w******e
发帖数: 1187 | 1 Tan group striked again...
http://www.pnas.org/content/early/2011/02/15/1016197108.abstrac
1. construct a conformation switch-based aptamer sensor which elicits
signal upon binding to specific target on cell(have been done many times);
2. use the sensor to quantify cancer cells down to a few hundred cells,
in both saline & serum, with a huge dynamic range (new to me, but
shouldn't be too hard)
3. use the sensor for in vivo imaging of cancer mouse model induced by
SC injected cancer cells. imagin... 阅读全帖 |
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z*****6
发帖数: 1486 | 2 exactly...specificty is the main concern... Charge is an important reason,
and besides, aptamer is too flexible, can frequently undergo structure
change in response to target or non-preferential target..there are several
papers talking about these issues.
Pegaptanib is a successful example of aptamer used as a drug...
I doubt the specificty of aptamer for a long time. However, in nature, the
special DNA structures, like ribosome RNA, hairpin structures, cloverleaf
structure...can act as specific... 阅读全帖 |
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r*******6
发帖数: 545 | 3 新手要用aptamer，有几个问题不清楚，求问专家：
1）RNA aptamer中A，C和G三个碱基中都含有NH2基。看的文献中都用EDC/NHS和基地的
COOH反应，COOH岂不是除了和3'和5'修饰的NH2反应还要和aptamer上的NH2反应？这不
就破坏aptamer的功能了？
2）如果把基地的功能团是NH2,没找到有公司可以在aptamer的3'和5'修饰COOH。这是什
么原因，不好修饰？ |
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r*******6
发帖数: 545 | 4 新手要用aptamer，有几个问题不清楚，求问专家：
1）RNA aptamer中A，C和G三个碱基中都含有NH2基。看的文献中都用EDC/NHS和基地的
COOH反应，COOH岂不是除了和3'和5'修饰的NH2反应还要和aptamer上的NH2反应？这不
就破坏aptamer的功能了？
2）如果把基地的功能团是NH2,没找到有公司可以在aptamer的3'和5'修饰COOH。这是什
么原因，不好修饰？ |
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T**********t
发帖数: 1604 | 5 Aptamer的优点是显而易见的，但也有难以克服的缺点。
理论上是可以针对任何target筛选aptamer,但实际上还是有困难的。因为aptamer是核
酸分子，带有大量的负电荷，所以它和target的作用受电荷作用的影响很大。亲和力强
的target分子往往都是带正电的，而且很多时候有特异性不够好的问题。所以aptamer
做了二十年了，还是没有能撼动抗体的主流地位。 |
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T**********t
发帖数: 1604 | 6 有没有人知道这些aptamer people的family tree啊。我就知道Aptamer大概最早是
Tuerk/Gold，Ellington/Szostak各自两个实验室开始搞的。后面怎么开枝散叶的就不
清楚了。Tan Weihong是师从什么人？最近几年的aptamer paper好像中国人很多啊，是
因为入门的门槛低么？ |
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w******e
发帖数: 1187 | 7 sullenger是gold的postdoc，famulok是szostak的postdoc。john lis不知。
shi hua和版上某牛人是john lis的postdoc/phd。tan weihong是半路出家的化学牛
人。li yingfu不知。谁给补充一下？
尤其tan组，现在基本dominate cell-SELEX并搞了n种cell-based aptamer
application
，很不容易，很佩服。打江山的shangguan回国了，现在还没有大paper面世，可能在
攒ing。
aptamer paper虽多，大部分都是拿个thrombin aptamer搞各种sensor platform，不
厚道说其实是打酱油的哈哈~作应用的只有tan组、langer组、ellington组、lu组、
li组我比较看好。 |
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w******e
发帖数: 1187 | 8 aptamer against thrombin protein.
I guess you are interested in application of aptamer. Lu Yi, Andrew
Ellington,
Tan weihong, Li Yingfu each published an aptamer-sensor review last year.
you can start from there. |
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z*****6
发帖数: 1486 | 9 advantages of aptamer: longer shelf-life, higher stability, easy to
synthesize....
