SmartFlare live RNA detection

I recently came across a booklet regarding SmartFlare for live cell RNA level detection. The basic idea, if i understand it correctly, is the use of gold nanoparticles that are conjugated to fluorescently tagged short double stranded probes. The “capture” strand is bound to the gold particle, is non-fluorescent and complementary to the target mRNA. The “reporter” strand is complementary to the capture strand but shorter. It is fluorescently labeled, but the gold is quenching the fluorescence.

This particle can enter the cells and if the target RNA is present, it replaces the “reporter” strand which is set free to the cytoplasm and fluoresce. This fluorescent can then be detected by microscopy or any other fluorescent detection.

SmartFlare concept. Source: Merck Millipore website

I haven’t used it yet because it clearly isn’t suited for single-molecule analysis. Furthermore, is does not give any data on RNA localization since the fluorescence appears only after the target RNA is bound and the “reporter” is a short oligo which probably diffuses very fast.

Furthermore, I’m concerned about the biological effects. Although they claim in their FAQ section that at the concentrations used it does not cause RNA knockdown and is non-toxic, I am concerned first about the short reported oligo which presumably will hybridize with antisense to the target RNA, if present and second, that capturing the target RNA will affect its gene expression (probably prevent its translation, maybe mis-localize it and can promote its decay thus actually affect its transcription rate as well).

Particularly I’m concerned due to the long incubation time they suggest in their protocol – 18 hours. Why so long? Shouldn’t it work at shorter time-scales?

This system can be a good reporter for relative expression levels for whole cell populations or single-cell analysis, but I’m hesitant about the use of these same cells for downstream assays.

Also, their illustrations always show complementary of the capture strand to the 5′ end of the target RNA. Does it have to be the 5′ end?

I am considering the use of this system as a reporter for my experimental purposes. Any thought? Did anyone use that and share their experience?

Nobel prize in chemistry 2014

The Nobel prize in chemistry 2014 was awarded to Eric Betzig, Stefan W. Hell & William E. Moerner for the development of Super resolution fluorescent microscopy!

See here for details on the history of this discovery.

As readers of this blog know, Super-resolution microscopy has made a revolution in the field of fluorescent microscopy in a very short time.

This is a justified award.

Eliminating mutated mitochondria during in-vitro fertilization

There are several genetic diseases which originate not from mutations in the nuclear genome but mutations in the mitochondrial genome. In humans, the threshold for disease occurrence is if 60% of the mitochondria has mutated mitochondrial DNA (mtDNA) (a mixed mitochondrial origin is called heteroplasmy). There is currently no cure, and no good way to prevent these genetic diseases.

Since the source of the mitochondria is solely from the oocyte, a lot of effort is invested in trying to get rid of mutated mitochondria by in-vitro fertilization (IVF) procedures – thus preventing the disease in the offspring. Two methods have been tried so far – pronuclear transfer (PNT) and spindle-chromosome transfer (ST).  The idea is to extract the nuclear genetic material of the oocyte or zygote and transfer it to an enucleated oocyte or zygote with healthy mitochondria. However, in both cases, there is still carry-over of some mutated mitochondria to levels that can be as high as 44%.

In this new paper published in Cell, a group of researchers from China suggest a different approach. During the oocyte, and later zygote development, a unique germ cell that is extruded. These are called polar body 1 (PB1), a diploid germ cell, which is extruded from the oocyte before ovulation and PB2, a haploid  germ cell, which is extruded from the zygote after fertilization. The authors reasoned that the nuclear genetic material in PB1 and PB2 is identical to the spindle chromosome and female pronuclear, respectively.  But, PB1 and PB2 are very small cells and therefore may contain very few mitochondria.

Levels of mitochondria in the different nuclei. Red - MitoTracker. Blue: Hoecht (stains DNA).

Levels of mitochondria in the different nuclei. Red – MitoTracker. Blue: Hoecht (stains DNA). Source: Wang T. et. al. (2014) Cell 157:1591-1604.

