Kuvvete duyarlı elemanlarda nanomekanik ölçümler için yüksek hızlı manyetik cımbız
1Department of Physics,Pohang University of Science and Technology (POSTECH), 2School of Biological Sciences,Seoul National University, 3Institute for Molecular Biology and Genetics,Seoul National University, 4School of Interdisciplinary Bioscience and Bioengineering,Pohang University of Science and Technology (POSTECH)
Summary
Burada, kuvvete duyarlı biyomoleküller üzerinde maksimum 1.2 kHz hızında nanomekanik ölçümler yapan yüksek hızlı bir manyetik cımbız kurulumunu açıklıyoruz. DNA saç tokalarına ve SNARE komplekslerine model sistemler olarak uygulanmasını tanıtıyoruz, ancak mekanobiyolojik olaylarda yer alan diğer moleküllere de uygulanabilecektir.
Abstract
Tek moleküllü manyetik cımbızlar (MT’ler), nükleik asitler ve proteinler gibi biyomolekülleri zorla sorgulamak için güçlü araçlar olarak hizmet etmiştir ve bu nedenle mekanobiyoloji alanında yararlı olmaya hazırdır. Yöntem genellikle manyetik boncukların görüntü tabanlı izlenmesine dayandığından, görüntülerin kaydedilmesi ve analiz edilmesindeki hız sınırının yanı sıra boncukların termal dalgalanmaları, hedef moleküllerdeki küçük ve hızlı yapısal değişiklikleri gözlemlemede uygulanmasını uzun zamandır engellemiştir. Bu makalede, biyomoleküllerin ve komplekslerinin nano ölçekli, milisaniyelik dinamiklerini çözebilen yüksek çözünürlüklü bir MT kurulumunun yapımı ve işletilmesi için ayrıntılı yöntemler açıklanmaktadır. Uygulama örnekleri olarak, DNA saç tokaları ve SNARE kompleksleri (membran-füzyon makineleri) ile yapılan deneyler, geçici durumlarının ve geçişlerinin pikonewton ölçekli kuvvetlerin varlığında nasıl tespit edilebileceğine odaklanarak gösterilmiştir. Yüksek hızlı MT’lerin, hücrelerdeki kuvvetleri algılayan, ileten ve üreten moleküller üzerinde yüksek hassasiyetli nanomekanik ölçümler yapmaya devam edeceğini ve böylece moleküler düzeyde mekanobiyoloji anlayışımızı derinleştireceğini umuyoruz.
Introduction
Hücreler mekanik uyaranları aktif olarak algılar ve bunlara yanıt verir. Bunu yaparken, birçok biyomolekül dinamik yapısal değişiklikleri mümkün kılan kuvvete bağımlı özellikler gösterir. İyi takdir edilen örnekler arasında, hücrelere çevrelerinden önemli mekanik bilgiler sağlayan mekanosensitivite iyon kanalları ve sitoiskelet elemanları bulunur.
Ek olarak, benzersiz bir kuvvet taşıyan nitelik gösteren moleküller, daha geniş anlamda mekanosensitif olarak da kabul edilebilir. Örneğin, nükleik asit dublekslerinin lokal oluşumu ve erimesi, ayrıca G-dörtlüleri gibi daha yüksek dereceli yapılar, replikasyon, transkripsiyon, rekombinasyon ve daha yakın zamanda genom düzenlemede çok önemli roller oynamaktadır. Dahası, sinaptik iletişimde yer alan bazı nöronal proteinler, tipik moleküller arası etkileşimlerin seviyelerini aşan fiziksel kuvvetler üreterek işlevlerini yerine getirirler. Hangi örnek üzerinde çalışılırsa çalışılsın, ilgili biyomoleküllerin nanomekaniğini yüksek uzaysal zamansal hassasiyetle araştırmak, ilişkili mekanobiyolojik süreçlerin moleküler mekanizmalarını ortaya çıkarmada oldukça yararlı olacaktır 1,2,3.
Tek moleküllü kuvvet spektroskopisi yöntemleri, biyomoleküllerin mekanik özelliklerini incelemek için güçlü araçlar olarak hizmet etmiştir 2,4,5,6. Nükleik asit ve proteinlerdeki yapısal değişiklikleri kuvvet uygulamasıyla eşzamanlı olarak izleyebilir, böylece kuvvete bağlı özellikleri inceleyebilirler. İyi bilinen iki kurulum, moleküllerimanipüle etmek için mikron boyutunda boncuklar kullanan optik cımbız ve manyetik cımbızdır (MT’ler), 5,6,7,8 moleküllerini manipüle eder. Bu platformlarda, polistiren (optik cımbız için) veya manyetik boncuklar (MT’ler için), tipik olarak çift sarmallı DNA’nın (dsDNA) kısa parçalarından yapılmış moleküler “tutamaklar” aracılığıyla hedef moleküllere (örneğin, nükleik asitler ve proteinler) bağlanır. Boncuklar daha sonra kuvvet uygulamak için hareket ettirilir ve hedef moleküllerdeki yapısal değişiklikleri bildiren konumlarını izlemek için görüntülenir. Optik ve manyetik cımbızlar uygulamalarında büyük ölçüde birbirinin yerine geçebilir, ancak kuvveti kontrol etme yaklaşımlarında önemli farklılıklar vardır. Optik cımbızlar, boncukları pozisyonda tutan doğal olarak pozisyon kelepçeli aletlerdir, çünkü bir hedef yapı şekil değişikliklerine uğradığında uygulanan kuvvet dalgalanır; Açılma gibi uzatma artışı, bağlantıyı gevşetir ve gerginliği azaltır ve bunun tersi de geçerlidir. Optik cımbızdaki kuvveti kontrol etmek için aktif geri bildirim uygulanabilse de, MT’ler aksine, doğal olarak çevresel bozulmaya da dayanabilen kalıcı mıknatısların kararlı, uzak alan manyetik kuvvetlerinden yararlanan bir kuvvet kelepçeleme cihazı olarak çalışır.