The most amazing fact of aptamer to me is, one can select aptamer against
almost any target! |
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w******e
发帖数: 1187 | 10 solution 1: super slow Koff, see somalogic;
solution 2: modified base, see somalogic;
solution 2: multivalent, multiple specificity check. remember aptamers are
only 1/10 the size of Abs
BTW, here is the aptamer affinity that somalogic claims to have achieved:
http://somalogic.com/science/agile.html
aptamer |
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a****o
发帖数: 1786 | 11 aptamer做了二十年了，还是没有能撼动抗体的主流地位
但是不是因为你说的原因，看一下AJCP / Editorial
Where Are All the Aptamers?
Geoffrey S. Baird, MD, PhD
可能会给你新的启示
新技术的使用有可能会影响公司的利润，培训宣传使医生了解这项新技术需要时间，，
太复杂
aptamer |
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w******e
发帖数: 1187 | 12 您有所不知，前几年粘PSMA aptamer的光n多文章上过好journal，从NP到Qdot到
dentrimer
，全都是换汤不换药的，包括我们敬爱的bob langer同志也混了几篇pnas，UIUC某位
同胞更是只在cell里搞了点NP，在去年还上了angewandte。。。
rossi同志好歹是自己搞了个aptamer，比只会追PSMA的同志们境界还是高一点点的呵呵 |
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m********8
发帖数: 314 | 13 感谢朋友们的介绍和看法。是不是因为aptamer相对Ab比较新颖，有很大的发展和推广
空间，所以，如果国内集中力量搞aptamer可能会有好的前景？ |
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w******e
发帖数: 1187 | 14 两个如此不靠谱的东西在一起居然能在老鼠里work，连我都没太敢信，但毕竟已经
是第三篇paper了。
btw谁能下全文麻烦发我一份pdf吧。email: q****[email protected]. 多谢！
An Aptamer-siRNA Chimera Suppresses HIV-1 Viral Loads and Protects from
Helper
CD4+ T Cell Decline in Humanized Mice
Charles Preston Neff, Jiehua Zhou, Leila Remling, Jes Kuruvilla, Jane Zhang
, Haitang Li, David D. Smith, Piotr Swiderski, John J. Rossi, and Ramesh
Akkina
Sci Transl Med 19 January 2011 3:66ra6 |
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w******e
发帖数: 1187 | 15 前两篇基本算是一个组做的（加上nature chemical biology那个结果
爆烂的in vivo SELEX lol），最近这篇是city of hope的John Rossi
出的，aptamer都是自力更生搞出来的，很是佩服 |
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T**********t
发帖数: 1604 | 16 哦，我没仔细看，以为都是Rossi组做的呢。他们已经发了两篇了。09年有一篇NAR也是
Aptamer-siRNA delivery的。我看paper不做笔记，看了白看。。。以后要改。
你等一下，我一会把这篇新的发给你。你说的另外两篇是什么？ |
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w******e
发帖数: 1187 | 17 aptamer的targeted delivery 也太hot了。我去年刚看到NB那篇paper时很fan了
一阵子。你现在做一个in vivo SELEX说不定还能上baby N/S呵呵 |
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h*****o
发帖数: 342 | 18 他那个ACPP放大信号，所以比antibody或者aptamer做的标记好。我不是很了解后两者
用来做optical imaging的时候对比啊分辨率啊情况到底怎么样。antibody用来做PET的
比较多 |
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h*****o
发帖数: 342 | 19 什么是thrombin aptamer，有没有入门文章看一下？ |
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m********8
发帖数: 314 | 20 aptamer探针在原理上是否比基于抗体的探针更优越?
谢谢。 |
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w******e
发帖数: 1187 | 21 what do you mean by 原理？
the status quo is, aptamer can't compete with abs on affinity in most cases,
but can handle some targets that abs can not (e.g., small molecules like
cocaine)
Things may change in the future -- read some somalogic's recent papers and
you'll see |
|
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k****o
发帖数: 728 | 23 好像主要用在RNA上吧。DNA的aptamer行吗？ |
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h**********r
发帖数: 671 | 24 LZ啊，在给我下一篇吧。同一期的：
Title:
Aptamers for allosteric regulation.
Nat Chem Biol. 2011 Jul 18;7(8):519-27. doi: 10.1038/nchembio.609.
