga
ccaaaatcccttaacgtgagttttcgttcca
ctgagcgtcagaccccgtagaaaagatcaaa
ggatcttcttgagatcctttttttctgcgcg
taatctgctgcttgcaaacaaaaaaaccacc
gctaccagcggtggtttgtttgccggatcaa
gagctaccaactctttttccgaaggtaactg
gcttcagcagagcgcagataccaaatactgt
ccttctagtgtagccgtagttaggccaccac
ttcaagaactctgtagcaccgcctacatacc
tcgctctgctaatcctgttaccagtggctgc
tgccagtggcgataagtcgtgtcttaccggg
ttggactcaagacgatagttaccggataagg
cgcagcggtcgggctgaacggggggttcgtg
cacacagcccagcttggagcgaacgacctac
accgaactgagatacctacagcgtgagctatg
agaaagcgccacgcttcccgaagggagaaa
ggcggacaggtatccggtaagcggcagggtc
ggaacaggagagcgcacgagggagcttcca
gggggaaacgcctggtatctttatagtcctgt
cgggtttcgccacctctgacttgagcgtcga
tttttgtgatgctcgtcaggggggcggagcc
tatggaaaaacgccagcaacgcggccttttt
acggttcctggccttttgctggccttttgct
cacatgttctttcctgcgttatcccctgatt
ctgtggataaccgtattaccgcctttgagtg
agctgataccgctcgccgcagccgaacgacc
gagcgcagcgagtcagtgagcgaggaagc
ggaagagcgcctgatgcggtattttctccttac
gcatctgtgcggtatttcacaccgcatatat
ggtgcactctcagtacaatctgctctgatgc
cgcatagttaagccagtatacactccgctat
cgctacgtgactgggtcatggctgcgccccg
acacccgccaacacccgctgacgcgccctga
cgggcttgtctgctcccggcatccgcttaca
gacaagctgtgaccgtctccgggagctgcat
gtgtcagaggttttcaccgtcatcaccgaaa
cgcgcgaggcagctgcggtaaagctcatcag
cgtggtcgtgaagcgattcacagatgtctgc
ctgttcatccgcgtccagctcgttgagtttc
tccagaagcgttaatgtctggcttctgataa
agcgggccatgttaagggcggttttttcctg
tttggtcactgatgcctccgtgtaaggggga
tttctgttcatgggggtaatgataccgatga
aacgagagaggatgctcacgatacgggttac
tgatgatgaacatgcccggttactggaacgt
tgtgagggtaaacaactggcggtatggatgc
ggcgggaccagagaaaaatcactcagggtc
aatgccagcgcttcgttaatacagatgtaggt
gttccacagggtagccagcagcatcctgcga
tgcagatccggaacataatggtgcagggcgc
tgacttccgcgtttccagactttacgaaaca
cggaaaccgaagaccattcatgttgttgctc
aggtcgcagacgttttgcagcagcagtcgct
tcacgttcgctcgcgtatcggtgattcattc
tgctaaccagtaaggcaaccccgccagccta
gccgggtcctcaacgacaggagcacgatcat
gcgcacccgtggggccgccatgccggcgata
atggcctgcttctcgccgaaacgtttggtgg
cgggaccagtgacgaaggcttgagcgagggc
gtgcaagattccgaataccgcaagcgacagg
ccgatcatcgtcgcgctccagcgaaagcggt
cctcgccgaaaatgacccagagcgctgccgg
cacctgtcctacgagttgcatgataaagaag
acagtcataagtgcggcgacgatagtcatgc
cccgcgcccaccggaaggagctgactgggtt
gaaggctctcaagggcatcggtcgagatccc
ggtgcctaatgagtgagctaacttacattaa
ttgcgttgcgctcactgcccgctttccagtc
gggaaacctgtcgtgccagctgcattaatga
atcggccaacgcgcggggagaggcggtttgc
gtattgggcgccagggtggtttttcttttca
ccagtgagacgggcaacagctgattgccctt
caccgcctggccctgagagagttgcagcaag
cggtccacgctggtttgccccagcaggcgaa
aatcctgtttgatggtggttaacggcgggat
ataacatgagctgtcttcggtatcgtcgtat
cccactaccgagatatccgcaccaacgcgca
gcccggactcggtaatggcgcgcattgcgcc
