نشكر جيمس واترز، وادز ورث غيبل وبو برودواتر لقراءة نقدية للمخطوطة. نشكر أيضا أربعة المراجعين المجهولين لتوفير تعليقات مفيدة. نحن نعترف الدعم المالي من معهد جورجيا للتكنولوجيا، وجائزة صندوق ويلكوم بوروز الوظيفي في واجهة العلمي، ومنحة شبكة البحوث طالب من جبهة الخلاص الوطني الفيزياء نظم المعيشة.

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الكتاب تعلن أي تضارب في المصالح.

واستخدمت مقايسة بسيطة جزيء واحد يعتمد على الحنق لدراسة حركية حلقات من السلطات الوطنية المعينة من الأشكال الجوهرية المختلفة. يمكن السلطات الوطنية المعينة منحني بتكرار تسلسل 10 مير في مرحلة مع الفترة حلزونية من 10.5 نقطة أساس على استعداد، ويمكن تقدير الانحرافات الخاصة ?...

فهم الخواص الميكانيكية للdsDNA وغير ذات أهمية أساسية في العلوم الأساسية والتطبيقات الهندسية. هيكل dsDNA وأكثر تعقيدا من سلم حلزوني على التوالي بسبب زوايا لفة، والميل، وتطور بين أزواج قاعدة المتعاقبة يمكن أن تختلف مع التسلسل. يمكن أن تسبب التقلبات الحرارية dsDNA والخضوع وسائط متنوعة من تقلبات بتكوين مثل الانحناء، واللف والتمدد. التحولات مثل ذوبان ومجعد يمكن أن يحدث أيضا في الظروف القاسية. من بين هذه الاقتراحات، dsDNA والانحناء له الأثر البيولوجي أكثر ما يلفت الانتباه 1. ويرتبط dsDNA والانحناء مع القمع الجينات أو التنشيط من خلال جلب اثنين من مواقع بعيدة قريبة من بعضها البعض. كما أنها تلعب دورا هاما في التعبئة و التغليف الحمض النووي داخل نواة الخلية أو قفيصة الفيروسية. الانحناء تشوه dsDNA ويمكن تصور تجريبيا بواسطة عالية الدقة المجهر (AFM 2 و 3 TEM)، وthermodynamics وحركية يمكن دراستها بواسطة حلقات المقايسات، التي تربط كيميائيا المواقع جنبا إلى جنب من dsDNA و. واحدة من هذه الاختبار هو تعتمد على يغاز سيكليزيشن 4. في هذا الاختبار، يتم circularized جزيئات dsDNA ومع &quot;لزجة&quot; (متماسكة) أو نهايات dimerized بواسطة يغاز الحمض النووي. من خلال مقارنة معدلات دائرة وتشكيل ديمر، يمكن للمرء الحصول على التركيز المولي الفعلي لنهاية واحدة من الحمض النووي في محيط الطرف الآخر، والذي يعرف باسم عامل J. هذا العامل J ما يعادل الأبعاد لكثافة احتمال العثور على واحدة نهاية من الحمض النووي على مسافة قصيرة من الطرف الآخر، وبالتالي يعكس المرونة من الحمض النووي. قياس عامل J بوصفها وظيفة من طول الحمض النووي يكشف خصائص كثيرة عن ميكانيكا الحمض النووي بما في ذلك طول استمرار 4،5. فقد كان ينظر على نطاق واسع سلسلة مثل دودة (WLC) نموذج كنموذج البوليمر الكنسي للميكانيكيين dsDNA وبناء على نجاحها في تفسيراining منحنيات القوة والإرشاد في الحمض النووي التي تم الحصول عليها سحب تجارب 6، وتوقع بشكل صحيح العوامل J من dsDNAs أطول من 200 سنة مضت 7. ومع ذلك، باستخدام مقايسة سيكليزيشن على جزيئات dsDNA وقصيرة بقدر 100 سنة مضت، ويدوم كلوتير وقياس العوامل J أن تكون عدة