多谢啊！同样有包子！ |
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r*******6
发帖数: 545 | 25 谢谢！还有一点不太清楚：
for 1）aptamer中碱基上的NH2受到共轭系统的影响是指所有NH2，还是只有成环部分的
NH2？直链部分的NH2反应活性和3',5'一样吗？ |
|
f***a
发帖数: 11477 | 26 多精细的要求啊？？
粗略的，紫外吸收。melting temperature
精细的，需要标记aptamer不同位点，测荧光共振能量差异。 |
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d*********e
发帖数: 15 | 27 How is RNA/DNA or protein aptamer in drug discovery ? Is it a promising
field to work on in pharmaceutical / Biotech companies ? |
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f********n
发帖数: 6465 | 28 Aptamers are oligonucleic acid or peptide molecules that bind to a specific
target molecule. Aptamers are usually created by selecting them from a large
random sequence pool, but natural aptamers also exist in riboswitches.
Aptamers can be used for both basic research and clinical purposes as
macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave
in the presence of their target molecule. These compound molecules have
additional research, industrial and clinical applications... 阅读全帖 |
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w******e
发帖数: 1187 | 29 hehe，跟aptamer沾边的人也一看就知道是gold同学的作品。你是做protein array的？
能不能评价一下，跟antibody array相比，performance上面高下如何？
实话讲，我虽然是做aptamer的，但自己都不敢相信突然冒出800多能跟antibody相媲美
的aptamer来。。。大部分做aptamer的组还都在一个aptamer做几年的阶段呢。。。
ARRAY |
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w******e
发帖数: 1187 | 30 it's very easy to couple aptamers to sensor. in fact sensor field
is dominated by aptamer as affinity agent as the stability &
reusability is a big deal for sensors.
back to your questions, you can do thiol-gold, EDC-NHS, biotin-SA...
all you need is to synthesize the aptamer w/ desired functional group.
so are you doing arrays? or beads, like luminex? you may want to check
out larry gold's recent publication
I'd certainly like to, if it doesn't take too much time. any suggestions?
I'm confiden... 阅读全帖 |
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s******y
发帖数: 28562 | 31 那么目前用多肽做出来的aptamer, 最高结合力有多少？
能不能给我推荐几个peptide aptamer and protein target pair? 最好是结合力比较
高一点的，而且靶蛋白本身是可溶的（最好不要是膜蛋白）。
我对这个一直有兴趣，很想知道有什么已知的 peptide aptamer kd <10E-8 的？
先谢谢了！
receptors
each
interaction |
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w******e
发帖数: 1187 | 32 I used native PAGE gel. I kinda hesitate to use competitors as I
don't know how that would affect aptamer band formation (e.g., would
there be an aptamer-BSA band if I add BSA?).
I also suspect oligomer, but even that can't explain why all my
aptamers are sucked onto it...@@
BTW, I actually tried doing ELISA against the same target using
biotinylated RNA, and that worked out fine. |
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a****o
发帖数: 1786 | 33 I think it is normal. Since you use pool, not all mmbers can bind to your
protein. if only 20% of the pool are real aptamers, it fit the result very
well. Even with real defined aptamer, you may not get 100% binding. RNA
aptamers can form different structures, maybe only one or a few of them bind
to your target. |
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g********d
发帖数: 4174 | 34 Posted on Advocate.com January 24, 2011
Calif. Doctor Develops HIV Smart Bomb
By Advocate.com Editors
Animal trials are showing promise for a new HIV treatment that attaches
itself to the virus like a magnet and then stops it from spreading.
John Rossi, chair of molecular and cellular biology at the City of Hope
hospital in Duarte, Calif., is the lead author on the study of the new
therapy, which utilized mice. Rossi describes the treatment as a "smart bomb
" against HIV.