cagcgccatctgatcgttggcaaccagcatc
gcagtgggaacgatgccctcattcagcattt
gcatggtttgttgaaaaccggacatggcact
ccagtcgccttcccgttccgctatcggctga
atttgattgcgagtgagatatttatgccagc
cagccagacgcagacgcgccgagacagaa
cttaatgggcccgctaacagcgcgatttgctgg
tgacccaatgcgaccagatgctccacgccca
gtcgcgtaccgtcttcatgggagaaaataat
actgttgatgggtgtctggtcagagacatca
agaaataacgccggaacattagtgcaggcag
cttccacagcaatggcatcctggtcatccag
cggatagttaatgatcagcccactgacgcgt
tgcgcgagaagattgtgcaccgccgctttac
aggcttcgacgccgcttcgttctaccatcga
caccaccacgctggcacccagttgatcggcg
cgagatttaatcgccgcgacaatttgcgacg
gcgcgtgcagggccagactggaggtggcaac
gccaatcagcaacgactgtttgcccgccagt
tgttgtgccacgcggttgggaatgtaattca
gctccgccatcgccgcttccactttttcccg
cgttttcgcagaaacgtggctggcctggttc
accacgcgggaaacggtctgataagagacac
cggcatactctgcgacatcgtataacgttac
tggtttcacattcaccaccctgaattgactc
tcttccgggcgctatcatgccataccgcgaa
aggttttgcgccattcgatggtgtccgggat
ctcgacgctctcccttatgcgactcctgcat
taggaagcagcccagtagtaggttgaggccg
ttgagcaccgccgccgcaaggaatggtgcat
gcaaggagatggcgcccaacagtcccccggc
cacggggcctgccaccatacccacgccgaaa
caagcgctcatgagcccgaagtggcgagccc
gatcttccccatcggtgatgtcggcgatata
ggcgccagcaaccgcacctgtggcgccggtg
atgccggccacgatgcgtccggcgtagagga
tcgagatctcgatcccgcgaaattaatacga
ctcactataggggaattgtgagcggataaca
attcccctctagaaataattttgtttaactt
taagaaggagatataccATGGGCAGC
AGCCATCATCATCATCATCAC
AGCAGCGGCCTGGTGCCGC
GCGGCAGCCATATGGCAGAT
CTCTCGGCTACCGCTGCCAC
CGTCCCGCCTGCCGCCCCG
GCCGGCGAGGGTGGCCCCC
CTGCACCTCCTCCAAATCTTA
CCAGTAACAGGAGATGCCAG
CAGACCCAGGCCCAGGTGG
ATGAGGTGGTGGACATCATG
AGGGTGAATGTGGACAAGGT
CCTGGAGCGAGACCAGAAG
CTATCGGAACTGGATGATCG
CGCAGATGCCCTCCAGGCA
GGGGCCTCCCAGTTTGAAA
CAAGTGCAGCCAAGCTCAA
GCGCAAATACTGGTGGAAA
AACCTCAAGATGATGTGCTA
Aggatccgaattcgagctccgtcg
acaagcttgcggccgcactcgagcaccacca
ccaccaccactgagatccggctgctaacaaa
gcccgaaaggaagctgagttggctgctgcca
ccgctgagcaataactagcataaccccttgg
ggcctctaaacgggtcttgaggggttttttg
ctgaaaggaggaactatatccggat
6×His-tagged ΔN-VAMP2 (49–96; capitalized) and Syntaxin-1A (191–267, I202C/I266C; capitalized) in pETDuet-1 homemade ggggaattgtgagcggataacaattcccctc
tagaaataattttgtttaactttaagaagga
gatataccATGGGCAGCAGCCATCA
TCATCATCATCACAGCAGCGG
CCTGGAAGTTCTGTTCCAGGG
GCCCGGTAATGTGGACAAGGT
CCTGGAGCGAGACCAGAAGCT
ATCGGAACTGGATGATCGCGC
AGATGCCCTCCAGGCAGGGGC
CTCCCAGTTTGAAACAAGTGC
AGCCAAGCTCAAGCGCAAATAC
TGGTGGAAAAACCTCAAGATGAT
GTAAgcggccgcataatgcttaagtcgaaca
gaaagtaatcgtattgtacacggccgcataa
tcgaaattaatacgactcactataggggaat
tgtgagcggataacaattccccatcttagta
tattagttaagtataagaaggagatatacat
ATGGCCCTCAGTGAGATCGAGA
CCAGGCACAGTGAGTGCATC