أوامر من حجم أعلى من التنبؤ WLC نموذج 8. وبعد ذلك بعام، دو وآخرون. أنتج عوامل J بالاتفاق مع نموذج WLC باستخدام مقايسة سيكليزيشن مع تركيزات أقل من يغاز وعزا نتيجة الشاذة من مجموعة ويدوم لتركيزات عالية تستخدم يغاز 9. هذا الجدل تجسد تأثير لا مفر منه من يغاز الحمض النووي على حركية سيكليزيشن عند استخدام الفحص التقليدية 9. علاوة على ذلك، يمكن أن تؤثر أيضا على الحمض النووي يغاز بنية الحمض النووي وصلابة عن طريق غير محددة 10،11 ملزمة. للقضاء على المخاوف من المقايسات الفنية التي تعتمد على البروتين حلقات، أثبتنا مؤخرا البروتوكول الاضافيعين خالية مقايسة حلقات على أساس نقل الإسفار الرنين الطاقة (الحنق) 12. في هذه الطريقة، يتم الكشف عن التشكل يحلق بواسطة الحنق بين المانحين ومتقبل تعلق بالقرب من نهايات لزجة من جزيء الحمض النووي. يستخدم هدفا من نوع مجموع المجهر مضان التأمل الداخلي (TIRFM) لتسجيل مسارات حلقات عكسها والأحداث unlooping من جزيئات الحمض النووي واحد يجمد السطح لفترة طويلة من الزمن. يتميز هذا الأسلوب التجمع PCR القائم من جزيئات الحمض النووي لتوليد جزيئات الحمض النووي خالية من عدم التوافق، وهو تحسن حاسم على أسلوب مماثل من قبل Vafabakhsh وها 13. على جانب واحد جزيء من هذا البروتوكول يسمح قياس توزيعات بالإضافة إلى فرقة المتوسطات في حين أن الجانب الحنق يسمح احد لقياس ديناميكية حلقات الحمض النووي مرارا وتكرارا من نفس الجزيء، حتى في الظروف التي يمكن أن يضعف النشاط يغاز. يظهر الإعداد TIRFM في الشكل رقم 1. A مخصصةيتم وضع تصميم المرحلة العينة على هيئة المجهر أوليمبوس IX61. يتم إدخال 532 نانومتر و 640 نانومتر ليزر من الجانب والتي تعكسها المرايا بيضاوي الشكل صغيرة 14 من زمن الهدف NA عالية لتحقيق زاوية حرجة من الإصابة في واجهة المياه ساترة. نلاحظ أن أكثر انتشارا النقل البري الدولي من خلال الهدف باستخدام المرايا مزدوج اللون أو الاجهزة النقل البري الدولي القائم على منظور يمكن أن تستخدم أيضا لهذا التطبيق الحنق. يتم تقسيم الصورة التي شكلتها مضان المجهر في الصور المانحة ومتقبل بواسطة مرآة مزدوج اللون. ثم يتم إعادة تصويرها، فهم على نصفين من EMCCD. وتستخدم مرشحات إضافية الانبعاثات تمريرة طويلة للحد من إشارة الخلفية. التحكم في درجة الحرارة أمر ضروري للحصول على البيانات الحركية استنساخه. لمراقبة درجة الحرارة، ويتم فصل الهدف من الأنفية من الجسم المجهر لتقليل انتقال الحرارة، والماء من درجة الحرارة التي تسيطر عليها المبرد / سخان يعمم من خلال طوق النحاس أن يناسب بإحكامحول المعدنية الداخلية تحت سترة الهدف. هذا الإعداد هو قادرة على تحقيق التحكم في درجة الحرارة قوية على السطح ساترة بين 15 و 50 درجة مئوية (الشكل 2). في هذا العمل، والحفاظ على درجة حرارة العينة في 24 درجة مئوية. ويقدم البروتوكول باتباع الإجراء خطوة بخطوة لبناء الحمض النووي، وتقدير شكل الحمض النووي، وتجربة واحدة جزيء، وJ تحديد عامل.

