SUMMARY Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insights. The comprehensive single cell dataset permits mapping of the mouse hematopoietic stem cell (HSC) differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems.

(3′-2′)-R-L-Threose nucleic acid (TNA, Figure 1A) is an unnatural nucleic acid that was identified during an extensive evaluation of alternative sugar-phosphate backbones aimed at explaining the structure of the biological nucleic acids. 1,2 TNA possesses the ability to specifically base-pair with RNA, DNA, and itself. 2 This capability, together with the chemical simplicity of threose relative to ribose, suggests that TNA could have acted as an evolutionary competitor of RNA or even have preceded RNA as the genetic molecule of life. We are attempting to investigate the functional potential of TNA by implementing an in vitro selection scheme for TNA. 3,4 Here, we show that a mutant archaeal family B DNA polymerase is capable of polymerizing more than 50 nucleotides of TNA on a DNA template. We also demonstrate the display of single-stranded TNA covalently linked to its encoding duplex DNA, thus enabling the selection of functional TNA sequences and the amplification or recovery of the attached DNA.We and others have previously shown that certain family B archaeal DNA polymerases possess the ability to synthesize limited stretches of TNA on a DNA template. 5,6 Our recent synthesis of all four TNA triphosphates (tNTPs) enabled us to test polymerases for more extensive activity. 7 The Therminator DNA polymerase is an engineered exonuclease-deficient form of "9°N" DNA polymerase containing an A485L mutation. 8 It is capable of efficiently incorporating a wide spectrum of modified nucleotides. We tested the ability of this polymerase to accept tNTPs as substrates using a DNA primer/template construct containing a 50-nucleotide singlestranded template region in which all four DNA nucleobases were represented ( Figure 1B). Since previous work had shown that pairing diaminopurine opposite thymine increases the efficiencies of both template-directed ligation and polymerization, 5,9 we used diaminopurine triphosphate (tDTP) instead of tATP. The Therminator polymerase catalyzed the synthesis of >20% full-length 50-nucleotide TNA product within 24 h ( Figure 1C).We reasoned that if transcribed TNA could be covalently linked to its DNA template, we could perform functional selections for TNA molecules and rescue the successful genotypes by PCR amplification of the attached DNA. This approach is analogous to the selection of functional peptides and proteins by mRNA display; 10 the use of DNA display for peptide and PNA selections has also been proposed. 10,11 By starting with a library of single-stranded DNA hairpins, the 3′ end of each hairpin could act as a primer for TNA transcription across the randomized DNA template region (Figure 2A). A primer annealed to the loop region of the hairpin could then initiate strand-displacement synthesis, liberating the TNA strand to allow folding and linearizing the DNA template by making it double-stranded.To test this idea, we synthesized a single-stranded DNA hairpin and transcribed the 60-nucleotide single-stranded region using the Therminator DNA polymerase and tNTPs ( Figure 2B). N...