AAGTTGGAGAACAGCATCCG
GGAGCTACACGATATGTTCAT
GGACATGGCCATGCTGGTGG
AGAGCCAGGGGGAGATGATT
GACAGGATCGAGTACAATGTG
GAACACGCTGTGGACTACGTG
GAGAGGGCCGTGTCTGACACC
AAGAAGGCCGTCAAGTACCAG
AGCAAGGCACGCAGGAAGAA
GTGCATGATCTAActcgagtc
tggtaaagaaaccgctgctgcgaaatttgaa
cgccagcacatggactcgtctactagcgcag
cttaattaacctaggctgctgccaccgctga
gcaataactagcataaccccttggggcctct
aaacgggtcttgaggggttttttgctgaaag
gaggaactatatccggattggcgaatgggac
gcgccctgtagcggcgcattaagcgcggcgg
gtgtggtggttacgcgcagcgtgaccgctac
acttgccagcgccctagcgcccgctcctttc
gctttcttcccttcctttctcgccacgttcg
ccggctttccccgtcaagctctaaatcgggg
gctccctttagggttccgatttagtgcttta
cggcacctcgaccccaaaaaacttgattagg
gtgatggttcacgtagtgggccatcgccctg
atagacggtttttcgccctttgacgttggag
tccacgttctttaatagtggactcttgttcc
aaactggaacaacactcaaccctatctcggt
ctattcttttgatttataagggattttgccg
atttcggcctattggttaaaaaatgagctga
tttaacaaaaatttaacgcgaattttaacaa
aatattaacgtttacaatttctggcggcacg
atggcatgagattatcaaaaaggatcttcac
ctagatccttttaaattaaaaatgaagtttt
aaatcaatctaaagtatatatgagtaaactt
ggtctgacagttaccaatgcttaatcagtga
ggcacctatctcagcgatctgtctatttcgt
tcatccatagttgcctgactccccgtcgtgt
agataactacgatacgggagggcttaccatc
tggccccagtgctgcaatgataccgcgagac
ccacgctcaccggctccagatttatcagcaa
taaaccagccagccggaagggccgagcgca
gaagtggtcctgcaactttatccgcctccatc
cagtctattaattgttgccgggaagctagag
taagtagttcgccagttaatagtttgcgcaa
cgttgttgccattgctacaggcatcgtggtg
tcacgctcgtcgtttggtatggcttcattca
gctccggttcccaacgatcaaggcgagttac
atgatcccccatgttgtgcaaaaaagcggtt
agctccttcggtcctccgatcgttgtcagaa
gtaagttggccgcagtgttatcactcatggt
tatggcagcactgcataattctcttactgtc
atgccatccgtaagatgcttttctgtgactg
gtgagtactcaaccaagtcattctgagaata
gtgtatgcggcgaccgagttgctcttgcccg
gcgtcaatacgggataataccgcgccacata
gcagaactttaaaagtgctcatcattggaaa
acgttcttcggggcgaaaactctcaaggatc
ttaccgctgttgagatccagttcgatgtaac
ccactcgtgcacccaactgatcttcagcatc
ttttactttcaccagcgtttctgggtgagcaaa
aacaggaaggcaaaatgccgcaaaaaagg
gaataagggcgacacggaaatgttgaatact
catactcttcctttttcaatcatgattgaag
catttatcagggttattgtctcatgagcgga
tacatatttgaatgtatttagaaaaataaac
aaataggtcatgaccaaaatcccttaacgtg
agttttcgttccactgagcgtcagaccccgt
agaaaagatcaaaggatcttcttgagatcct
ttttttctgcgcgtaatctgctgcttgcaaa
caaaaaaaccaccgctaccagcggtggtttg
tttgccggatcaagagctaccaactcttttt
ccgaaggtaactggcttcagcagagcgcaga
taccaaatactgtccttctagtgtagccgta
gttaggccaccacttcaagaactctgtagca
ccgcctacatacctcgctctgctaatcctgt
taccagtggctgctgccagtggcgataagtc
gtgtcttaccgggttggactcaagacgatag
ttaccggataaggcgcagcggtcgggctgaa
cggggggttcgtgcacacagcccagcttgga
gcgaacgacctacaccgaactgagataccta
cagcgtgagctatgagaaagcgccacgcttccc