<table border="0" cellpadding="0" cellspacing="0" width="793">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Small DNA FRAG Extract Kit-100PR</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">97060-558</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Acrylamide 40% solution 500 mL</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">97064-522</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bis-acrylamide 2% (w/v) solution 500 mL</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">97063-948</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">GeneRuler 100 bp DNA Ladder, 100-1000 bp</td>
			<td style="width:124px;">Fermentas</td>
			<td>SM0241</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Mini Vertical PAGE System</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">89032-300</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Syringe filter 0.2um CS50</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">A2666</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Trolox</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">238813-1G</td>
			<td>triplet state quencher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Protocatechuic acid (PCA)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">08992-50MG</td>
			<td>oxygen scavenging system</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Protocatechuate 3,4-Dioxygenase (PCD)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">P8279-25UN</td>
			<td>oxygen scavenging system</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">mPEG-silane, MW 2000 1g</td>
			<td style="width:124px;">Laysan Bio</td>
			<td style="width:135px;">MPEG-SIL-2000-1g</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Biotin-PEG-Silane, MW 3400</td>
			<td style="width:124px;">Laysan Bio</td>
			<td style="width:135px;">Biotin-PEG-SIL-3400-1g</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Avidin, NeutrAvidin Biotin-binding Protein</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:135px;">A2666</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Phusion Hot Start High-Fidelity DNA Polymerase</td>
			<td style="width:124px;">New England Biolabs</td>
			<td style="width:135px;">F-540L</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gel/PCR DNA Fragments Extraction Kit</td>
			<td>IBI Scientific</td>
			<td>IB47020</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Premium plain glass microscope slides</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:135px;">12-544-1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">VWR micro cover glass, rectangular, no. 1</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">48404-456</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Fisher Scientific Isotemp 1006s Recirculating Chiller/Heater</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:135px;"></td>
			<td>temperature control</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Objective Cooling Collar</td>
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			<td style="width:135px;">150303</td>
			<td>temperature control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">KMI53 Biological Micrometer Measuring Stage</td>
			<td style="width:124px;">Semprex</td>
			<td style="width:135px;">KMI53</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">High Performance DPSS Laser 532nm 50mW&#160;</td>
			<td style="width:124px;">Edmund optics</td>
			<td style="width:135px;">NT66-968</td>
			<td>Cy3 excitation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:351px;">CUBE Fiber Pigtailed 640 nm, 30mW, Fiber, FC/APC Connector</td>
			<td style="width:124px;">Coherent</td>
			<td style="width:135px;">1139604</td>
			<td>Cy5 excitation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">650 nm BrightLine Dichroic Beamsplitter</td>
			<td style="width:124px;">Semrock</td>
			<td style="width:135px;">FF650-Di01-25x36</td>
			<td>splitting dichroic</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:351px;">LaserMUX Beam Combiner, reflects 514.5, 532, &#38; 543.5 nm lasers, 25 mm</td>
			<td style="width:124px;">Semrock</td>
			<td style="width:135px;">LM01-552-25</td>
			<td>combining dichroic</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Brightline Fluorescence Filter 593/40</td>
			<td style="width:124px;">Semrock</td>
			<td>FF01-593/40-25</td>
			<td>Cy3 emission filter</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">635 nm EdgeBasic LWP longpass Filter, 25 mm</td>
			<td style="width:124px;">Semrock</td>
			<td style="width:135px;">BLP01-635R-25</td>
			<td>Cy5 emission filter</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">EMCCD iXon+</td>
			<td style="width:124px;">Andor Technology</td>
			<td style="width:135px;">DU-897E-CS0-#BV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">IX51 inverted microscope frame</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Objective UApo N 100x/1.49 Oil TIRF</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Immersion oil type-F for fluorescence microscopy</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:135px;">IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2mm Diameter 45&#176; Rod Lens Aluminum Coated&#160;</td>
			<td style="width:124px;">Edmund optics</td>
			<td style="width:135px;">54-092</td>
			<td>miniature mirror</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1/4&#34; Travel Single-Axis Translation Stage</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">MS-1</td>
			<td>translation of miniature mirror</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#216;1&#34; Achromatic Doublet, ARC: 400-700 nm, f=200 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">AC254-200-A</td>
			<td>focusing lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Adjustable Mechanical Slit</td>
			<td style="width:124px;">Thorlabs</td>
			<td>VA100</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Dielectric Mirror</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">BB1-E02</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">&#216;1&#34; Achromatic Doublet, f = 100 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">AC254-100-A</td>
			<td>relay lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Lens Mount for &#216;1&#34; Optics</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">LMR1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Dichroic Filter Mount</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">FFM1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Fixed Cage Cube Platform</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">B3C</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Kinematic Mount for &#216;1&#34; Optics</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">KM100</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">N-BK7 Plano-Convex Lens, &#216;1&#34;, f = 40 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">LA1422-A</td>
			<td>collimating lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">N-BK7 Plano-Convex Lense, &#216;6.0 mm, f = 15 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">LA1222-A</td>
			<td>telescope lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">N-BK7 Plano-Convex Lense, &#216;6.0 mm, f = 150 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">LA1433-A</td>
			<td>telescope lens</td>
		</tr>
	</tbody>
</table>