"This particular approa... 阅读全帖 |
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s*****i
发帖数: 74 | 35 【 以下文字转载自 I140 讨论区 】
发信人: aptamer (aptamer), 信区: I140
标 题: TSC EB1A (12/10/2014) Approved non-PP
发信站: BBS 未名空间站 (Mon Apr 20 23:24:37 2015, 美东)
-Timeline:
Jun. 2014 signed contract
Sep. 2014 finished research summary
End of Sep. 2014, all recommendation letters drafted
End of Oct. 2014: collected all of 6 recommendation letters
Nov. 2014: petition letter drafted and prepared all documents
Dec.1st 2014: e-File
Dec.10th 2014: receipt received
Apr.16th 2015: Approval email from USCIS
Apr.20th 2015... 阅读全帖 |
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w******e
发帖数: 1187 | 36 首先得能发现个体的特异性差别。DNA/RNA的估计能指望NGS，protein要指望antibody
array/MS似乎还有很长一段路（希望aptamer array能迎头赶上呵呵）。小分子（
glycan
，metabolite，etc。最近出了个sarcosine）呢？lectin特异性太烂。希望aptamer
能顶上？不然也许只能指望HPLC之类能high throughput了？
其次得能把差别和各种physiological/pathological condition联系起来。这个ms进
展也太太太太慢了吧？就说cancer吧，有几个大家都服气的diagnostic/prognostic
/endpoint biomarker？一个PSA用了这么久，还一直被批。
最后要能根据这些差别来调节therapeutic regimen。Y/N型的或差别大的好办，学
herceptin
附赠个kit就是。检测分辨率不够的话呢？最好还是能够下一样的药，对不同的人达
到不同的效果，controlled release？也许就要在delivery method上做文章了？各
种deliv |
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w******e
发帖数: 1187 | 37 I hope I knew this earlier...:)
I guess you are talking about endosomal escape? or chemical conjugation
simply
abrogate the drug property?
I'm hooked with the simplicity of conjugating aptamer with siRNA. both can
achieve certain level of specificity; both can be synthesized in a single
run; the toxicity profile of ONs are widely available due to the gold
rush in anti-sense field... Yet it's a shame that you only have that few
aptamers to choose from (well, only 1 or 2, according to my knowledge |
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w******e
发帖数: 1187 | 38 同意你说的问题。特异性方面，主要是biomarker的validation严重滞后于
discovery。查文献可以找出一堆特异性marker，鬼都不follow up。。。
AACR上听了一个early marker validation的initiative，很多negative data。。。
deliver方面，问题的根源在于大家都柿子捡软的捏，能只做cell culture
绝不做老鼠，能只做intratumor injection决不做systemic administration，
能只做subcutaeous model by tumor cell injection决不做orthotopic/GEM model，
一旦热点过了就换一个概念接着从最低级的model做起（话说到了2006用aptamer
还能只做cell culture就发nature biotech，到了2009还能用aptamer+NP只做
cell culture发angwandte chemie。。。可惜等我跳进来好日子就到头了。。。），
当然对real worldproblem指导意义有限。。。 |
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w******e
发帖数: 1187 | 39 用radiolabeled RNA aptamer+varying amount of protein，gel shift测affinity，
develop出来所有的protein concentration都只有一条很靠近加样口的band（很
怀疑根本没进gel），而且确定跟aptamer alone的band不是一条。请教有人遇到
这种情况没有？and如果确定了那条band根本就是在加样口附近，问题怎么解决？
protein确实挺大（250kd），但不应该进不了gel。
//bow |
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a****o
发帖数: 1786 | 40 One extra lane will tell you if your aptamer bind to BSA. This is kind of
necessary control. you need to show ur aptamer not bind to any unrelated
proteins.
you can try 0.5x TBE, lower concentrration of acrylamide, I usually use 6%.
I tried 4% before, it is not a big deal.
silicate only one plate, it is very easy to separate your gel from that
plate, but stick to the other plate. put one a piece of DE filter will
transfer the whole gel to filter.
Good luck.
