Создание эффективных ингибиторов генной экспрессии на основе олигонуклеотидов с природным сахаро-фосфатным остовом

выводы.

1. Предложена стратегия модификации концевых фосфатных групп олигонуклеотидов в водной среде, позволяющая получать фосфамидные и фосфодиэфирные производные незащищенных олигонуклеотидов с различными соединениями: флуоресцентными красителями, интеркаляторами, гидрофобными молекулами, аминокислотами, пептидами. Эта стратегия основана на активации фосфомоноэфирной группы олигонуклеотидов водорастворимым карбодиимидом или бромцианом с М-замещенным морфолином в присутствии различных нуклеофильных соединений.

2. Получено несколько новых типов антисмысловых олигонуклеотидов: химерные олигонуклеотиды, содержащие а-аномерные фрагменты, циклические, псевдоциклические и структурированные олигонуклеотиды. Изучено влияние их структуры на эффективность и специфичность их гибридизации с РНК-мишенями и их устойчивость к нуклеазному гидролизу в различных биологических системах.

Показано, что для химерных а-(3-олигонуклеотидов оптимальной с точки гибридизационной способности, гидролитической устойчивости и специфичности ингибирующего действия является структура, состоящая из одного аи одного^{-фрагмента,} соединенных между собой З'-концами. Циклические олигонуклеотиды обладают повышенной устойчивостью к нуклеазному гидролизу в биологических средах, особенно в тех, которые не имеют высокой эндонуклеазной активности, и способны образовывать с РНК достаточно устойчивые дуплексы. Однако в случае протяженных РНК, обладающих сложной пространственной структурой, эффективность их гидролиза РНКазой Н в комплексах с циклическими олигонуклеотидами существенно ниже, чем с линейными. Это можно объяснить тем, что циклическая структура олигонуклеотида затрудняет его взаимодействие со структурированной РНК. Кроме того, удлинение одноцепочечной части олигонуклеотида повышает возможность образования им нежелательных комплексов и тем самым может снизить специфичность действия циклических оли го нуклеотидо в.

Олигонуклеотиды, концы которых вовлечены в образование внутримолекулярных дуплексов, значительно устойчивее к ферментативному гидролизу, чем линейные олигонуклеотиды. Они способны эффективно гибридизоваться с комплементарными РНК, при этом происходит денатурация их внутримолекулярной структуры и образуются совершенные комплексы с мишенью. Учитывая доступность и простоту синтеза этих соединений, их наиболее целесообразно использовать в качестве антисмысловых реагентов.

3. Изучена способность двух коммерческих реагентов: полиамидоаминного дендримера SuperFect™ и катионных липосом DAC-30™ — обеспечивать транспорт фосфодиэфирных олигонуклеотидов через цитоплазматическую мембрану и предохранять их от ферментативного гидролиза в клетках HeLa и NIH ЗТЗ. Установлено, что SuperFect™ является более эффективным носителем для используемых олигонуклеотидов и клеток, чем DAC-30™.

4. Методом тушения флуоресценции изучено взаимодействие структурированных олигонуклеотидов с РНК-мишенью in vitro и ex vivo. Установлено, что скорость и эффективность этого взаимодействия снижаются с увеличением длины и усложнением структуры РНК. SuperFect™ не влияет на гибридизацию олигонуклеотида с протяженной мРНК, но полностью блокирует ее в случае короткой РНК-мишени. Оптимальной для антисмыслового олигонуклеотида с точки зрения эффективности и специфичности его взаимодействия с целевой мРНК является структура типа «ракетки» с небольшой петлей и коротким внутримолекулярным дуплексом с Тш, в районе 45−50°С.

5. Изучена зависимость между характером внутриклеточного распределения олигонуклеотидов, векторизованных SuperFect™, и тем, в какой фазе находится клетка. Обнаружена преимущественная ядерная локализация олигонуклеотида в клетках в фазе G2/M. Для клеток, находящихся в фазах G1 и S, характерно гетерогенное распределение олигонуклеотида с преимущественной его локализацией в эндосомах и других везикулах с кислой средой. Установлено, что невекторизованные фосфодиэфирные олигонуклеотиды могут проникать в облученные опухолевые клетки, при этом эффективность их проникновения зависит от дозы облучения.

6. Для оценки эффективности и специфичности действия, а также токсичности различным образом модифицированных антисмысловых олигонуклеотидов и систем-носителей разработана простая тест-система, которая позволяет быстро и количественно определить все перечисленные параметры. Она представляет собой два разных репортерных гена под контролем одного и того же промотора, уровень экспрессии которых можно количественно измерить. Один из этих генов (ген зеленого флуоресцирующего белка) используется как мишень для антисмыслового олигонуклеотида, второй (ген (3-галактозидазы) — как контроль его специфичности и токсичности. С использованием этой тест-системы измерено ингибирующее действие олигонуклеотидов различной структуры в присутствии двух носителей: БирегРе^&tradeи БАС-ЗО™.

7. Установлено, что фосфодиэфирные олигонуклеотиды, имеющие структуру типа «ракетки» и содержащие по две тиофосфатные группы на концах, способны специфично подавлять экспрессию целевого гена зеленого флуоресцирующего белка (ОБР) с эффективности 70−80%. Степень ингибирования зависит от нескольких факторов: эффективности проникновения векторизованного олигонуклеотида в клетку, расположения участка его узнавания в структуре мРНК, стабильности вторичной структуры самого олигонуклеотида. Снижение уровня синтеза ОБР связано с непосредственным действием антисмысловых олигонуклеотидов на его мРНК, а не какими-либо вариациями в уровне трансфекции плазмид или изменениями в клеточном метаболизме.

8. По результатам ингибирования экспрессии ОБР выбран наиболее эффективный олигонуклеотид-ингибитор и изучен механизм его действия. Установлено, что подавление белкового синтеза связано с тем, что в присутствии антисмыслового олигонуклеотида происходит специфичное расщепление мРНК гена %[р РНКазой Н. Эффективность гидролиза мРНК увеличивается с увеличением концентрации олигонуклеотида. Степень гидролиза мРНК соответствует степени ингибирования белкового синтеза.

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