Инструменты нанотехнологий ДНК — от дизайна до приложений

Набор устройств с ДНК-нанотехнологиями, созданных для преодоления определенных узких мест в разработке новых методов лечения, диагностики и понимания молекулярных структур

Наноструктуры ДНК с их потенциалом для проницаемости клеток и тканей, биосовместимости и высокой программируемости на наномасштабном уровне являются многообещающими кандидатами в качестве новых типов средств доставки лекарств, высокоспецифичных диагностических устройств и инструментов для расшифровки того, как биомолекулы динамически изменяют свою форму и взаимодействуют с ними. друг друга и кандидатов в препараты. Исследователи Wyss Institute предоставляют набор разнообразных, многофункциональных ДНК-нанотехнологических инструментов с уникальными возможностями и потенциалом для широкого круга клинических и биомедицинских исследований.

ДНК-нанотехнологические устройства для доставки терапевтических лекарств

У наноструктур ДНК в будущем есть потенциал для широкого использования для транспортировки и презентации различных биологически активных молекул, таких как лекарственные препараты, а также иммуностимулирующие антигены и адъюванты, для нацеливания на клетки и ткани человеческого тела.

ДНК-оригами как компоненты высокоточной доставки противораковых вакцин
Институт Висса разработал противораковые вакцины для улучшения иммунотерапии. В этих подходах используются имплантируемые или инъецируемые каркасы на основе биоматериалов, которые представляют опухолеспецифические антигены и биомолекулы, которые привлекают дендритные иммунные клетки (ДК) в каркас и активируют их, чтобы после их высвобождения они могли управлять противоопухолевыми Т-клеточными ответами против опухолей. несущие те же антигены. Чтобы активироваться наиболее эффективно, DC, вероятно, должны испытать опухолевые антигены и иммуностимулирующие молекулы адъюванта CpG в определенных соотношениях (стехиометриях) и конфигурациях, которые соответствуют плотности и распределению молекул рецепторов на их клеточной поверхности.

Специально разработанное ДНК-оригами, запрограммированное на сборку в блоки с жесткой квадратной решеткой, которые совместно представляют опухолевые антигены и адъюванты для DC в каркасах биоматериалов с точностью до нанометра, имеют потенциал для повышения эффективности терапевтических противораковых вакцин и могут быть дополнительно функционализированы с помощью анти- противораковые препараты.

Стратегия химической модификации для защиты наноструктур
ДНК, доставляющих лекарства. Наноструктуры ДНК, такие как самособирающиеся ДНК-оригами, являются многообещающими средствами доставки лекарств и диагностических средств. Их можно гибко функционализировать с помощью низкомолекулярных и белковых лекарств, а также функций, облегчающих их доставку к конкретным клеткам-мишеням и тканям. Однако их потенциал ограничен их ограниченной стабильностью в тканях и крови организма. Чтобы помочь реализовать необычайные перспективы наноструктур ДНК, исследователи Wyss разработали простой, эффективный и масштабируемый подход к химическому перекрестному связыванию, который может обеспечить наноструктурам ДНК стабильность, которая им необходима в качестве эффективных носителей для лекарств и диагностики.

В двух простых и экономически эффективных шагах подход Wyss сначала использует маломолекулярный ненавязчивый нейтрализующий агент , ПЭГ-олиголизин, который несет несколько положительных зарядов, чтобы покрыть структуры ДНК-оригами. В отличие от обычно используемых ионов Mg 2+ , каждый из которых нейтрализует только два отрицательных изменения в структурах ДНК, ПЭГ-олиголизин покрывает несколько отрицательных зарядов на одном, образуя, таким образом, стабильную «электростатическую сеть», которая увеличивает стабильность наноструктур ДНК примерно в 400 раз. . Затем, применяя химическое сшивание реагент, известный как глутаральдегид, дополнительные стабилизирующие связи вводятся в электростатическую сеть, что увеличивает стабильность наноструктур ДНК еще в 250 раз, продлевая период их полураспада до диапазона, совместимого с широким спектром клинических применений.

ДНК-нанотехнологические устройства как сверхчувствительные диагностические и аналитические инструменты

Создание детектируемых наноструктур ДНК в ответ на болезнь или патоген-специфические нуклеиновые кислоты, в принципе, предлагает средства для высокоэффективного обнаружения биомаркеров в различных образцах. Событие связывания одной молекулы синтетического олигонуклеотида с нуклеиновой кислотой-мишенью может привести к созданию гораздо более крупных структур за счет совместной сборки меньших синтетических единиц ДНК, таких как плитки или кирпичики ДНК, в более крупные структуры, которые затем можно визуализировать с помощью простых лабораторных анализов. Однако основным препятствием для этих подходов является возникновение (1) неспецифического связывания и (2) событий неспецифической нуклеации в отсутствие конкретной нуклеиновой кислоты-мишени, что может привести к ложноположительным результатам. Нанотехнологи Wyss DNA разработали два отдельно применимых, но комбинируемых решения этих проблем.

Цифровой подсчет молекул биомаркеров с катенанами нанопереключателей ДНК
Чтобы обеспечить первоначальное обнаружение (связывание) биомаркеров со сверхвысокой чувствительностью и специфичностью, исследователи Wyss разработали тип нанопереключателя ДНК, который спроектирован как более крупная катенана (латинскоеслово catena означает цепь), Он собран из механически связанных кольцевых подструктур со специфическими функциями, которые вместе позволяют обнаруживать и подсчитывать отдельные молекулы биомаркеров. В структуре «DNA Nanoswitch Catenane» оба конца более длинной синтетической цепи ДНК связаны с двумя фрагментами антител, каждый из которых специфически связывается с разными частями одной и той же интересующей молекулы биомаркера, что обеспечивает высокую целевую специфичность и чувствительность.

This bridging-event causes the strand to close into a “host ring,” which it is interlocked at different regions with different “guest rings.” Closing of the host ring switches the guest rings into a configuration that allows the synthesis of a new DNA strand. The newly synthesized diagnostic strand then can be unambiguously detected as a single digital molecule count, while disrupting the antibody fragment/biomarker complex starts a new biomarker counting cycle. Both, the target binding specificity and the synthesis of a target-specific DNA strand also enable the combination of multiple DNA nanoswitch catenanes to simultaneously count different biomarker molecules in a single multiplexed reaction.

Быстрое платформа усиления для различных биомаркеров быстрый, недорогой и фермент , свободной от обнаружения и амплификации платформы
позволяет избежать неспецифического зародышеобразования и амплификации и позволяет самосборку гораздо более крупных структур микронного размера из одной затравки всего за несколько минут. Метод, названный «Обнаружение перекрестных наноразмеров», позволяет осуществлять сверхкооперативную сборку лент, начиная с одного события связывания биомаркера. Структуры микронного размера плотно сотканы из одноцепочечных «планок ДНК», в результате чего входящая планка змеится над и под шестью или более ранее захваченными планками на конце растущей ленты «крест-накрест», образуя слабые, но высокоспецифичные взаимодействия. с его взаимодействующими планками ДНК. Зарождение процесса сборки строго специфично для целевых семян, и сборку можно провести в одностадийной реакции примерно за 15 минут без добавления дополнительных реагентов и в широком диапазоне температур.

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