gaagggagaaaggcggacaggtatccggta
agcggcagggtcggaacaggagagcgcac
gagggagcttccagggggaaacgcctggtatc
tttatagtcctgtcgggtttcgccacctctg
acttgagcgtcgatttttgtgatgctcgtca
ggggggcggagcctatggaaaaacgccagc
aacgcggcctttttacggttcctggccttttg
ctggccttttgctcacatgttctttcctgcg
ttatcccctgattctgtggataaccgtatta
ccgcctttgagtgagctgataccgctcgccgc
agccgaacgaccgagcgcagcgagtcagtg
agcgaggaagcggaagagcgcctgatgcgg
tattttctccttacgcatctgtgcggtatttc
acaccgcatatatggtgcactctcagtacaa
tctgctctgatgccgcatagttaagccagta
tacactccgctatcgctacgtgactgggtca
tggctgcgccccgacacccgccaacacccgc
tgacgcgccctgacgggcttgtctgctcccg
gcatccgcttacagacaagctgtgaccgtct
ccgggagctgcatgtgtcagaggttttcacc
gtcatcaccgaaacgcgcgaggcagctgcgg
taaagctcatcagcgtggtcgtgaagcgatt
cacagatgtctgcctgttcatccgcgtccag
ctcgttgagtttctccagaagcgttaatgtc
tggcttctgataaagcgggccatgttaaggg
cggttttttcctgtttggtcactgatgcctc
cgtgtaagggggatttctgttcatgggggta
atgataccgatgaaacgagagaggatgctca
cgatacgggttactgatgatgaacatgcccg
gttactggaacgttgtgagggtaaacaactg
gcggtatggatgcggcgggaccagagaaaaa
tcactcagggtcaatgccagcgcttcgttaa
tacagatgtaggtgttccacagggtagccag
cagcatcctgcgatgcagatccggaacataa
tggtgcagggcgctgacttccgcgtttccag
actttacgaaacacggaaaccgaagaccatt
catgttgttgctcaggtcgcagacgttttgc
agcagcagtcgcttcacgttcgctcgcgtat
cggtgattcattctgctaaccagtaaggcaa
ccccgccagcctagccgggtcctcaacgaca
ggagcacgatcatgctagtcatgccccgcgc
ccaccggaaggagctgactgggttgaaggct
ctcaagggcatcggtcgagatcccggtgcct
aatgagtgagctaacttacattaattgcgtt
gcgctcactgcccgctttccagtcgggaaac
ctgtcgtgccagctgcattaatgaatcggcc
aacgcgcggggagaggcggtttgcgtattgg
gcgccagggtggtttttcttttcaccagtga
gacgggcaacagctgattgcccttcaccgcc
tggccctgagagagttgcagcaagcggtcca
cgctggtttgccccagcaggcgaaaatcctg
tttgatggtggttaacggcgggatataacat
gagctgtcttcggtatcgtcgtatcccacta
ccgagatgtccgcaccaacgcgcagcccgga
ctcggtaatggcgcgcattgcgcccagcgcc
atctgatcgttggcaaccagcatcgcagtgg
gaacgatgccctcattcagcatttgcatggt
ttgttgaaaaccggacatggcactccagtcg
ccttcccgttccgctatcggctgaatttgat
tgcgagtgagatatttatgccagccagccag
acgcagacgcgccgagacagaacttaatggg
cccgctaacagcgcgatttgctggtgaccca
atgcgaccagatgctccacgcccagtcgcgt
accgtcttcatgggagaaaataatactgttg
atgggtgtctggtcagagacatcaagaaata
acgccggaacattagtgcaggcagcttccac
agcaatggcatcctggtcatccagcggatag
ttaatgatcagcccactgacgcgttgcgcga
gaagattgtgcaccgccgctttacaggcttc
gacgccgcttcgttctaccatcgacaccacc
acgctggcacccagttgatcggcgcgagatt
taatcgccgcgacaatttgcgacggcgcgtg
cagggccagactggaggtggcaacgccaatc
agcaacgactgtttgcccgccagttgttgtg
ccacgcggttgggaatgtaattcagctccgc
catcgccgcttccactttttcccgcgttttc
gcagaaacgtggctggcctggttcaccacgc
gggaaacggtctgataagagacaccggcata
