Single Nucleic Acid Molecule Manipulation And Single Cell Sequencing

Nucleic acid is well known as the genetic material of all living organisms.And its double-helical structure has become one of the cultural symbols of our civilization.The key feature that makes nucleic acid so well suited to its biological role is the complementarity relationships that form hydrogen-bonded Watson-Crick base pairs:Adenine pairs with thymine(uracil in RNA),and guanine pairs with cytosine.The principle of base pairing make nucleic acid as the most predictable and readily programmed material on nano scale.Based on the principle of base pairing,various complex 2D or 3D nanostructures and molecular machines with different functions have been designed,which can be used for biological analysis,nanoreactor,nanomachine,and drug delivery.The principle of base pairing of the semi-conservative DNA replication,has a special significance,which provides a molecular basis for passing the consistent genetic information from the parent generation to the offspring.As the proverb says,"There are no two identical leaves," and this is truly reflected in the fundamental unit of life-the cell.And there are more and more evidence showing that single cells,even those derived from the same cell line or individual,can present different genomes,transcriptomes and epigenomes due to reprogramming of the genome and epigenome,and DNA replication error during cell division and differentiation,which is"Heterogeneity".Heterogeneity is widely exist in the individual,tissue,and even single cell level.Heterogeneity at single cell level plays an important role in some key life processes,such as embryonic development,stem cell differentiation,cancer generation and development.Single cell DNA/RNA sequencing can reveal the heterogeneity at single cell level,and elucidate the mechanism of cell heterogeneity,which plays a key role in the field of developmental biology,oncology,neurology,microbiology etc.Based on the key functions of nucleic acid,such as nano-assembly,the heredity function and the heterogeneity in single cells,the main work composed by four parts are listed as follows:1.Single-molecule photon-fueled DNA nanoscissors for DNA cleavageA new type of single-molecule photo-responsive DNA nanoscissors for DNA cleavage based on the regulation of substrate binding affinity was designed and fabricated.The DNA nanoscissors were created by attaching complementary azobenzene-modified sequence as a regulatory domain to one end of a DNAzyme.The unimolecular DNA nanodevice can be easily and precisely regulated via the change of irradiation wavelength.The reversible light induced trans-cis transition of azobenzene leads to a dramatic change in substrate binding affintiy,and a sharp difference in the enzymatic activity.Compared with other DNA nanomachines,our DNA nanoscissors have the advantages of a clean switching mechanism,as well as robust and highly reversible operation.Our proposed strategy of substrate binding regulation is also applicable to other DNAzymes,allowing the construction of many other photo-responsive DNAzymes for different substrates.The newly constructed photon-fueled DNA nanoscissors provide a new method for photo-regulation of DNA cleavage with high spatial and temporal resolution,which will be useful for many applications in biotechnology and biomedicine.2.Barcode beads synthesis for single cell sequencingEach barcode bead has four parts:(a)universal primer,a constant sequence for downstream PCR and sequencing;(b)cell barcode,identical for each one bead,but different from the cell barcodes on other beads;(c)a unique molecular identifier(UMI),different from each oligo;and(d)capture probe,an oligo-dT sequence for capturing polyadenylated mRNAs and priming reverse transcription.We develop two methods for barcode beads synthesis.First one is split and pool method based on combinatorial chemistry.After 12 cycles split and pooling,barcode beads with large number of cell barcode can be synthesized with DNA synthesizer based on solid phase synthesis.The other one is emulsion PCR(ePCR)method based on digital PCR.One cell label and one bead is assigned to a tiny droplet with PCR reagents to amplify the cell label on the beads.With hundreds thousands of individual droplets reaction,a large barcode beads pool can be synthesized by one step.Thereby,every lab with PCR machine can make barcode beads.It is simpler and more universal.3.High-throughput single cell mRNA sequencingWe developed a PAIR-CHIP for high-throughput single cell and barcode bead manipulation.Massively parallel manipulation of single cells,including capture,cell and bead pairing,separation,lysis,and mRNA capture et al,can be done by using PAIR-CHIP.The PAIR-CHIP has many advantages,such as high single cell capture efficiency,especially for a small amount of cells or samples(such as CTCs),and high pairing efficiency,easy manipulation,low cost.Then the mRNA transcripts from each cell can be tagged by barcode bead pairing.The pooling tagged mRNAs can be amplified together.After sequencing,the mRNAs for individual cell can be identified by cell barcode,and single cell mRNA transcripts can be counted by UMIs to avoid double-counting sequence reads that arose from the same mRNA transcript.Thereby,a matrix of digital gene-expression measurements(one measurement per gene per cell)can be created for further analysis.With barcoding beads and PAIR-CHIP,we developed a high-throughput single cell mRNA sequencing,which can be used for solving the problems of biological heterogeneity and low quantities of available biological materials.