multi-step-variable-height-photolithography-for-valved-multilayer

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Stanford University

The ability of kinesin to travel long distances on its microtubule track without dissociating has led to a variety of models to explain how this remarkable degree of processivity is maintained. All of these require that the two motor domains remain enzymatically "out of phase," a behavior that would ensure that, at any given time, one motor is strongly attached to the microtubule. The maintenance of this coordination over many mechanochemical cycles has never been explained, because key steps in the cycle could not be directly observed. We have addressed this issue by applying several novel spectroscopic approaches to monitor motor dissociation, phosphate release, and nucleotide binding during processive movement by a dimeric kinesin construct. Our data argue that the major effect of the internal strain generated when both motor domains of kinesin bind the microtubule is to block ATP from binding to the leading motor. This effect guarantees the two motor domains remain out of phase for many mechanochemical cycles and provides an efficient and adaptable mechanism for the maintenance of processive movement.

Stepping and stretching. How kinesin uses internal strain to walk processively.

&#x5149;&#x9631;&#x548C;&#x5355;&#x5206;&#x5B50;&#x8367;&#x5149;&#x5355;&#x5206;&#x5B50;&#x7814;&#x7A76;&#x4E2D;&#x7684;&#x4E3B;&#x6D41;&#x6280;&#x672F;&#x7684;&#x4E24;&#x4E2A;&#x3002;&#x4EE5;&#x524D;&#x66FE;&#x8BD5;&#x56FE;&#x7ED3;&#x5408;&#x8FD9;&#x4E9B;&#x6280;&#x672F;&#xFF0C;&#x5728;&#x5355;&#x4E00;&#x7684;&#x5B9E;&#x9A8C; &mdash; &mdash; &#x548C;&#x5229;&#x606F;-&#x5355;&#x4E2A;&#x9AD8;&#x5206;&#x5B50;&#x4F1A;&#x89C1;&#x4E86;&#x5C0F;&#x5C0F;&#x7684;&#x6210;&#x529F;&#xFF0C;&#x56E0;&#x4E3A;&#x5728;&#x5149;&#x5B66;&#x9677;&#x9631;&#x5185;&#x7684;&#x5149;&#x7EBF;&#x4EAE;&#x5EA6;&#x662F;&#x5341;&#x591A;&#x4E2A;&#x6570;&#x91CF;&#x7EA7;&#x5927;&#x4E8E;&#x5355;&#x4E2A;&#x8367;&#x5149;&#x53D1;&#x51FA;&#x7684;&#x706F;&#x5149;&#x3002;&#x76F8;&#x53CD;&#xFF0C;&#x8FD9;&#x4E24;&#x79CD;&#x6280;&#x672F;&#x91C7;&#x7528;&#x4E86;&#x6309;&#x987A;&#x5E8F;&#xFF0C;&#x6216;&#x7A7A;&#x95F4;&#x4E0A;&#x9694;&#x5F00;&#x7684;&#x793A;&#x4F8B;&#x4E2D;&#xFF0C;&#x65BD;&#x52A0;&#x9650;&#x5236;&#x7684;&#x5408;&#x5E76;&#x65B9;&#x6CD5;&#x7684;&#x5B9E;&#x7528;&#x7A0B;&#x5E8F;&#x7684;&#x5B9E;&#x9A8C;&#x9650;&#x5236;&#x5185;&#x7684;&#x51E0;&#x4E2A;&#x5FAE;&#x7C73;&#x7684;&#x8DDD;&#x79BB;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x62A5;&#x544A;&#x80FD;&#x591F;&#x771F;&#x5B9E;&#x3001; &#x540C;&#x65F6;&#x3001; &#x7A7A;&#x95F4;&#x91CD;&#x5408;&#x5149;&#x9631;&#x548C;&#x5355;&#x5206;&#x5B50;&#x8367;&#x5149;&#x4EEA;&#x7684;&#x7814;&#x5236;&#x3002;

&#x5149;&#x9631;&#x548C;&#x5355;&#x5206;&#x5B50;&#x8367;&#x5149;&#x7ED3;&#x5408;&#x8D77;&#x6765;&#x3002;

Two of the mainstay techniques in single-molecule research are optical trapping and single-molecule fluorescence. Previous attempts to combine these techniques in a single experiment - and on a single macromolecule of interest - have met with little success, because the light intensity within an optical trap is more than ten orders of magnitude greater than the light emitted by a single fluorophore. Instead, the two techniques have been employed sequentially, or spatially separated by distances of several micrometers within the sample, imposing experimental restrictions that limit the utility of the combined method. Here, we report the development of an instrument capable of true, simultaneous, spatially coincident optical trapping and single-molecule fluorescence.

Combined optical trapping and single-molecule fluorescence.

Deux des techniques des piliers dans la recherche de mol&eacute;cules simples sont le pi&eacute;geage optique et fluorescence de simple-mol&eacute;cule. Les tentatives pr&eacute;c&eacute;dentes de combiner ces techniques en une seule exp&eacute;rience - et sur une macromol&eacute;cule unique d&#39;int&eacute;r&ecirc;t - ont rencontr&eacute; peu de succ&egrave;s, parce que l&#39;intensit&eacute; lumineuse dans un pi&egrave;ge optique est plus de dix ordres de grandeur sup&eacute;rieurs &agrave; la lumi&egrave;re &eacute;mise par un fluorophore seul. Au lieu de cela, les deux techniques ont &eacute;t&eacute; employ&eacute;es dans l&#39;ordre, ou dans l&#39;espace, s&eacute;par&eacute;es par des distances de plusieurs microm&egrave;tres au sein de l&#39;&eacute;chantillon, en imposant des restrictions exp&eacute;rimentales qui limitent l&#39;utilit&eacute; de la m&eacute;thode combin&eacute;e. Les auteurs rapportent ici le d&eacute;veloppement d&#39;un instrument capable de vraie, simultan&eacute;e, spatialement co&iuml;ncidente de pi&eacute;geage optique et de fluorescence de simple-mol&eacute;cule.

Combin&eacute; pi&eacute;geage optique et fluorescence de simple-mol&eacute;cule.

Kombinierter optisch-Trapping und Einzelmolek&#xFC;l-Fluoreszenz.

Due delle tecniche cardine nella ricerca singola molecola sono cattura ottica e fluorescenza di singola molecola. Precedenti tentativi di combinare queste tecniche in un singolo esperimento - e su una singola macromolecola di interesse - si sono incontrati con poco successo, perch&eacute; l&#39;intensit&agrave; della luce all&#39;interno di una trappola ottica &egrave; pi&ugrave; di dieci ordini di grandezza maggiori della luce emessa da un singolo fluorophore. Invece, le due tecniche sono state impiegate in sequenza, o spazialmente separate da distanze di parecchi micrometri all&#39;interno del campione, che impone restrizioni sperimentali che limitano l&#39;utilit&agrave; del metodo combinato. Riportiamo qui, lo sviluppo di uno strumento capace di intrappolamento ottico vero, simultanea, spazialmente coincidente e fluorescenza di singola molecola.