ctctgcgacatcgtataacgttactggtttc
acattcaccaccctgaattgactctcttccg
ggcgctatcatgccataccgcgaaaggtttt
gcgccattcgatggtgtccgggatctcgacg
ctctcccttatgcgactcctgcattaggaag
cagcccagtagtaggttgaggccgttgagca
ccgccgccgcaaggaatggtgcatgcaagga
gatggcgcccaacagtcccccggccacgggg
cctgccaccatacccacgccgaaacaagcgc
tcatgagcccgaagtggcgagcccgatcttc
cccatcggtgatgtcggcgatataggcgcca
gcaaccgcacctgtggcgccggtgatgccgg
ccacgatgcgtccggcgtagaggatcgagat
cgatctcgatcccgcgaaattaatacgactc
actata
SNAP-25b (1–206, all C to A; capitalized) in pET28a homemade tggcgaatgggacgcgccctgtagcggcgca
ttaagcgcggcgggtgtggtggttacgcgca
gcgtgaccgctacacttgccagcgccctagc
gcccgctcctttcgctttcttcccttccttt
ctcgccacgttcgccggctttccccgtcaag
ctctaaatcgggggctccctttagggttccg
atttagtgctttacggcacctcgaccccaaa
aaacttgattagggtgatggttcacgtagtg
ggccatcgccctgatagacggtttttcgccc
tttgacgttggagtccacgttctttaatagt
ggactcttgttccaaactggaacaacactca
accctatctcggtctattcttttgatttata
agggattttgccgatttcggcctattggtta
aaaaatgagctgatttaacaaaaatttaacg
cgaattttaacaaaatattaacgtttacaat
ttcaggtggcacttttcggggaaatgtgcgc
ggaacccctatttgtttatttttctaaatac
attcaaatatgtatccgctcatgaattaatt
cttagaaaaactcatcgagcatcaaatgaaa
ctgcaatttattcatatcaggattatcaata
ccatatttttgaaaaagccgtttctgtaatg
aaggagaaaactcaccgaggcagttccatag
gatggcaagatcctggtatcggtctgcgatt
ccgactcgtccaacatcaatacaacctatta
atttcccctcgtcaaaaataaggttatcaag
tgagaaatcaccatgagtgacgactgaatcc
ggtgagaatggcaaaagtttatgcatttctt
tccagacttgttcaacaggccagccattacg
ctcgtcatcaaaatcactcgcatcaaccaaa
ccgttattcattcgtgattgcgcctgagcga
gacgaaatacgcgatcgctgttaaaaggaca
attacaaacaggaatcgaatgcaaccggcgc
aggaacactgccagcgcatcaacaatatttt
cacctgaatcaggatattcttctaatacctg
gaatgctgttttcccggggatcgcagtggtg
agtaaccatgcatcatcaggagtacggataa
aatgcttgatggtcggaagaggcataaattc
cgtcagccagtttagtctgaccatctcatct
gtaacatcattggcaacgctacctttgccat
gtttcagaaacaactctggcgcatcgggctt
cccatacaatcgatagattgtcgcacctgat
tgcccgacattatcgcgagcccatttatacc
catataaatcagcatccatgttggaatttaa
tcgcggcctagagcaagacgtttcccgttga
atatggctcataacaccccttgtattactgt
ttatgtaagcagacagttttattgttcatga
ccaaaatcccttaacgtgagttttcgttcca
ctgagcgtcagaccccgtagaaaagatcaaa
ggatcttcttgagatcctttttttctgcgcg
taatctgctgcttgcaaacaaaaaaaccacc
gctaccagcggtggtttgtttgccggatcaa
gagctaccaactctttttccgaaggtaactg
gcttcagcagagcgcagataccaaatactgt
ccttctagtgtagccgtagttaggccaccac
ttcaagaactctgtagcaccgcctacatacc
tcgctctgctaatcctgttaccagtggctgc
tgccagtggcgataagtcgtgtcttaccggg
ttggactcaagacgatagttaccggataagg
cgcagcggtcgggctgaacggggggttcgtg
cacacagcccagcttggagcgaacgacctac
accgaactgagatacctacagcgtgagctatg