Indeed, staining with MitoTracker (a dye specific for mitochondria) shows only a small amount of mitochondria in PB1 and PB2.  So, after several tested to see if the PB1 and PB2 has characteristics similar to the corresponding oocyte/zygote nuclei (using immunofluorescence against specific nuclear and epigenetic markers) they compared the four different methods (PNT, ST and PB1 or PB2 transfer) to prevent mitochondrial transfer in IVF.

IF staining with antibody against Lamin B1 (green) shows the nuclear envelope of the different nuclie at distinct stages of zygote development. Red- propidium iodide (PI) stains DNA. Source: Wang T. et. al. (2014) Cell 157:1591-1604

IF staining with antibody against Lamin B1 (green) shows the nuclear envelope of the different nuclie at distinct stages of zygote development. Red- propidium iodide (PI) stains DNA. Source: Wang T. et. al. (2014) Cell 157:1591-1604

Their results are pretty amazing.

All methods have similar success rates of the IVF procedure and give similar number of offspring, with normal development. However, PNT still maintains high levels of heteroplasmy (20-60% in 2nd generation (F2) mice). ST maintains moderate-low heteroplasmy (5-20% in F2). PB2T has even lower heteroplasmy rates (<5% in F2). But PB1T has no heteroplasmy (at least, below the detection level).

This work, done in mice, is a real breakthrough , because if this also works in humans then we can have genetic screens for heteroplasmy and women with high levels of heteroplasmy (or already suffering from an inherited mitochondrial disease) could use PB1T-IVF to give birth to healthy children.

Pretty awesome.
ResearchBlogging.orgWang, T., Sha, H., Ji, D., Zhang, H., Chen, D., Cao, Y., & Zhu, J. (2014). Polar Body Genome Transfer for Preventing the Transmission of Inherited Mitochondrial Diseases Cell, 157 (7), 1591-1604 DOI: 10.1016/j.cell.2014.04.042

An excelent new tool for comparing fluorescent protein properties

Screenshot of the new tool

Screenshot of the new tool

Two very useful tools for visualizations of many fluorescent and photoswitchable proteins have been developed by Talley Lambert and Kurt Thorn (UCSF).

With these tools you can compare the properties of the different FPs by changing the X & Y axes parameters & range on the left. Play with it. Its fun!

If you want exact numbers, there are extensive tables and references below the graph.

Thanks to Optical Nanoscopy Blog for pointing this out.

 

Announcements

I am aware that it has been a while since I updated new posts on fluorescent microscopy. I blame life. And work.
Anyway, I have several papers piled up, waiting to get blogged.

In other news, I wrote a blog post for Addgene blog. Addgene, for those of you who do not know, is a non-profit organization that helps biologists share plasmids. Briefly, researchers can send Addgene their plasmids (which were featured in their publications) and other researches can order these plasmids from Addgene at a relatively small fee (relative to the amount invested in cloning a plasmid). My post is about how to choose the best suited fluorescent protein, something I wrote about in this blog sporadically. It should get published soon.

On May 22 I’m going to present my work at the annual meeting of the Israeli Society for Cancer Research.

On July 12-13 I’m going to present my work at the Gordon-Merck Research Seminar on Post-Transcriptional Gene Regulation  and the very next day at the Gordon Research conference on the same topic. You can still apply (for poster presentation only) for both of these.

On the publications’s front, a revised version of a paper I co-authored (which continues my work on the decay-transcription story) was recently sent back to the editors. We’re hoping for good news soon.

Am co-authoring review paper on mRNA localization for Nature Reviews Molecular & Cell Biology. Hope to submit within a couple of weeks.

And, the research I’ve been working on for the past two years is finally starting to take shape as a paper. Hope to submit within a month or so.

Also, for the first time, I’m now an official reviewer of a peer-reviewed journal ( RNA journal ).

The next evolutionary step

Human have always tried to improve on nature, from domestication of plants & animals through directed evolution in the test tube and GMO and up to Craig Venter’s synthetic bacteria and the expansion of the genetic code.
Today, another step was taken towards creating completely artificial life.

The natural genome is composed of four bases, which form two pairs: A-T and G-C. This pairing allows the double-helical DNA to maintain the information upon replication (the  semi-conservative replication).