Intrappolamento ottico combinato e fluorescenza di singola molecola.

&#x5358;&#x4E00;&#x5206;&#x5B50;&#x7814;&#x7A76;&#x306B;&#x304A;&#x3051;&#x308B;&#x4E3B;&#x529B;&#x6280;&#x8853;&#x306E;&#x5149;&#x30C8;&#x30E9;&#x30C3;&#x30D4;&#x30F3;&#x30B0;&#x3068;&#x5358;&#x4E00;&#x5206;&#x5B50;&#x86CD;&#x5149;&#x3067;&#x3059;&#x3002;&#x5149;&#x30C8;&#x30E9;&#x30C3;&#x30D7;&#x5185;&#x306E;&#x5149;&#x5F37;&#x5EA6;&#x5358;&#x4E00; fluorophore &#x306B;&#x3088;&#x3063;&#x3066;&#x653E;&#x51FA;&#x3055;&#x308C;&#x308B;&#x5149;&#x3088;&#x308A;&#x5927;&#x304D;&#x3044;&#x4EE5;&#x4E0A; 10 &#x6841;&#x3067;&#x3042;&#x308B;&#x305F;&#x3081;&#x5358;&#x4E00;&#x306E;&#x5B9F;&#x9A13;&#x3067;&#x306F; - &#x3068;&#x5358;&#x4E00;&#x9AD8;&#x5206;&#x5B50;&#x306E;&#x8208;&#x5473; - &#x3053;&#x308C;&#x3089;&#x306E;&#x6280;&#x8853;&#x3092;&#x7D50;&#x5408;&#x3059;&#x308B;&#x4EE5;&#x524D;&#x306E;&#x8A66;&#x307F;&#x306F;&#x5C11;&#x3057;&#x306E;&#x6210;&#x529F;&#x3068;&#x4F1A;&#x3044;&#x307E;&#x3057;&#x305F;&#x3002;&#x4EE3;&#x308F;&#x308A;&#x306B;&#x3001;&#x9806;&#x756A;&#x306B;&#x3001;&#x307E;&#x305F;&#x306F;&#x7A7A;&#x9593;&#x7684;&#x5408;&#x6210;&#x6CD5;&#x306E;&#x6709;&#x7528;&#x6027;&#x3092;&#x5236;&#x9650;&#x3059;&#x308B;&#x5B9F;&#x9A13;&#x306E;&#x5236;&#x9650;&#x3092;&#x8AB2;&#x3059;&#x3001;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x5185;&#x306E;&#x6570; &mu; m &#x306E;&#x8DDD;&#x96E2;&#x3067;&#x533A;&#x5207;&#x3089;&#x308C;&#x305F; 2 &#x3064;&#x306E;&#x30C6;&#x30AF;&#x30CB;&#x30C3;&#x30AF;&#x3092;&#x63A1;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;true&#x3001;&#x540C;&#x6642;&#x3001;&#x7A7A;&#x9593;&#x7684;&#x306B;&#x4E00;&#x81F4;&#x306E;&#x5149;&#x30C8;&#x30E9;&#x30C3;&#x30D4;&#x30F3;&#x30B0;&#x5358;&#x4E00;&#x5206;&#x5B50;&#x86CD;&#x5149;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x88C5;&#x7F6E;&#x306E;&#x958B;&#x767A;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;

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Dos de las t&eacute;cnicas de base en la investigaci&oacute;n de una sola mol&eacute;cula son interceptaci&oacute;n &oacute;ptica y fluorescencia de una sola mol&eacute;cula. Intentos previos para combinar estas t&eacute;cnicas en un experimento &uacute;nico - y en una sola macromol&eacute;cula de inter&eacute;s - han tenido poco &eacute;xito, debido a la intensidad de la luz dentro de una trampa &oacute;ptica es m&aacute;s de diez &oacute;rdenes de magnitud mayores que la luz emitida por un fluor&oacute;foro solo. En cambio, las dos t&eacute;cnicas han sido empleadas secuencialmente, o espacialmente separadas por distancias de varios micr&oacute;metros dentro de la muestra, imponiendo restricciones experimentales que limitan la utilidad del m&eacute;todo combinado. Aqu&iacute;, se presenta el desarrollo de un instrumento capaz de interceptaci&oacute;n &oacute;ptica verdadera, simult&aacute;nea, espacialmente coincidente y fluorescencia de una sola mol&eacute;cula.

Interceptaci&oacute;n &oacute;ptica combinada y fluorescencia de una sola mol&eacute;cula.

Construimos un instrumento basado en microscopio capaz de interceptaci&oacute;n &oacute;ptica simult&aacute;nea, espacialmente coincidente y fluorescencia de una sola mol&eacute;cula. Las capacidades de este aparato fueron demostrados por estudiar la separaci&oacute;n de filamento inducida por fuerza de un marcado tinte, 15-base-par regi&oacute;n de ADN de doble hebra (dsDNA), con la fuerza aplicada perpendicular (modo &#39;corte&#39;) o URL (modo &#39;descomprimir&#39;) al eje largo de la regi&oacute;n. Transiciones mec&aacute;nicas correspondiente a la ruptura de ADN h&iacute;brido ocurrieron simult&aacute;neamente con cambios discontinuos en la emisi&oacute;n de fluorescencia. La fuerza de la ruptura fue fuertemente dependiente de la direcci&oacute;n de la fuerza aplicada, indicando la existencia de distintas v&iacute;as de desvinculaci&oacute;n para los dos modos de carga de fuerza. De los histogramas de fuerza de ruptura, determinamos la distancia hasta el estado termodin&aacute;mico de la transici&oacute;n y la t&eacute;rmica sobre precios en ausencia de carga para ambos procesos.

Interceptaci&oacute;n &oacute;ptica simult&aacute;nea, coincidente y fluorescencia de una sola mol&eacute;cula.