Uzun geçmişlerine ve basit tasarımlarına rağmen, MT’ler, büyük ölçüde hızlı boncuk izlemedeki teknik zorluklar nedeniyle, yüksek hassasiyetli ölçümlere uygulamalarında optik cımbızların gerisinde kalmıştır. Bununla birlikte, son zamanlarda, birkaç grup MT enstrümanları için hem donanım hem de yazılımın çok yönlü bir şekilde geliştirilmesine öncülük etmiştir 2,9,10,11,12,13,14,15,16,17,18,19 . Bu çalışmada, 1.2 kHz’de çalışan böyle bir kurulumun bir örneğini tanıtıyoruz ve kuvvete duyarlı biyomoleküller üzerinde nanomekanik ölçümler yapmak için nasıl kullanılacağını açıklıyoruz. Model sistemler olarak, DNA saç tokalarını ve nöronal SNARE komplekslerini kullanıyoruz ve pikonewton rejimindeki hızlı, yapısal değişikliklerini inceliyoruz. DNA saç tokaları, iyi tanımlanmış bir kuvvet aralığı20,21’de basit iki durumlu geçişler sergiler ve bu nedenle cımbız kurulumunun performansını doğrulamak için oyuncak modeller olarak hizmet eder. SNARE proteinleri, membran füzyonu22’yi yönlendiren kuvvete duyarlı bir kompleks halinde bir araya geldikçe, tek moleküllü kuvvet spektroskopisi 14,23,24,25 ile de kapsamlı bir şekilde incelenmiştir. Verileri analiz etmek ve termodinamik ve kinetik hakkında yararlı bilgiler çıkarmak için standart yaklaşımlar sunulmaktadır. Bu makalenin, mekanobiyolojik çalışmalarda yüksek hassasiyetli MT’lerin benimsenmesini kolaylaştırabileceğini ve okuyucuları kendi kuvvete duyarlı ilgi sistemlerini keşfetmeye motive edebileceğini umuyoruz.
Protocol
Bu protokolde açıklanan tüm malzeme ve ekipmanlar Malzeme Tablosunda listelenmiştir. Aşağıda açıklanan yüksek hızlı MT kurulumunu çalıştırmak için LabVIEW yazılımı ve örnek verileri analiz etmek için MATLAB betikleri GitHub’da (https://github.com/ShonLab/Magnetic-Tweezers) depolanır ve genel kullanıma sunulur. 1. Aparat yapımı NOT: Yüksek hızlı MT yapısının genel prensibi, yüksek hızlı tamamlayıcı …
Representative Results
Kuvvet kalibrasyonuİki kuvvet ölçüm yönteminden (boncukların yanal yer değiştirme varyansı ve güç spektrumu analizi) elde edilen sonuçlar 0-2 pN arasında farklılık göstermiştir (Şekil 2G). Şekil 2F’deki sonuçlara göre, normal neodimyum mıknatıslarla 30 pN’ye kadar güvenilir bir şekilde ulaşabiliriz. 8 bp DNA saç tokasının iki durumlu geçişleriİlk önce kısa bir…
Discussion
Bu çalışmada, biyomoleküllerin yapısal değişikliklerini yüksek uzaysal zamansal hassasiyette gözlemleyebilen tek moleküllü bir kuvvet spektroskopisi kurulumunu tanıttık. Kullanılan yüksek hızlı CMOS kamera, 1.280 x 1.024 çözünürlükte 1.200 kare s−1 elde ederek 1,2 kHz boncuk izleme sağlar. Bununla birlikte, ölçümlerin hızı şu anda boncuk izleme yazılımı ile sınırlıdır, bu nedenle yatırım getirisi genellikle yüksek hızlı ölçümlerde daha küçük alanlara indirgenir….
Disclosures
The authors have nothing to disclose.
Acknowledgements
Bu çalışma, Kore hükümeti (MSIT) tarafından finanse edilen Kore Ulusal Araştırma Vakfı (NRF) hibesi tarafından desteklenmiştir (NRF-2022R1C1C1012176, NRF-2021R1A4A1031754 ve NRF- 2021R1A6A1A10042944). S.-H.R. NMG hibesi (2021R1C1C2009717) tarafından desteklenmiştir.
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 |
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| 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 |
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| 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 |
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| 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 |
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Tags
Magnetic TweezersNanomechanical MeasurementsForce-sensitive ElementsBiomoleculesStructural ChangesNucleic AcidsProteinsMechanosensitive ProteinsCardiovascular DisordersMusculoskeletal DisordersHigh-speed CMOS CameraMotorized Linear StageRotary Stepper MotorSuper Luminescent DiodePiezo Lens ScannerCoverslip PreparationPegylation
Cite This Article
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).