The group of Floyd Romesberg from the Scripps institute now published a paper in Nature titled “A semi-synthetic organism with an expanded genetic alphabet”. I think that the title is a bit misleading, since its not really semi-synthetic, but is still awesome.

Roemsberg have previously published several papers on a synthetic deoxyribonucleotide pair: d5SICS and dNaM (I have failed to discover what these abbreviations stand for). These bases create an unnatural base-pair, yet it is still recognized, in vitro, by DNA and RNA polymerases and is maintained upon semiconservative replication (e.g. by PCR).

Now, they incorporated this pair into the bacteria E. coli to see if this pair can be maintained in vivo.

Unnatural (d5SICS-dNaM) and natural Watson–Crick (dC-dG) base pairs. Source: Malyshev et al. (2012) PNAS vol 109:12005-12010.

Chemical structures and the unnatural (d5SICS-dNaM) and natural Watson–Crick (dC-dG) base pairs. Source: Malyshev et al. (2012) PNAS vol 109:12005-12010.

The main problem going in vivo is how to get these bases into the bacteria, and how to get achieve the tri-phosphate (tri-P) form that can be incorporated by the DNA polymerase. They decided to use synthetic tri-P forms and screen to a transported that can import them into the cell. Having found a suitable nucleotide triphosphate transporters (NTT) from Phaeodactylum tricornutum and solved a problem of de-phosphorylation of the tri-P bases before they enter the cell, they set out to test if the bases-pair can be maintained in the E. coli cell.

To this end, they synthetically created a plasmid that harbors one d5SIC-dNaM base pair at a specific location. After transformation, they show that this base-pair is maintained at 95-97% of the plasmids for 15hr (24 generations). If they let the bacteria grow to stationary phase without supplementing them with fresh media (with fresh synthetic bases) then they slowly lose this pair to an A-T pair. but even after 6 days, ~10% of plasmids maintain this pair. This suggests that the replacement is not due to repair but due to the polymerase errors, and depletion of the bases in the media (though they don’t actually prove its not repair).

At this point, the paper ends. So, first of all, having one base pair is not semi-synthetic. I was disappointment :-)

Second, I hope these experiments can be repeated by other labs, and we will not get another “arsenic life” scandal.  But by the looks of it, it seems more reliable.

Third, this could be a platform for real synthetic biology, with a 6-letter code, instead of the 4 we have now, (that’s 216 3-letter codons, instead of the 64 we naturally have.  If we go to 4-letter codons, that’s 1296 codons to use.)

These could be used to create synthetic, unnatural proteins to produce new products; ribozymes and other RNA structures; imagine gene therapy that can only be maintained by ingesting these synthetic bases and I’m sure there are many options I haven’t thought of. In terms of this blog – maybe fluorescent proteins or RNA aptamers with interesting or useful properties (e.g. fast folding, very bright, etc..).

And last, this is the first proof that a DNA molecule does not have to be composed by the bases we’re used to. Aliens from other planets could have DNA molecules with similar double-helix Watson-Crick structure, semiconservative replication, but made out of other ribo nucleotide bases.

At the very least, we can revive dinosaurs without fearing they will run off the island and get their Lysine contingency supplemented elsewhere.

 
ResearchBlogging.orgMalyshev, D., Dhami, K., Lavergne, T., Chen, T., Dai, N., Foster, J., Corrêa, I., & Romesberg, F. (2014). A semi-synthetic organism with an expanded genetic alphabet Nature DOI: 10.1038/nature13314

iBiology Microscopy Course

Originally posted on Optical Nanoscopy Blog:

Light microscopy has become one of the most useful tools in the life sciences. Following the traditions of great courses on light microscopy, such as those offered by the Marine Biological Laboratory, EMBO, and the NCBS in Bangalore, this free online comprehensive course begins with the basics of optics, proceeds through transmitted light microscopy, covers the various methods of imaging fluorescent samples, describes how cameras work and image processing, and concludes with some of the latest advances in light microscopy. In addition to lectures, they also provide labs (filmed at a microscope) and short tips, so as to cover pragmatics of how to use microscopes. Assessments are provided for each lecture. Enjoy learning microscopy!

ibiologyMicroscopyCourseLong

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