Jeffrey C F Lam

McMaster University, Hamilton, Ontario, Canada

Are you Jeffrey C F Lam?

Claim your profile

Publications (10)62.3 Total impact

  • Jeffrey C F Lam, Samantha O Kwan, Yingfu Li
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8-17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8-17 sequences obtained from a previous in vitro selection experiment wherein the 8-17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (k(obs) from 0.003 to 1 min(-1)), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.
    Molecular BioSystems 01/2011; 7(7):2139-46. · 3.35 Impact Factor
  • Jeffrey C F Lam, Yingfu Li
    [Show abstract] [Hide abstract]
    ABSTRACT: 8-17 is a DNAzyme with metal-dependent endoribonuclease activity. Recently, a variant termed 8-17NG was reported as the first nucleic acid enzyme capable of cleaving all 16 dinucleotide junctions of RNA with rate enhancements ranging from 1000- to 1,000,000,000-fold over background activity. We attributed this broad-ranging cleavage efficiency to global folding of the DNAzyme. We sought to examine the influence of dinucleotides at the cleavage site of 8-17NG on global folding by using three-color (3c) FRET. By comparing the folding of 8-17NG with all 16 possible dinucleotide junctions, we found all examined DNAzyme-substrate constructs adopted a two-step folding process in the presence of Mn(2+), which was consistent with previous metal-induced folding studies of 8-17. Interestingly, Mn(2+) titration experiments also suggest that the second folding step is dependent on dinucleotide identity: purine-purine junctions allowed 8-17NG to fold at lower concentrations than pyrimidine-pyrimidine linkages. This finding was corroborated by RNA cleavage assays, in which the largest improvement in cleavage yield was observed in pyrimidine-pyrimidine junctions when [Mn(2+)] was increased. Taken together, these results support the previously observed hierarchy of 8-17 activity for different cleavage sites. Complemented by earlier sequence and structure-function studies, this investigation allowed for the first detailed examination of crucial relationships between the structural influence and junction preferences of nucleic acid-catalyzed RNA cleavage reactions.
    ChemBioChem 08/2010; 11(12):1710-9. · 3.74 Impact Factor
  • Jeffrey C F Lam, Johanna B Withers, Yingfu Li
    [Show abstract] [Hide abstract]
    ABSTRACT: Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k(obs) of approximately 0.001 min-1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k(obs) of approximately 0.1 min-1 were obtained from a DNAzyme pool. The most efficient motif, denoted "CT10-3.29," was found to have a catalytic core of approximately 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k(obs) of 0.3-1.4 min-1 against the rC-T junction. CT10-3.29 also shows strong activity (k(obs)>0.1 min-1) for rU-A and rU-T junctions, medium activity (>0.01 min-1) for rC-A and rA-T junctions, and weak activity (>0.001 min-1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.
    Journal of Molecular Biology 07/2010; 400(4):689-701. · 3.91 Impact Factor
  • Source
    Jeffrey C. F. Lam, Sergio Aguirre, Yingfu Li
    05/2010: pages 401 - 431; , ISBN: 9780470664001
  • Source
    Kenny Schlosser, Jeffrey C F Lam, Yingfu Li
    [Show abstract] [Hide abstract]
    ABSTRACT: Herein, we describe a case study into the population dynamics of in vitro selection, using RNA-cleaving DNAzymes as a model system. We sought to understand how the composition of the population can change over time in response to different levels of selection pressure, and how well these changes are correlated with selection of the target phenotype. The model population is composed of 857 DNAzyme clones representing 215 discrete sequence classes, which had previously been identified from two parallel selection experiments, conducted under an increasingly stringent, or permissive and constant selection time pressure. In this report, we determined the principal phenotypic properties (i.e. k(obs), maximum cleavage yield and PCR efficiency) from a sample of 58 clones representing 46 different major and minor sequence classes from various rounds of each selection experiment. Interestingly, a positive correlation between the catalytic rate constant and the corresponding frequency and temporal position of a given DNAzyme was not consistently observed; however, the strength of the correlation was qualitatively higher under conditions of more stringent selection time pressure. These results suggest that the selective sampling paradigm on which in vitro selection is based, may underestimate the true functional capacity of any given random-sequence library.
    Nucleic Acids Research 05/2009; 37(11):3545-57. · 8.81 Impact Factor
  • Source
    Kenny Schlosser, Jimmy Gu, Jeffrey C F Lam, Yingfu Li
    [Show abstract] [Hide abstract]
    ABSTRACT: Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine-pyrimidine (pyr-pyr) junctions, which are cleaved 1-3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr-pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited k(obs) values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited approximately 1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.
    Nucleic Acids Research 08/2008; 36(14):4768-77. · 8.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.
    Small 07/2008; 4(6):810-6. · 7.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.
    Journal of the American Chemical Society 04/2008; 130(11):3610-8. · 10.68 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Non-crosslinking gold nanoparticle (AuNP) aggregation induced by the loss (or screen) of surface charges is applied for enzymatic activity sensing and potentially inhibitor screening.
    Chemical Communications 10/2007; · 6.38 Impact Factor
  • Source
    Kenny Schlosser, Jeffrey C F Lam, Yingfu Li
    [Show abstract] [Hide abstract]
    ABSTRACT: We previously conducted an in vitro selection experiment for RNA-cleaving deoxyribozymes, using a combinatorial DNA library containing 80 random nucleotides. Ultimately, 110 different sequence classes were isolated, but the vast majority contained a short14-15 nt catalytic DNA motif commonly known as 8-17. Herein, we report extensive truncation experiments conducted on multiple sequence classes to confirm the suspected catalytic role played by 8-17 and to determine the effect of excess sequence elements on the activity of this motif and the outcome of selection. Although we observed beneficial, detrimental and neutral consequences for activity, the magnitude of the effect rarely exceeded 2-fold. These deoxyribozymes appear to have survived increasing selection pressure despite the presence of additional sequence elements, rather than because of them. A new deoxyribozyme with comparable activity, called G15-30, was approximately 2.5-fold larger and experienced a approximately 4-fold greater inhibitory effect from excess sequence elements than the average 8-17 motif. Our results suggest that 8-17 may be less susceptible to the potential inhibitory effects of excess arbitrary sequence than larger motifs, which represents a previously unappreciated selective advantage that may contribute to its widespread recurrence.
    Nucleic Acids Research 02/2006; 34(8):2445-54. · 8.81 Impact Factor

Publication Stats

191 Citations
62.30 Total Impact Points

Institutions

  • 2006–2011
    • McMaster University
      • • Department of Biochemistry and Biomedical Sciences
      • • Department of Chemistry and Chemical Biology
      Hamilton, Ontario, Canada
  • 2010
    • University of Strathclyde
      • Strathclyde Institute of Pharmacy and Biomedical Sciences
      Glasgow, SCT, United Kingdom