&#x6211;&#x4EEC;&#x5EFA;&#x9020;&#x80FD;&#x529B;&#x7684;&#x540C;&#x65F6;&#xFF0C;&#x7A7A;&#x95F4;&#x91CD;&#x5408;&#x5149;&#x9631;&#x548C;&#x5355;&#x5206;&#x5B50;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x57FA;&#x7840;&#x6587;&#x4E66;&#x3002;&#x8FD9;&#x79CD;&#x4EEA;&#x5668;&#x7684;&#x529F;&#x80FD;&#x662F;&#x901A;&#x8FC7;&#x7814;&#x7A76;&#x8BF1;&#x5BFC;&#x529B;&#x94A2;&#x7EDE;&#x7EBF;&#x5206;&#x79BB;&#x7684;&#x67D3;&#x6599;&#x6807;&#x8BB0;&#xFF0C;&#x8868;&#x660E; 15 &#x78B1;&#x57FA;&#x5BF9;&#x7684;&#x53CC;&#x94FE; DNA (&#x4F5C;&#x7528;)&#xFF0C;&#x4E0E;&#x90E8;&#x961F;&#x533A;&#x57DF;&#x5E76;&#x884C; &#xFF08;&#x89E3;&#x538B;&#x7F29;&#x6A21;&#x5F0F;&#xFF09; &#x6216;&#x5782;&#x76F4; &#xFF08;&#39;&#x526A;&#39; &#x6A21;&#x5F0F;&#xFF09; &#x5E94;&#x7528;&#x5230;&#x8BE5;&#x533A;&#x57DF;&#x7684;&#x957F;&#x8F74;&#x3002;&#x5BF9;&#x5E94;&#x4E8E; DNA &#x6742;&#x4EA4;&#x65AD;&#x88C2;&#x529B;&#x5B66;&#x8FC7;&#x6E21;&#x7684;&#x8367;&#x5149;&#x53D1;&#x5C04;&#x4E0D;&#x8FDE;&#x7EED;&#x53D8;&#x5316;&#x540C;&#x65F6;&#x53D1;&#x751F;&#x3002;&#x7834;&#x88C2;&#x529B;&#x662F;&#x4F5C;&#x7528;&#x529B;&#x7684;&#x5F3A;&#x70C8;&#x4F9D;&#x8D56;&#x4E8E;&#x65B9;&#x5411;&#xFF0C;&#x8868;&#x660E;&#x5B58;&#x5728;&#x8FD9;&#x4E24;&#x79CD;&#x529B;&#x6A21;&#x5F0F;&#x7684;&#x4E0D;&#x540C;&#x89E3;&#x9664;&#x7ED1;&#x5B9A;&#x7684;&#x9014;&#x5F84;&#x3002;&#x4ECE;&#x7834;&#x88C2;&#x529B;&#x76F4;&#x65B9;&#x56FE;&#xFF0C;&#x6211;&#x4EEC;&#x51B3;&#x5FC3;&#x5230;&#x70ED;&#x529B;&#x5B66;&#x8FC7;&#x6E21;&#x72B6;&#x6001;&#x548C;&#x5173;&#x95ED;&#x5728;&#x6CA1;&#x6709;&#x8FD9;&#x4E24;&#x4E2A;&#x8FC7;&#x7A0B;&#x7684;&#x8D1F;&#x8F7D;&#x7387;&#x70ED;&#x4E4B;&#x95F4;&#x7684;&#x8DDD;&#x79BB;&#x3002;

&#x540C;&#x65F6;&#xFF0C;&#x91CD;&#x5408;&#x5149;&#x9631;&#x548C;&#x5355;&#x5206;&#x5B50;&#x8367;&#x5149;&#x3002;

We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.

Simultaneous, coincident optical trapping and single-molecule fluorescence.

Nous avons construit un instrument microscope capable de pi&eacute;geage optique simultan&eacute;e, spatialement co&iuml;ncidente et fluorescence des mol&eacute;cules simples. Les capacit&eacute;s de cet appareil ont &eacute;t&eacute; d&eacute;montr&eacute;s par l&#39;&eacute;tude de la s&eacute;paration de brin induite par la force d&#39;un colorant-&eacute;tiquet&eacute;, r&eacute;gion 15-paires de bases d&#39;ADN double brin (dsDNA), avec une force appliqu&eacute;e parall&egrave;lement (mode de &laquo; d&eacute;compression &raquo;) ou perpendiculaire (mode &laquo; cisaillement &raquo;) &agrave; l&#39;axe longitudinal de la r&eacute;gion. Transitions m&eacute;caniques correspondant &agrave; la rupture de l&#39;ADN hybride se produisent simultan&eacute;ment avec les changements discontinus dans l&#39;&eacute;mission de fluorescence. La force de rupture &eacute;tait fortement d&eacute;pendante de la direction de la force appliqu&eacute;e, indiquant l&#39;existence de voies sans engagement distinctes pour les deux modes de chargement par force. Les histogrammes de force de rupture, nous avons d&eacute;termin&eacute; la distance &agrave; l&#39;&eacute;tat de transition thermodynamique et thermique sur le tarif en l&#39;absence de charge pour les deux proc&eacute;d&eacute;s.

Simultan&eacute;e, co&iuml;ncidant de pi&eacute;geage optique et fluorescence des mol&eacute;cules simples.

Gleichzeitige, zusammenfallend optische Fallen und Einzelmolek&#xFC;l-Fluoreszenz.

Abbiamo costruito un microscopio-based strumento capace di intrappolamento ottico simultanea, spazialmente coincidente e fluorescenza di singola molecola. Le funzionalit&agrave; di questo apparecchio sono state dimostrate da studiare la separazione strand forza-indotta di un colorante-etichettati, regione 15-coppia di basi di DNA double-stranded (dsDNA), con la forza applicata o parallelo (modalit&agrave; &#39;decompressione&#39;) o perpendicolare (modalit&agrave; &#39;taglio&#39;) all&#39;asse lungo della regione. Transizioni meccaniche corrispondente alla rottura ibrido DNA si sono verificati contemporaneamente con cambiamenti discontinui dell&#39;emissione di fluorescenza. La forza di rottura &egrave; stato fortemente dipendente sulla direzione della forza applicata, che indica l&#39;esistenza di distinti percorsi non vincolante per le due modalit&agrave; di caricamento a forza. Dagli istogrammi di forza di rottura, abbiamo determinato la distanza dello stato di transizione termodinamico e termico off tariffe in assenza di carico per entrambi i processi.

Intrappolamento ottico simultanea, coincidente e fluorescenza di singola molecola.