agaaagcgccacgcttcccgaagggagaaa
ggcggacaggtatccggtaagcggcagggtc
ggaacaggagagcgcacgagggagcttcc
agggggaaacgcctggtatctttatagtcctgt
cgggtttcgccacctctgacttgagcgtcga
tttttgtgatgctcgtcaggggggcggagcc
tatggaaaaacgccagcaacgcggccttttt
acggttcctggccttttgctggccttttgct
cacatgttctttcctgcgttatcccctgatt
ctgtggataaccgtattaccgcctttgagtg
agctgataccgctcgccgcagccgaacgacc
gagcgcagcgagtcagtgagcgaggaagc
ggaagagcgcctgatgcggtattttctccttac
gcatctgtgcggtatttcacaccgcatatat
ggtgcactctcagtacaatctgctctgatgc
cgcatagttaagccagtatacactccgctat
cgctacgtgactgggtcatggctgcgccccg
acacccgccaacacccgctgacgcgccctga
cgggcttgtctgctcccggcatccgcttaca
gacaagctgtgaccgtctccgggagctgcat
gtgtcagaggttttcaccgtcatcaccgaaa
cgcgcgaggcagctgcggtaaagctcatcag
cgtggtcgtgaagcgattcacagatgtctgc
ctgttcatccgcgtccagctcgttgagtttc
tccagaagcgttaatgtctggcttctgataa
agcgggccatgttaagggcggttttttcctg
tttggtcactgatgcctccgtgtaaggggga
tttctgttcatgggggtaatgataccgatga
aacgagagaggatgctcacgatacgggttac
tgatgatgaacatgcccggttactggaacgt
tgtgagggtaaacaactggcggtatggatgc
ggcgggaccagagaaaaatcactcagggtc
aatgccagcgcttcgttaatacagatgtaggt
gttccacagggtagccagcagcatcctgcga
tgcagatccggaacataatggtgcagggcgc
tgacttccgcgtttccagactttacgaaaca
cggaaaccgaagaccattcatgttgttgctc
aggtcgcagacgttttgcagcagcagtcgct
tcacgttcgctcgcgtatcggtgattcattc
tgctaaccagtaaggcaaccccgccagccta
gccgggtcctcaacgacaggagcacgatcat
gcgcacccgtggggccgccatgccggcgata
atggcctgcttctcgccgaaacgtttggtgg
cgggaccagtgacgaaggcttgagcgagggc
gtgcaagattccgaataccgcaagcgacagg
ccgatcatcgtcgcgctccagcgaaagcggt
cctcgccgaaaatgacccagagcgctgccgg
cacctgtcctacgagttgcatgataaagaag
acagtcataagtgcggcgacgatagtcatgc
cccgcgcccaccggaaggagctgactgggtt
gaaggctctcaagggcatcggtcgagatccc
ggtgcctaatgagtgagctaacttacattaa
ttgcgttgcgctcactgcccgctttccagtc
gggaaacctgtcgtgccagctgcattaatga
atcggccaacgcgcggggagaggcggtttgc
gtattgggcgccagggtggtttttcttttca
ccagtgagacgggcaacagctgattgccctt
caccgcctggccctgagagagttgcagcaag
cggtccacgctggtttgccccagcaggcgaa
aatcctgtttgatggtggttaacggcgggat
ataacatgagctgtcttcggtatcgtcgtat
cccactaccgagatatccgcaccaacgcgca
gcccggactcggtaatggcgcgcattgcgcc
cagcgccatctgatcgttggcaaccagcatc
gcagtgggaacgatgccctcattcagcattt
gcatggtttgttgaaaaccggacatggcact
ccagtcgccttcccgttccgctatcggctga
atttgattgcgagtgagatatttatgccagc
cagccagacgcagacgcgccgagacagaa
cttaatgggcccgctaacagcgcgatttgctgg
tgacccaatgcgaccagatgctccacgccca
gtcgcgtaccgtcttcatgggagaaaataat
actgttgatgggtgtctggtcagagacatca
agaaataacgccggaacattagtgcaggcag
cttccacagcaatggcatcctggtcatccag
cggatagttaatgatcagcccactgacgcgt
tgcgcgagaagattgtgcaccgccgctttac
aggcttcgacgccgcttcgttctaccatcga