I suspect the protein gel system will |
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w******e
发帖数: 1187 | 41 nod，我进lab之后也经过一阵郁闷期，后来想通了：比aptamer更忽悠的东西多了去了，
加上现在好歹aptamer专利快过期＋借siRNA的东风RNA的PKPD study会越来越多，RNA
chemistry会越来越成熟廉价，现在逢低买入，观望个一两年，实在不行尽早止损~ |
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w******e
发帖数: 1187 | 42
sorry, 没看懂-_-
我的目的是测一个蛋白，想用该蛋白的一个interacting peptide做capturing agent，
用aptamer做detection agent，用peptide instead of Ab做capturing agent的
原因是1. 担心aptamer跟ab有steric hindrance；2. 不想用ab呵呵 |
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w******e
发帖数: 1187 | 43 多谢啦。aptamer在价格方面肯定有竞争里，performance嘛，现在跟
commercial kit比不太现实，我也就是做点proof of concept，证明
aptamer在各种ELISA scheme下都work呵呵 |
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w******e
发帖数: 1187 | 44 我做的integrin的aptamer转让给你吧呵呵。要我做你想要的aptamer的话你得赞助哈哈~ |
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w******e
发帖数: 1187 | 45 1. Applied Immunohistochemistry & Molecular Morphology:
POST AUTHOR CORRECTIONS, 7 January 2011
doi: 10.1097/PAI.0b013e3182008c29
Rapid Histochemistry Using Slow Off-rate Modified Aptamers With Anionic
Competition
2.BIOLOGICAL MICROARRAYS
Methods in Molecular Biology?, 2011, Volume 671, Part 1, 35-54, DOI: 10.1007
/978-1-59745-551-0_2
Aptamer Arrays
3. Functional Nucleic Acids as Molecular Recognition Elements for Small
Organic and Biological Molecules
Authors: Sai Lau, Pui; Li, Yingfu
Source: C... 阅读全帖 |
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w******e
发帖数: 1187 | 46 其实一个biotin跟SA的binding ms没想象的NB。我把biotinylated aptamer
coat到SA beads上做过sandwich assay，结果发现heat后掉下来的
aptamer造成极大background。杯具。。。 |
|
w******e
发帖数: 1187 | 47 Expert Opinion on Medical Diagnostics
Aptamer amplification: divide and signal
December 2008, Vol. 2, No. 12 , Pages 1333-1346 (doi:10.1517/
17530050802562016
)
Supriya Pai1 BS, Ana Roberts1 BS & Andrew D Ellington
2.Expert Opin Drug Discov. 2011 Jan 1;6(1):75-87.
Strategies for the discovery of therapeutic Aptamers.
Yang X, Li N, Gorenstein DG.
email: q************[email protected]
Thank you! |
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a********n
发帖数: 844 | 48 如果lz在讲附上的胶，我的看法是：
1.上面那个带是well里边滞留的，下面那个是free CTP还是free RNA不好说。但整个胶
曝光时间不够长。我做过无数EMSA，也是RNA aptamer，RNA 从MAXIscript得到后不用
纯化，直接取1 ul去跑denaturing gel，看看有没有transcripts，产量有可能因为变
化的，所以每批都要检验。我组里有个博后不信邪，在这上面浪费了几个星期，一直以
为是这个那个仪器不好用。
2.lz提到protine minus也有多条带，这在native gel里边太正常了，因为RNA有多种
folding的可能性，即使mfold只给出一种结构，我在胶里也能看到很多条带。蛋白特异
性的带可能跟RNA一下带superimpose，但是你会看到下边一条带变弱，上面一条带变强
，这样你要换种浓度的胶跑跑。
3.制胶和电泳液可以用1/4xTBE或者1/2xTGB，前者我用的比较多，这个每个蛋白性质有
关，一般都要试试。
4.ARGA胶有些时候可以替代page，想B52的RNA APTAMER只能在ARGA胶才能看到shift，
原因不... 阅读全帖 |
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w******e
发帖数: 1187 | 49 en，技术上没什么创新，但点子不错。
话说aptamer领域打酱油的人太多了，既有生物头脑又自己做selex的太少，
而用现成的aptamer就很被掣肘 |
|
s******s
发帖数: 13035 | 50 大家参考一下。其实我是没有精力做的，有人成功的话给我acknowledge就够了。
找你要研究的基因的BAC，然后再E.Coli里面通过homologous recombination在
有兴趣的片段（当然要比较长的）两头加rare的RE site或者ZFN一类的，随便折
腾；里面再加点strong的aptamer, 或者变态点，加一堆lacO.
这玩意儿做成转基因IPS或者ES，好处是可以多copy, 十个八个没问题吧，更多
应该也行吧? 然后做成转基因小鼠，取你有兴趣的特定组织，或者细胞培养，
做ChIP的前面部分就是fix，洗掉细胞质，然后instead of sonication, 用RE
一顿乱切，配合aptamer purification. 你可以先size select然后affinity,
或者先affinity然后size select, 或者几种不同的方法series affinity，
anyway, 拿到东西decrosslinking上ms spec, 当然前面crosslink的时候不能
用glycine quench.
这个思路欢迎大家补充。 |
|