&#x6211;&#x3005; &#x306F;&#x3001;&#x540C;&#x6642;&#x3001;&#x7A7A;&#x9593;&#x7684;&#x306B;&#x4E00;&#x81F4;&#x306E;&#x5149;&#x30C8;&#x30E9;&#x30C3;&#x30D4;&#x30F3;&#x30B0;&#x5358;&#x4E00;&#x5206;&#x5B50;&#x86CD;&#x5149;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x9855;&#x5FAE;&#x93E1;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x697D;&#x5668;&#x3092;&#x69CB;&#x7BC9;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x88C5;&#x7F6E;&#x306E;&#x6A5F;&#x80FD;&#x3055;&#x308C;&#x305F;&#x5B9F;&#x8A3C;&#x8272;&#x7D20;&#x6A19;&#x8B58;&#x306E;&#x529B;&#x8A98;&#x8D77;&#x30B9;&#x30C8;&#x30E9;&#x30F3;&#x30C9;&#x5206;&#x96E2;&#x52C9;&#x5F37;&#x3057;&#x3066; 15 &#x5869;&#x57FA;&#x5BFE;&#x5730;&#x57DF;&#x529B;&#x3068;&#x4E8C;&#x672C;&#x9396; DNA (dsDNA) &#x306E;&#x4E26;&#x5217; (&#39;&#x89E3;&#x51CD;&#39; &#x30E2;&#x30FC;&#x30C9;) &#x307E;&#x305F;&#x306F;&#x5782;&#x76F4; (&#39;&#x305B;&#x3093;&#x65AD;&#39; &#x30E2;&#x30FC;&#x30C9;) &#x306E;&#x3044;&#x305A;&#x308C;&#x304B;&#x9069;&#x7528;&#x5730;&#x57DF;&#x306E;&#x9577;&#x3044;&#x8EF8;&#x306B;&#x3002;DNA &#x30CF;&#x30A4;&#x30D6;&#x30EA;&#x30C3;&#x30C9;&#x7834;&#x88C2;&#x306B;&#x5BFE;&#x5FDC;&#x3059;&#x308B;&#x30E1;&#x30AB;&#x30CB;&#x30AB;&#x30EB;&#x9077;&#x79FB;&#x540C;&#x6642;&#x86CD;&#x5149;&#x767A;&#x5149;&#x306E;&#x4E0D;&#x9023;&#x7D9A;&#x5909;&#x5316;&#x304C;&#x767A;&#x751F;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7834;&#x65AD;&#x529B;&#x5F37;&#x304F;&#x5F37;&#x5236;&#x8AAD;&#x307F;&#x8FBC;&#x307F;&#x30E2;&#x30FC;&#x30C9;&#x306E; 2 &#x3064;&#x306E;&#x7570;&#x306A;&#x308B;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x89E3;&#x9664;&#x7D4C;&#x8DEF;&#x306E;&#x5B58;&#x5728;&#x3092;&#x793A;&#x3059;&#x3001;&#x5FDC;&#x7528;&#x306E;&#x529B;&#x306E;&#x65B9;&#x5411;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x305F;&#x3002;&#x7834;&#x65AD;&#x529B;&#x30D2;&#x30B9;&#x30C8;&#x30B0;&#x30E9;&#x30E0;&#x304B;&#x3089;&#x3001;&#x71B1;&#x529B;&#x5B66;&#x7684;&#x9077;&#x79FB;&#x72B6;&#x614B;&#x3068;&#x71B1;&#x8CA0;&#x8377;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x305F;&#x3081;&#x306E;&#x4E0D;&#x5728;&#x3067;&#x6599;&#x91D1;&#x30AA;&#x30D5;&#x307E;&#x3067;&#x306E;&#x8DDD;&#x96E2;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3059;&#x3002;

&#x540C;&#x6642;, &#x30B3;&#x30A4;&#x30F3;&#x30B7;&#x30C7;&#x30F3;&#x30B9;&#x5149;&#x30C8;&#x30E9;&#x30C3;&#x30D4;&#x30F3;&#x30B0;&#x3068;&#x5358;&#x4E00;&#x5206;&#x5B50;&#x86CD;&#x5149;&#x3002;

&#xC6B0;&#xB9AC;&#xB294; &#xB3D9;&#xC2DC;, &#xACF5;&#xAC04;&#xC801; &#xC77C;&#xCE58; &#xAD11;&#xD559; &#xD2B8;&#xB798;&#xD551; &#xD558; &#xACE0; &#xB2E8;&#xC77C; &#xBD84;&#xC790; &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; &#xAE30;&#xBC18; &#xC545;&#xAE30;&#xB97C; &#xAC74;&#xC124; &#xD55C;&#xB2E4;. &#xC774; &#xAE30;&#xAD6C;&#xC758; &#xAE30;&#xB2A5; &#xBB34;&#xB825;&#xC73C;&#xB85C; &#xC774;&#xC911; &#xAC00;&#xB2E5; DNA (dsDNA)&#xC758; 15-&#xAE30;&#xC9C0;-&#xC30D; &#xC9C0;&#xC5ED; &#xC9C0;&#xC5ED;&#xC758; &#xAE34; &#xCD95;&#xC5D0; &#xBCD1;&#xB82C; (&#39;&#xC9C0;&#xD37C;&#39; &#xBAA8;&#xB4DC;) &#xB610;&#xB294; &#xC218;&#xC9C1; (&#39;&#xC804;&#xB2E8;&#39; &#xBAA8;&#xB4DC;) &#xC801;&#xC6A9; &#xC5FC;&#xB8CC; &#xBD84;&#xB958;&#xC758; &#xD798;&#xC744; &#xC774;&#xC6A9;&#xD55C; &#xAC00;&#xB2E5; &#xBD84;&#xB9AC;&#xB97C; &#xACF5;&#xBD80; &#xD558; &#xC5EC; &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;. &#xD574;&#xB2F9; DNA &#xD558;&#xC774;&#xBE0C;&#xB9AC;&#xB4DC; &#xD30C;&#xC5F4; &#xD558;&#xB294; &#xAE30;&#xACC4;&#xC801; &#xC804;&#xD658; &#xB3D9;&#xC2DC;&#xC5D0; &#xD615;&#xAD11; &#xBC29;&#xCD9C;&#xC758; &#xBD88;&#xC5F0;&#xC18D;&#xC801;&#xC778; &#xBCC0;&#xD654; &#xBC1C;&#xC0DD;. &#xD30C;&#xC5F4; &#xD798; &#xAC15;&#xB825; &#xD558; &#xAC8C; &#xB450; &#xD798; &#xB85C;&#xB529; &#xBAA8;&#xB4DC;&#xC5D0; &#xB300; &#xD55C; &#xACE0;&#xC720;&#xD55C; &#xBC14;&#xC778;&#xB529; &#xD574;&#xC81C; &#xD1B5;&#xB85C;&#xC758; &#xC874;&#xC7AC;&#xB97C; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xC801;&#xC6A9; &#xB41C; &#xD798;&#xC758; &#xBC29;&#xD5A5;&#xC5D0; &#xC758;&#xC874; &#xD588;&#xB2E4;. &#xD30C;&#xC5F4; &#xD798; &#xD788;&#xC2A4;&#xD1A0;&#xADF8;&#xB7A8;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC; &#xC5F4;&#xC5ED;&#xD559;&#xC801; &#xC804;&#xD658; &#xC0C1;&#xD0DC;&#xC640; &#xC5F4; &#xB450; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xB300; &#xD55C; &#xBD80;&#xD558;&#xC758; &#xBD80;&#xC7AC;&#xC5D0;&#xC11C; &#xC694;&#xAE08;&#xC744; &#xAC70;&#xB9AC;&#xB97C; &#xACB0;&#xC815; &#xD569;&#xB2C8;&#xB2E4;.

&#xB3D9;&#xC2DC;, &#xC77C;&#xCE58; &#xAD11;&#xD559; &#xD2B8;&#xB798;&#xD551; &#xBC0F; &#xB2E8;&#xC77C; &#xBD84;&#xC790; &#xD615;&#xAD11;.

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.

Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro.