caccaccacgctggcacccagttgatcggcg
cgagatttaatcgccgcgacaatttgcgacg
gcgcgtgcagggccagactggaggtggcaac
gccaatcagcaacgactgtttgcccgccagt
tgttgtgccacgcggttgggaatgtaattca
gctccgccatcgccgcttccactttttcccg
cgttttcgcagaaacgtggctggcctggttc
accacgcgggaaacggtctgataagagacac
cggcatactctgcgacatcgtataacgttac
tggtttcacattcaccaccctgaattgactc
tcttccgggcgctatcatgccataccgcgaa
aggttttgcgccattcgatggtgtccgggat
ctcgacgctctcccttatgcgactcctgcat
taggaagcagcccagtagtaggttgaggccg
ttgagcaccgccgccgcaaggaatggtgcat
gcaaggagatggcgcccaacagtcccccggc
cacggggcctgccaccatacccacgccgaaa
caagcgctcatgagcccgaagtggcgagccc
gatcttccccatcggtgatgtcggcgatata
ggcgccagcaaccgcacctgtggcgccggtg
atgccggccacgatgcgtccggcgtagagga
tcgagatctcgatcccgcgaaattaatacga
ctcactataggggaattgtgagcggataaca
attcccctctagaaataattttgtttaactt
taagaaggagatataccATGGCCGA
GGACGCAGACATGCGCAATG
AGCTGGAGGAGATGCAGAGG
AGGGCTGACCAGCTGGCTGA
TGAGTCCCTGGAAAGCACCC
GTCGCATGCTGCAGCTGGTT
GAAGAGAGTAAAGATGCTGG
CATCAGGACTTTGGTTATGTT
GGATGAGCAAGGCGAACAAC
TGGAACGCATTGAGGAAGGG
ATGGACCAAATCAATAAGGAC
ATGAAAGAAGCAGAAAAGAAT
TTGACGGACCTAGGAAAATTC
GCCGGCCTTGCCGTGGCCCC
CGCCAACAAGCTTAAATCCAG
TGATGCTTACAAAAAAGCCTG
GGGCAATAATCAGGATGGAGT
AGTGGCCAGCCAGCCTGCCC
GTGTGGTGGATGAACGGGAG
CAGATGGCCATCAGTGGTGGC
TTCATCCGCAGGGTAACAAAT
GATGCCCGGGAAAATGAGATG
GATGAGAACCTGGAGCAGGT
GAGCGGCATCATCGGAAACCT
CCGCCACATGGCTCTAGACAT
GGGCAATGAGATTGACACCCA
GAATCGCCAGATCGACAGGAT
CATGGAGAAGGCTGATTCCAA
CAAAACCAGAATTGATGAAGC
CAACCAACGTGCAACAAAGAT
GCTGGGAAGTGGTTAA
ctcgagcaccaccaccaccaccactgag
atccggctgctaacaaagcccgaaagga
agctgagttggctgctgccaccgctgagc
aataactagcataaccccttggggcctc
taaacgggtcttgaggggttttttgctgaa
aggaggaactatatccggat
Materials for protein purificaiton
2-Mercaptoethanol SIGMA M3148-25ML
Agar LPS Solution AGA500
Ampicillin, Sodium salt PLS AC1043-005-00
Chloramphenicol PLS CR1023-050-00
Competent cells (E. coli) Novagen 70956 Rosetta(DE3)pLysS
Glycerol SIGMA G5516-500ML
HEPES SIGMA H4034-100G
Hydrochloric acid / HCl SIGMA 320331-500ML
Imidazole SIGMA I2399-100G
Isopropyl β-D-1-thiogalactopyranoside / IPTG SIGMA 10724815001
Kanamycin Sulfate PLS KC1001-005-02
Luria-Bertani (LB) Broth LPS Solution LB-05
Ni-NTA resin Qiagen 30210
PD MiniTrap G-25 (desalting column) Cytiva GE28-9180-07 For instructions, see: https://www.cytivalifesciences.com/en/us/shop/chromatography/prepacked-columns/desalting-and-buffer-exchange/pd-minitrap-desalting-columns-with-sephadex-g-25-resin-p-06174
Phenylmethylsulfonyl fluoride / PMSF ThermoFisher Scientific 36978
Plasmids for SNARE proteins cloned in house N/A Available upon request