studying dna looping by single-molecule fret

الانحناء من الحمض النووي المزدوج تقطعت بهم السبل (dsDNA و) ويرتبط مع العديد من العمليات البيولوجية الهامة مثل التعرف على الحمض النووي والبروتينات والحمض النووي في التعبئة والتغليف Nucleosomes لل. وقد تمت دراسة الديناميكا الحرارية من dsDNA والانحناء بطريقة تسمى سيكليزيشن التي تعتمد على الحمض النووي يغاز للانضمام تساهميا نهايات لزجة قصيرة من dsDNA و. ومع ذلك، يمكن أن تتأثر كفاءة ربط العديد من العوامل التي لا ترتبط dsDNA وحلقات مثل بنية الحمض النووي المحيطة نهايات لزجة انضم، ويغاز يمكن أن يؤثر أيضا على معدل حلقات اضحا من خلال آليات مثل الربط غير محددة. هنا، وتبين لنا كيفية قياس حركية dsDNA وحلقات من دون يغاز عن طريق الكشف عابرة تشكيل حلقة الحمض النووي عن طريق الحنق (الإسفار الرنين نقل الطاقة). هي التي شيدت جزيئات dsDNA وباستخدام بروتوكول بسيطة PCR القائم مع زوج الحنق ورابط البيوتين. يتم استخراج كثافة الاحتمال حلقات المعروفة باسم عامل J من معدل حلقات ومعدل الصلب بين اثنين disconnecتيد نهايات لزجة. عن طريق اختبار اثنين dsDNAs مع الانحرافات الجوهرية المختلفة، وتبين لنا أن العامل J حساسة لشكل جوهري من dsDNA و.

تتكون جزيئات الحمض النووي المستخدمة في الدراسة حلقات من المنطقة المزدوجة من تسلسل متغير وطول واحد يتدلى الذين تقطعت بهم السبل التي هي مكملة لبعضها البعض. ويتدلى، والتي هي 7 قاعدة طويلة، يمكن أن يصلب مع بعضها البعض لالتقاط حالة يحلق. كل عبء يحتوي إما CY3 أو Cy5 مرتبط في ال...

Watch this Scientific Journal Video about Studying DNA Looping by Single-Molecule FRET at JoVE.com

Studying DNA Looping by Single-Molecule FRET

تقدم هذه الدراسة إجراء التجارب مفصلة لقياس ديناميكية حلقات من الحمض النووي المزدوج تقطعت بهم السبل باستخدام جزيء واحد نقل الإسفار الرنين الطاقة (الحنق). يصف البروتوكول أيضا كيفية استخراج كثافة الاحتمال حلقات يسمى عامل J.

دراسة الحمض النووي عن طريق حلقات جزيء واحد الحنق

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

studying-dna-looping-by-single-molecule-fret

Research

JoVE Journal

Biology

15.3K Views.  Georgia Institute of Technology.  تقدم هذه الدراسة إجراء التجارب مفصلة لقياس ديناميكية حلقات من الحمض النووي المزدوج تقطعت بهم السبل باستخدام جزيء واحد نقل الإسفار الرنين الطاقة (الحنق). يصف البروتوكول أيضا كيفية استخراج كثافة الاحتمال حلقات يسمى عامل J. 

Video: Studying DNA Looping by Single-Molecule FRET

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

طرق جزيء واحد لرصد التغيرات في خصائص طبقة ثنائية مطاطا

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

الجمع بين QD - الحنق و microfluidics الحمض النووي لرصد Nanocomplex الذاتي الجمعية في الوقت الحقيقي

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

تصور تكرار الحمض النووي جزيء واحدة مع مضان المجهري

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

النظام الآلي للقياسات واحدة الإسفار جزيء من الجزيئات الحيوية سطح يجمد

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

الملاحظة المباشرة من الحمض النووي الانزيمات تكرار استخدام الحمض النووي جزيء واحدة تمتد الفحص

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

المفرد جزيء التصوير النقل النووية

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

رصد كيناز والفوسفاتيز الأنشطة من خلال دورة الخلية التي Ratiometric الحنق

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

واحدة جزيء التصوير من تنظيم الجينات<em&gt; في الجسم الحي</em&gt; استخدام التنشيط بواسطة Cotranslational انشقاق (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

الجمع بين التلاعب جزيء واحدة والتصوير لدراسة التفاعلات-DNA البروتين

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

الحنق التصوير في الهلاميات المائية ثلاثية الأبعاد

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...