Understanding how molecular motors generate force and move microtubules in mitosis is essential to understanding the physical mechanism of cell division. Recent measurements have shown that one mitotic kinesin superfamily member, Eg5, is mechanically processive and capable of crosslinking and sliding microtubules in vitro. In this review, we highlight recent work that explores how Eg5 functions under load, with an emphasis on the nanomechanical properties of single enzymes.

Eg5 steps it up!

&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x662F;&#x90E8;&#x5206;&#x53D7;&#x7ED1;&#x5B9A;&#x76EE;&#x6807;&#x8C03;&#x63A7; DNA &#x5E8F;&#x5217;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x3002;&#x9884;&#x6D4B; DNA &#x7ED3;&#x5408;&#x4F4D;&#x70B9;&#x548C;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x5E8F;&#x5217;&#x6216;&#x7ED3;&#x6784;&#x4ECE;&#x4EB2;&#x7F18;&#x5173;&#x7CFB;&#x662F;&#x5F88;&#x96BE; &#xFF1B;&#x56E0;&#x6B64;&#xFF0C;&#x5B9E;&#x9A8C;&#x6570;&#x636E;&#x88AB;&#x9700;&#x8981;&#x94FE;&#x63A5;&#x5230;&#x76EE;&#x6807;&#x5E8F;&#x5217;&#x7684;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x3002;&#x6211;&#x4EEC;&#x63D0;&#x51FA;&#x4E00;&#x79CD;&#x57FA;&#x4E8E;&#x5FAE;&#x6D41;&#x4F53; de novo &#x53D1;&#x73B0;&#x548C;&#x5B9A;&#x91CF;&#x751F;&#x7269;&#x7269;&#x7406;&#x8868;&#x5F81; DNA &#x76EE;&#x6807;&#x5E8F;&#x5217;&#x7684;&#x65B9;&#x6CD5;&#x3002;&#x6211;&#x4EEC;&#x901A;&#x8FC7;&#x6D4B;&#x91CF;&#x5E8F;&#x5217; 28 &#x917F;&#x9152;&#x9175;&#x6BCD;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x4E0E; DNA &#x7ED3;&#x5408;&#x57DF;&#xFF0C;&#x5305;&#x62EC;&#x51E0;&#x4E2A;&#x4E8B;&#x5B9E;&#x8BC1;&#x660E;&#x96BE;&#x4EE5;&#x901A;&#x8FC7;&#x5176;&#x4ED6;&#x6280;&#x672F;&#x7814;&#x7A76;&#x5404;&#x79CD;&#x9996;&#x9009;&#x9879;&#x9A8C;&#x8BC1;&#x6211;&#x4EEC;&#x7684;&#x6280;&#x672F;&#x3002;&#x4E3A;&#x6BCF;&#x4E2A;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#xFF0C;&#x6211;&#x4EEC;&#x6D4B;&#x91CF;&#x5230;&#x6DB5;&#x76D6;&#x6240;&#x6709;&#x53EF;&#x80FD;&#x7684; 8 bp DNA &#x5E8F;&#x5217;&#xFF0C;&#x4EE5;&#x521B;&#x5EFA;&#x5E8F;&#x5217;&#x7684;&#x9996;&#x9009;&#x9879; &#xFF1B; &#x7EFC;&#x5408;&#x5730;&#x56FE;&#x7684;&#x5BE1;&#x6838;&#x82F7;&#x9178;&#x76F8;&#x5BF9;&#x4EB2;&#x548C;&#x529B;&#x56DB;&#x4E2A;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#xFF0C;&#x6211;&#x4EEC;&#x4E5F;&#x575A;&#x51B3;&#x7EDD;&#x5BF9;&#x7684;&#x4EB2;&#x7F18;&#x5173;&#x7CFB;&#x3002;&#x6211;&#x4EEC;&#x671F;&#x671B;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x548C;&#x5C06;&#x6765;&#x4F7F;&#x7528;&#x8FD9;&#x79CD;&#x6280;&#x672F;&#x5C06;&#x63D0;&#x4F9B;&#x4E86;&#x89E3;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x7279;&#x5F02;&#x6027;&#x3001; &#x6539;&#x5584;&#x8C03;&#x63A7;&#x4F4D;&#x70B9;&#x9274;&#x5B9A;&#x548C;&#x91CD;&#x6784;&#x76D1;&#x7BA1;&#x4E92;&#x52A8;&#x7684;&#x91CD;&#x8981;&#x8D44;&#x6599;&#x3002;

&#x5FB7;&#x8BFA;&#x7684;&#x9274;&#x5B9A;&#x53CA;&#x751F;&#x7269;&#x7269;&#x7406;&#x7279;&#x5F81;&#x4E0E;&#x5FAE;&#x6D41;&#x63A7;&#x4EB2;&#x548C;&#x5206;&#x6790;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x7ED3;&#x5408;&#x4F4D;&#x70B9;&#x3002;

Gene expression is regulated in part by protein transcription factors that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link transcription factors to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 Saccharomyces cerevisiae transcription factors with a variety of DNA-binding domains, including several that have proven difficult to study by other techniques. For each transcription factor, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for four transcription factors, we also determined absolute affinities. We expect that these data and future use of this technique will provide information essential for understanding transcription factor specificity, improving identification of regulatory sites and reconstructing regulatory interactions.

De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis.

L&#39;expression des g&egrave;nes est r&eacute;gul&eacute;e en partie par des facteurs de transcription des prot&eacute;ines qui lient les s&eacute;quences r&eacute;gulatrices de l&#39;ADN cible. Pr&eacute;dire les sites de fixation de l&#39;ADN et les affinit&eacute;s de la s&eacute;quence de facteur de transcription ou une structure est difficile ; par cons&eacute;quent, les donn&eacute;es exp&eacute;rimentales sont tenues de lier des facteurs de transcription &agrave; s&eacute;quences cibles. Nous pr&eacute;sentons une approche ax&eacute;e sur la microfluidique de novo d&eacute;couverte et caract&eacute;risation biophysique quantitative des s&eacute;quences d&#39;ADN cible. Nous avons valid&eacute; notre technique en mesurant les pr&eacute;f&eacute;rences de s&eacute;quence pour les facteurs de transcription de 28 Saccharomyces cerevisiae avec une vari&eacute;t&eacute; de domaines de liaison &agrave; l&#39;ADN, y compris plusieurs qui se sont av&eacute;r&eacute;es difficiles &agrave; &eacute;tudier par d&#39;autres techniques. Pour chaque facteur de transcription, nous avons mesur&eacute; les affinit&eacute;s de liaison relative aux oligonucl&eacute;otides couvrant toutes les s&eacute;quences d&#39;ADN 8-bp possibles pour cr&eacute;er une carte exhaustive des pr&eacute;f&eacute;rences de la s&eacute;quence ; pour les quatre facteurs de transcription, nous avons &eacute;galement d&eacute;termin&eacute; les affinit&eacute;s absolues. Nous esp&eacute;rons que ces donn&eacute;es et l&#39;utilisation future de cette technique fournira des informations essentielles pour comprendre la sp&eacute;cificit&eacute; de facteur de transcription, am&eacute;liorant l&#39;identification des sites r&eacute;glementaires et reconstruire les interactions r&eacute;glementaires.