Protease inhibitor cocktail genDEPOT P3100
Sodium chloride SIGMA S5886-500G
Sodium phosphate dibasic / Na2HPO4 SIGMA S7907-100G
Sodium phosphate monobasic / NaH2PO4 SIGMA S3139-250G
Tris(2-carboxyethyl)phosphine / TCEP SIGMA C4706-2G
Trizma base SIGMA T1503-250G
Materials for sample assembly
Biotin-PEG-SVA LAYSAN BIO BIO-PEG-SVA-5K-100MG & MPEG-SVA-5K-1g For PEGylation
Dibenzocyclooctyne-amine / DBCO-NH2 SIGMA 761540-10MG For bead coating
Double-sided tape 3M 136 For flow cell assembly
Epoxy glue DEVCON S-208 For flow cell assembly
Glass coverslip for bottom surface VWR 48393-251 Rectangular, 60×24 mm, #1.5
Glass coverslip for top surface VWR 48393-241 Rectangular, 50×24 mm, #1.5
Magnetic bead ThermoFisher Scientific 14301 Dynabeads M-270 Epoxy, 2.8 μm
mPEG-SVA LAYSAN BIO mPEG-SVA 1g For PEGylation
N,N-Dimethylformamide / DMF SIGMA D4551-250ML For bead coating
N-[3-(trimethoxysilyl)propyl]ethylenediamine SIGMA 104884-100ML For PEGylation
Neutravidin ThermoFisher Scientific 31000 For sample tethering
Phosphate buffered saline / PBS, pH 7.2 PLS PR2007-100-00
Plastic syringe Norm-ject A5 5 ml, luer tip
Polyethylene Tubing SCI BB31695-PE/4 PE-60
Reference bead SPHEROTECH SVP-30-5 Streptavidin-coated Polystyrene Particles; 3.0-3.4 µm
Syringe needle Kovax 21G-1 1/4'' 21 G
Syringe pump KD SCIENTIFIC 788210
Equipment for magnetic tweezer instrument
1-axis motorized microtranslation stage PI M-126.PD1 For vertical positioning of magnets
2-axis manual translation stage ST1 LEE400 For alignment of magnets to the optical axis
Acrylic holder for magnets DaiKwang Precision custum order Drawing available upon request
Frame grabber Active Silicon AS-FBD-4XCXP6-2PE8
High-speed CMOS camera Mikrotron EoSens 3CXP
Inverted microscope Olympus IX73P2F-1-2
Neodymium magnets LG magnet ND 10x10x12t Dimension: 10 mm × 10 mm × 12 mm; two needed
Objective lens Olympus UPLXAPO100XO Oil-immersion, NA 1.45
Objective lens nanopositioner Mad City Labs Nano-F100S
Rotation stepper motor AUTONICS A3K-S545W For rotating magnets
Superluminescent diode QPHOTONICS QSDM-680-2 680 nm
Software
LabVIEW National Instruments v20.0f1
MATLAB MathWorks v2021a

Referencias

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Park, C., Yang, T., Rah, S., Kim, H. G., Yoon, T., Shon, M. J. High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements. J. Vis. Exp. (195), e65137, doi:10.3791/65137 (2023).

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