De novo identification et caract&eacute;risation biophysique des sites de liaison du facteur de transcription analyse des affinit&eacute;s microfluidique.

Genexpression ist teilweise durch Protein-Transkriptionsfaktoren geregelt, die eine regulatorische DNA-Sequenzen zu binden. Vorhersage DNA-Bindungsstellen und Affinit&auml;ten von Transkription Faktor Reihenfolge oder Struktur ist schwierig; Daher sind experimentelle Daten ben&ouml;tigt, um Transkriptionsfaktoren mit Ziel-Sequenzen zu verkn&uuml;pfen. Wir pr&auml;sentieren einen Mikrofluidik-Ansatz f&uuml;r de-Novo-Entdeckung und quantitative biophysikalische Charakterisierung der Ziel-DNA-Sequenzen. Wir haben unsere Technik durch Mess-Sequenz Einstellungen f&uuml;r 28 Saccharomyces Cerevisiae Transkriptionsfaktoren mit einer Vielzahl von DNA-bindenden Dom&auml;nen, darunter mehrere, die schwer durch andere Techniken zu studieren erwiesen haben &uuml;berpr&uuml;ft. F&uuml;r jede Transkriptionsfaktor gemessen wir relative verbindliche Affinit&auml;t zu Oligonukleotide, die f&uuml;r alle m&ouml;gliche 8-bp DNA-Sequenzen um eine umfassende Karte der Sequenz Einstellungen erstellen; f&uuml;r vier Transkriptionsfaktoren bestimmen wir auch absolute Affinit&auml;ten. Wir erwarten, dass diese Daten und zur k&uuml;nftigen Verwendung dieser Technik wesentlich zum Verst&auml;ndnis Transkription Faktor Spezifit&auml;t, Ermittlung der regulatorischen Websites zu verbessern und rekonstruieren von regulatorische Interaktionen informieren werden.

De-Novo-Identifikation und biophysikalische Charakterisierung von Transkriptionsfaktor Bindungsstellen mit mikrofluidische Affinit&auml;t Analyse.

Espressione genica &egrave; regolata in parte da fattori di trascrizione di proteine che legano sequenze regolatrici di DNA bersaglio. Predire i siti di legame del DNA e affinit&agrave; dalla sequenza di fattore di trascrizione o struttura &egrave; difficile; Pertanto, dati sperimentali sono tenuti a fattori di trascrizione di collegamento alle sequenze target. Vi presentiamo un approccio basato su microfluidics per de novo scoperta e la caratterizzazione biofisica quantitativa delle sequenze di DNA bersaglio. Abbiamo convalidato la nostra tecnica di preferenze di sequenza per fattori di trascrizione 28 Saccharomyces cerevisiae con una variet&agrave; di domini di DNA-binding, tra cui diversi che si sono rivelate difficili da studiare da altre tecniche di misurazione. Per ogni fattore di trascrizione, abbiamo misurato l&#39;affinit&agrave; di legame relativo di oligonucleotidi che coprono tutte le possibili sequenze di DNA 8-bp per creare una mappa completa delle preferenze di sequenza; per i quattro fattori di trascrizione, abbiamo determinato anche assoluta affinit&agrave;. Ci aspettiamo che questi dati e futuro impiego di questa tecnica fornir&agrave; informazioni essenziali per comprendere la specificit&agrave; di fattore di trascrizione, migliorando l&#39;identificazione dei siti di regolamentazione e ricostruire le interazioni normative.

De novo identificazione e caratterizzazione biofisica di siti di legame del fattore di trascrizione con analisi di affinit&agrave; microfluidica.

&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306F;&#x4E00;&#x90E8;&#x898F;&#x5236;&#x306E; DNA &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x30D0;&#x30A4;&#x30F3;&#x30C9; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x306B;&#x3088;&#x3063;&#x3066;&#x898F;&#x5236;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;DNA &#x7D50;&#x5408;&#x90E8;&#x4F4D;&#x3068;&#x89AA;&#x548C;&#x6027;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x307E;&#x305F;&#x306F;&#x69CB;&#x9020;&#x304B;&#x3089;&#x306E;&#x4E88;&#x6E2C;&#x306F;&#x56F0;&#x96E3;&#x3067;&#x3042;&#x308B;;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x5B9F;&#x9A13;&#x30C7;&#x30FC;&#x30BF;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30B7;&#x30E7;&#x30F3;&#x8981;&#x56E0;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;De novo &#x306E;&#x767A;&#x898B;&#x3068;&#x5B9A;&#x91CF;&#x7684;&#x5E74;&#x4F1A;&#x30AD;&#x30E3;&#x30E9;&#x30AF;&#x30BF;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3; DNA &#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306E;&#x305F;&#x3081;&#x306E;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x63D0;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x597D;&#x307F;&#x306B;&#x3088;&#x3063;&#x3066;&#x4ED6;&#x306E;&#x6280;&#x8853;&#x3092;&#x52C9;&#x5F37;&#x3059;&#x308B;&#x306F;&#x96E3;&#x3057;&#x3044;&#x3068;&#x5224;&#x660E;&#x3057;&#x305F;&#x3053;&#x3068;&#x3092;&#x3044;&#x304F;&#x3064;&#x304B;&#x542B;&#x3080; DNA &#x7D50;&#x5408;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x69D8;&#x3005; &#x306A; 28 &#x30B5;&#x30C3;&#x30AB;&#x30ED; &#x30DF;&#x30BB;&#x30B9;&#x9175;&#x6BCD;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x3092;&#x6E2C;&#x5B9A;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x79C1;&#x305F;&#x3061;&#x306E;&#x30C6;&#x30AF;&#x30CB;&#x30C3;&#x30AF;&#x3092;&#x691C;&#x8A3C;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x5404;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x306E;&#x30AA;&#x30EA;&#x30B4;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x8A2D;&#x5B9A;&#x306E;&#x5305;&#x62EC;&#x7684;&#x306A;&#x30DE;&#x30C3;&#x30D7;&#x3092;&#x4F5C;&#x6210;&#x3059;&#x308B;&#x3059;&#x3079;&#x3066;&#x306E;&#x53EF;&#x80FD;&#x306A; 8 bp DNA &#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x30AB;&#x30D0;&#x30FC;&#x3059;&#x308B;&#x76F8;&#x5BFE;&#x7D50;&#x5408;&#x89AA;&#x548C;&#x6027;&#x3092;&#x6E2C;&#x5B9A;&#x3057;&#x305F;;4 &#x3064;&#x306E;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x306E;&#x6211;&#x3005; &#x3082;&#x7D76;&#x5BFE;&#x7684;&#x306A;&#x89AA;&#x548C;&#x6027;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x3068;&#x5C06;&#x6765;&#x306E;&#x4F7F;&#x7528;&#x306E;&#x3053;&#x306E;&#x30C6;&#x30AF;&#x30CB;&#x30C3;&#x30AF;&#x306E;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x306E;&#x7279;&#x7570;&#x6027;&#x3092;&#x7406;&#x89E3;&#x3001;&#x8ABF;&#x7BC0;&#x90E8;&#x4F4D;&#x306E;&#x540C;&#x5B9A;&#x306E;&#x6539;&#x5584;&#x3001;&#x898F;&#x5236;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x518D;&#x69CB;&#x7BC9;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x60C5;&#x5831;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x671F;&#x5F85;&#x3057;&#x307E;&#x3059;&#x3002;

De novo &#x540C;&#x5B9A;&#x304A;&#x3088;&#x3073;&#x5E74;&#x4F1A;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x306E;&#x89AA;&#x548C;&#x6027;&#x306E;&#x5206;&#x6790;&#x3068;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x7D50;&#x5408;&#x90E8;&#x4F4D;&#x3002;

&#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC740; &#xBC14;&#xC778;&#xB529; &#xB300;&#xC0C1; &#xADDC;&#xC81C; DNA &#xC2DC;&#xD000;&#xC2A4; &#xB2E8;&#xBC31;&#xC9C8; &#xC804;&#xC0AC; &#xC778;&#xC790;&#xC5D0; &#xC758;&#xD574; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xD1B5;&#xC81C; &#xB41C;&#xB2E4;. DNA &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8; &#xBC0F; &#xC804;&#xC0AC; &#xC694;&#xC18C; &#xC2DC;&#xD000;&#xC2A4; &#xB610;&#xB294; &#xAD6C;&#xC870;&#xC5D0;&#xC11C; &#xB3D9;&#xC9C8;&#xC131;&#xC744; &#xC608;&#xCE21; &#xD558;&#xB294; &#xAC83;&#xC740; &#xC5B4;&#xB835;&#xB2E4;; &#xB530;&#xB77C;&#xC11C;, &#xC2E4;&#xD5D8; &#xB370;&#xC774;&#xD130; &#xB300;&#xC0C1; &#xC2DC;&#xD000;&#xC2A4;&#xC5D0; &#xC804;&#xC0AC; &#xC778;&#xC790;&#xB97C; &#xC5F0;&#xACB0; &#xD574;&#xC57C; &#xD569;&#xB2C8;&#xB2E4;. &#xB4DC; &#xB178; &#xBCF4; &#xAC80;&#xC0C9; &#xBC0F; DNA &#xB300;&#xC0C1; &#xC2DC;&#xD000;&#xC2A4;&#xC758; &#xC591;&#xC801; &#xC0DD;&#xBB3C; &#xB9AC; &#xD2B9;&#xC131;&#xD654; microfluidics &#xAE30;&#xBC18; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC744; &#xC18C;&#xAC1C;&#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC6B0;&#xB9AC;&#xC758; &#xAE30;&#xC220;&#xC744; &#xCE21;&#xC815; &#xB2E4;&#xC591; &#xD55C; DNA &#xBC14;&#xC778;&#xB529; &#xB3C4;&#xBA54;&#xC778;, &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC5EC;&#xB7EC; &#xAC00;&#xC9C0; &#xB2E4;&#xB978; &#xAE30;&#xC220;&#xC744; &#xD558; &#xC5EC; &#xACF5;&#xBD80; &#xD558;&#xAE30; &#xC5B4;&#xB824;&#xC6B4; &#xC785;&#xC99D; &#xD588;&#xB2E4; 28 Saccharomyces cerevisiae &#xC804;&#xC0AC; &#xC778;&#xC790;&#xC5D0; &#xB300; &#xD55C; &#xC2DC;&#xD000;&#xC2A4; &#xAE30;&#xBCF8; &#xC124;&#xC815;&#xC5D0;&#xC11C; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xAC80;&#xC0AC;. &#xAC01; &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC694;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xC0C1;&#xB300;&#xC801; &#xBC14;&#xC778;&#xB529; affinities oligonucleotides &#xCDE8;&#xC7AC; &#xC21C;&#xC11C; &#xD658;&#xACBD; &#xC124;&#xC815;;&#xC758; &#xD3EC;&#xAD04;&#xC801;&#xC778; &#xC9C0;&#xB3C4;&#xB97C; &#xB9CC;&#xB4E4; &#xBAA8;&#xB4E0; &#xAC00;&#xB2A5;&#xD55C; 8 bp DNA &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xCE21;&#xC815; 4 &#xC804;&#xC0AC; &#xC778;&#xC790;&#xC5D0; &#xB300; &#xD55C; &#xC6B0;&#xB9AC;&#xB294; &#xC808;&#xB300; &#xB3D9;&#xC9C8;&#xC131;&#xC744; &#xACB0;&#xC815;. &#xC6B0;&#xB9AC;&#xAC00; &#xAE30;&#xB300; &#xD558;&#xB294; &#xB370;&#xC774;&#xD130; &#xBC0F; &#xB098;&#xC911;&#xC5D0;&#xC774; &#xAE30;&#xC220;&#xC758; &#xC0AC;&#xC6A9;&#xC774; &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC694;&#xC778; &#xD2B9;&#xC774;&#xC131; &#xC774;&#xD574; &#xADDC;&#xC81C; &#xC0AC;&#xC774;&#xD2B8;&#xC758; &#xD5A5;&#xC0C1;&#xACFC; &#xADDC;&#xC81C;&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xC7AC;&#xAD6C;&#xC131; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xD544;&#xC218; &#xC815;&#xBCF4;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

&#xB4DC; &#xB178; &#xBCF4; &#xC2DD;&#xBCC4; &#xBC0F; &#xC120;&#xD638;&#xB3C4; &#xBD84;&#xC11D;&#xC744; &#xACB0;&#xD569; &#xC804;&#xC0AC; &#xC778;&#xC790; &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;&#xC758; &#xC0DD;&#xBB3C; &#xB9AC; &#xD2B9;&#xC131;&#xD654;.

Expresi&oacute;n g&eacute;nica es regulada en parte por factores de transcripci&oacute;n de prote&iacute;nas que se unen a secuencias reguladoras de ADN diana. Predicci&oacute;n de sitios de uni&oacute;n de ADN y afinidades de secuencia del factor de transcripci&oacute;n o estructura es dif&iacute;cil; por lo tanto, los datos experimentales deben relacionar factores de transcripci&oacute;n con secuencias diana. Presentamos un enfoque microflu&iacute;dica de novo descubrimiento y caracterizaci&oacute;n Biof&iacute;sica cuantitativa de secuencias de ADN diana. Validamos nuestra t&eacute;cnica mediante la medici&oacute;n de las preferencias de la secuencia de 28 factores de transcripci&oacute;n Saccharomyces cerevisiae con una variedad de dominios de uni&oacute;n al ADN, incluyendo varios que han demostrado ser dif&iacute;ciles de estudiar por otras t&eacute;cnicas. Para cada factor de transcripci&oacute;n, medimos las afinidades de uni&oacute;n relativa a oligonucle&oacute;tidos cubriendo todas secuencias de ADN 8-bp posibles para crear un mapa comprensivo de las preferencias de la secuencia; para los cuatro factores de transcripci&oacute;n, tambi&eacute;n determinamos afinidades absolutas. Esperamos que estos datos y el uso futuro de esta t&eacute;cnica proporcionar&aacute; informaci&oacute;n esencial para comprender la especificidad del factor de transcripci&oacute;n, mejorar la identificaci&oacute;n de sitios regulatorios y reconstruir las interacciones reguladoras.

De novo identificaci&oacute;n y caracterizaci&oacute;n Biof&iacute;sica de sitios de uni&oacute;n del factor de transcripci&oacute;n con an&aacute;lisis de afinidad de microfluidos.

A quantitative understanding of how transcription factors interact with genomic target sites is crucial for reconstructing transcriptional networks in vivo. Here, we use Hac1, a well-characterized basic leucine zipper (bZIP) transcription factor involved in the unfolded protein response (UPR) as a model to investigate interactions between bZIP transcription factors and their target sites. During the UPR, the accumulation of unfolded proteins leads to unconventional splicing and subsequent translation of HAC1 mRNA, followed by transcription of UPR target genes. Initial candidate-based approaches identified a canonical cis-acting unfolded protein response element (UPRE-1) within target gene promoters; however, subsequent studies identified a large set of Hac1 target genes lacking this UPRE-1 and containing a different motif (UPRE-2). Using a combination of unbiased and directed microfluidic DNA binding assays, we established that Hac1 binds in two distinct modes: (i) to short (6-7 bp) UPRE-2-like motifs and (ii) to significantly longer (11-13 bp) extended UPRE-1-like motifs. Using a genetic screen, we demonstrate that a region of extended homology N-terminal to the basic DNA binding domain is required for this dual site recognition. These results establish Hac1 as the first bZIP transcription factor known to adopt more than one binding mode and unify previously conflicting and discrepant observations of Hac1 function into a cohesive model of UPR target gene activation. Our results also suggest that even structurally simple transcription factors can recognize multiple divergent target sites of very different lengths, potentially enriching their downstream target repertoire.

Basic leucine zipper transcription factor Hac1 binds DNA in two distinct modes as revealed by microfluidic analyses.

Sequence-specific DNA-binding proteins are among the most important classes of gene regulatory proteins, controlling changes in transcription that underlie many aspects of biology. In this work, we identify a transcriptional regulator from the human fungal pathogen Candida albicans that binds DNA specifically but has no detectable homology with any previously described DNA- or RNA-binding protein. This protein, named White-Opaque Regulator 3 (Wor3), regulates white-opaque switching, the ability of C. albicans to switch between two heritable cell types. We demonstrate that ectopic overexpression of WOR3 results in mass conversion of white cells to opaque cells and that deletion of WOR3 affects the stability of opaque cells at physiological temperatures. Genome-wide chromatin immunoprecipitation of Wor3 and gene expression profiling of a wor3 deletion mutant strain indicate that Wor3 is highly integrated into the previously described circuit regulating white-opaque switching and that it controls a subset of the opaque transcriptional program. We show by biochemical, genetic, and microfluidic experiments that Wor3 binds directly to DNA in a sequence-specific manner, and we identify the set of cis-regulatory sequences recognized by Wor3. Bioinformatic analyses indicate that the Wor3 family arose more recently in evolutionary time than most previously described DNA-binding domains; it is restricted to a small number of fungi that include the major fungal pathogens of humans. These observations show that new families of sequence-specific DNA-binding proteins may be restricted to small clades and suggest that current annotations--which rely on deep conservation--underestimate the fraction of genes coding for transcriptional regulators.

Identification and characterization of a previously undescribed family of sequence-specific DNA-binding domains.

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein's DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2's-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets.

The human fungal pathogen Candida albicans can switch between two phenotypic cell types, termed 'white' and 'opaque'. Both cell types are heritable for many generations, and the switch between the two types occurs epigenetically, that is, without a change in the primary DNA sequence of the genome. Previous work identified six key transcriptional regulators important for white-opaque switching: Wor1, Wor2, Wor3, Czf1, Efg1, and Ahr1. In this work, we describe the structure of the transcriptional network that specifies the white and opaque cell types and governs the ability to switch between them. In particular, we use a combination of genome-wide chromatin immunoprecipitation, gene expression profiling, and microfluidics-based DNA binding experiments to determine the direct and indirect regulatory interactions that form the switch network. The six regulators are arranged together in a complex, interlocking network with many seemingly redundant and overlapping connections. We propose that the structure (or topology) of this network is responsible for the epigenetic maintenance of the white and opaque states, the switching between them, and the specialized properties of each state.

Structure of the transcriptional network controlling white-opaque switching in Candida albicans.

The duplication of transcription regulators can elicit major regulatory network rearrangements over evolutionary timescales. However, few examples of duplications resulting in gene network expansions are understood in molecular detail. Here we show that four Candida albicans transcription regulators that arose by successive duplications have differentiated from one another by acquiring different intrinsic DNA-binding specificities, different preferences for half-site spacing, and different associations with cofactors. The combination of these three mechanisms resulted in each of the four regulators controlling a distinct set of target genes, which likely contributed to the adaption of this fungus to its human host. Our results illustrate how successive duplications and diversification of an ancestral transcription regulator can underlie major changes in an organism's regulatory circuitry.

Article	Year
Stepping and stretching. How kinesin uses internal strain to walk processively. The Journal of biological chemistry\| PubMed ID: 12626516	2003
Combined optical trapping and single-molecule fluorescence. Journal of biology\| PubMed ID: 12733997	2003
Simultaneous, coincident optical trapping and single-molecule fluorescence. Nature methods\| PubMed ID: 15782176	2004
Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro. Nature cell biology\| PubMed ID: 16604065	2006
Eg5 steps it up! Cell division\| PubMed ID: 17173688	2006
De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis. Nature biotechnology\| PubMed ID: 20802496	2010
Basic leucine zipper transcription factor Hac1 binds DNA in two distinct modes as revealed by microfluidic analyses. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23054834	2012
Identification and characterization of a previously undescribed family of sequence-specific DNA-binding domains. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23610392	2013
Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets. Nucleic acids research\| PubMed ID: 23625967	2013
Structure of the transcriptional network controlling white-opaque switching in Candida albicans. Molecular microbiology\| PubMed ID: 23855748	2013
How duplicated transcription regulators can diversify to govern the expression of nonoverlapping sets of genes. Genes & development\| PubMed ID: 24874988	2014

Polly M. Fordyce

Biography

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